Engineered stem cell derived grafts and methods of use thereof

Abstract

Provided herein are compositions, kits, and methods related to in vivo monitoring and controlling of cell therapies. In some aspects, the methods relate to administration of cell compositions comprising insulin producing cells (e.g., an insulin producing stem cell-derived pancreatic cell) for treatment of diabetes.

Description

ENGINEERED STEM CELL DERIVED GRAFTS AND METHODS OF USETHEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 662,937 filed on June 21, 2024, the contents of which are incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (MINU_013_01WO_SeqList_ST26.xml; Size: 42,670 bytes; and Date of Creation: June 20, 2025) are herein incorporated by reference in its entirety.BACKGROUND

[0003] A primary aim in regenerative medicine is to transplant cell or tissue grafts that integrate and replace function of tissues or organs that have lost function due to age, disease (e.g., autoimmunity), damage, or congenital defects. Success of such therapies depends, at least in part, on the isolation and generation of stem cell-derived (SCD) cells that can be administered to restore lost tissue or organ function. Pluripotent stem cells (e.g., embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are a primary source of SCD cells as they have the ability to self-renew and differentiate into any cell type in the body, including whatever cell type has been damaged due to disease or injury. For example, SCD neurons, insulin producing cells, heart cells, and retinal cells are of interest for treating neurological disorders (e.g., Alzheimer’s disease or Parkinson’s disease), diabetes, heart disease, and macular degeneration respectively.

[0004] However, administration of SCD cells is associated with numerous safety and efficacy concerns. Safety concerns arise, for example, due to the risk of a SCD cell graft having residual undifferentiated pluripotent stem cells or de-differentiated cells that can grow uncontrollably and form teratomas (i.e., tumors containing all three germ layers). Poor efficacy can result from immune rejection of the SCD cell graft or failure of the graft to receive adequate nutrient and oxygen supply to support engraftment and function. Clinical interventions can be made to promote engraftment (e.g., administering immunosuppressantsto prevent graft rejection), however it is challenging to identify when and to what extent such interventions should be made without a means to monitor the status of the cell graft.

[0005] Thus, there is a need for SCD cell compositions that can be controlled and monitored to ensure adequate and timely clinical interventions are made to support engraftment and to protect against uncontrolled proliferation and immune attack against the graft.SUMMARY

[0006] In some aspects, the disclosure provides a nanoprobe for detecting a target nucleic acid, comprising: (i) a plasmonic-active nanoparticle; (ii) a nucleic acid probe comprising a sequence that forms a stem loop, a first end attached to the nanoparticle, and a second end attached to an optical label; and (iii) a placeholder sequence comprising a first region complementary to a sequence in the target nucleic acid and a second region complementary to the sequence that forms a stem loop or a portion thereof, wherein the second region is shorter than and overlapping the first region, and wherein the target nucleic acid is a microRNA (miRNA) biomarker for inflammation and / or hypoxia.

[0007] In some embodiments of the foregoing or related aspects, the nanoprobe further comprises a second optical label operably linked to the nanoparticle. In some embodiments, the optical label emits a signal at a first Raman shift (cm'1), wherein the second optical label emits a signal at a second, different Raman shift (cm'1). In some embodiments, the signal of the optical label is increased in the presence of the target nucleic acid, wherein the signal of the second optical label is substantially similar in the presence or absence of the target nucleic acid.

[0008] In some aspects, the disclosure provides a nanoprobe, comprising: (i) a plasmonic- active nanoparticle; (ii) a nucleic acid probe comprising (a) a sequence that forms a stem loop, wherein the stem loop has an open and a closed configuration, and wherein the stem loop forms the open configuration in the presence of a second, complementary sequence, (b) a first end attached to the nanoparticle, and (c) a second end attached to an optical label, wherein the optical label emits a signal at a first Raman shift (cm'1), and wherein the signal is increased in the closed configuration relative to the open configuration; and (iii) a second optical label operably linked to the nanoparticle, wherein the second optical label emits a signal at a second, different Raman shift (cm-1) that is substantially similar in the open and closed configuration.

[0009] In some embodiments of the foregoing or related aspects, the second, complementary sequence comprises a target nucleic acid. In some embodiments, the second, complementary sequence comprises a placeholder sequence, wherein the placeholder sequence comprises a first region complementary to a sequence in a target nucleic acid and a second region complementary to the sequence that forms a stem loop or a portion thereof, and wherein the second region is shorter than and overlapping the first region. In some embodiments, the target nucleic acid comprises a DNA or an RNA. In some embodiments, the RNA comprises a pre-mRNA, and mRNA, or a non-coding RNA. In some embodiments, the non-coding RNA is selected from a miRNA, a small interfering RNA (siRNA), a pi wi -interacting RNA (piRNA), a small nuclear RNA (snRNA), a small nucleolar RNA, a ribosomal RNA (rRNA), a transfer RNA (tRNA), a small cajal body-specific RNA (scaRNA), and a circular RNA (circRNA). In some embodiments, the target nucleic acid is a biomarker of proliferation, cell death, inflammation, hypoxia, nutrient deprivation, differentiation, teratoma formation, metabolic stress, endoplasmic reticulum stress, oxidative stress, and / or activation of a cytokine signaling pathway.

[0010] In some embodiments of the foregoing or related aspects, the target nucleic acid is a miRNA biomarker for inflammation. In some embodiments, the target nucleic acid is a miRNA biomarker for cytokine-dependent inflammation. In some embodiments, the miRNA biomarker is selected from the group consisting of miR-203b, miR-302a, miR-155, miR- 146a, miR-302a, miR-21, and a combination thereof. In some embodiments, the miRNA biomarker is selected from the group consisting of miR-203b-3p, miR-302a-5p, miR-155-5p, miR-146a-5p, miR-302a-3p, miR-21-5p, and a combination thereof. In some embodiments, the miRNA biomarker is miR-146a-5p. In some embodiments, the target nucleic acid is a miRNA biomarker for interferon-dependent inflammation. In some embodiments, the miRNA biomarker is selected from the group consisting of miR-224, miR-302b, miR-302a, miR-203a, miR-146b, miR-21, and a combination thereof. In some embodiments, the miRNA biomarker is selected from the group consisting of miR-224-5p, miR-302b-3p, miR-302a-5p, miR-203a-3p, miR-146b-5p, and a combination thereof. In some embodiments, the miRNA biomarker is miR-224-5p. In some embodiments, the target nucleic acid is a miRNA biomarker for hypoxia. In some embodiments, the miRNA biomarker is a 5 'arm (5p) or a 3'arm (3p) of a miRNA selected from the group consisting of miR-210, miR-124, miR-1246, miR-155, miR-205, and a combination thereof. In some embodiments, the miRNA biomarker is selected from the group consisting of miR-210-3p, miR-124-5p, miR-1246-3p, miR-1246-5p, miR-155-5p, miR-205-5p, and a combination thereof. In some embodiments, the miRNA biomarker is miR-210-3p.

[0011] In some embodiments of the foregoing or related aspects, (i) the miRNA biomarker is miR-146a, wherein the nucleic acid probe comprises SEQ ID NO: 24 or 26 or a sequence having at least 80% identity to SEQ ID NO: 24 or 26, and wherein the placeholder sequence comprises SEQ ID NO: 25 or 27 or a sequence having at least 80% identity to SEQ ID NO: 25 or 27; (ii) the miRNA biomarker is miR-224, wherein the nucleic acid probe comprises SEQ ID NO: 28 or a sequence having at least 80% identity to SEQ ID NO: 28, and wherein the placeholder sequence comprises SEQ ID NO: 29 or a sequence having at least 80% identity to SEQ ID NO: 29; (iii) the miRNA biomarker is miR-210, wherein the nucleic acid probe comprises any one of SEQ ID NOS: 32-35, or a sequence having at least 80% identity to SEQ ID NO: 32-35, and wherein the placeholder sequence comprises SEQ ID NO: 36 or a sequence having at least 80% identity to SEQ ID NO: 36; (iv) the miRNA biomarker is miR- 21, wherein the nucleic acid probe comprises SEQ ID NOS: 20 or 22, or a sequence having at least 80% identity to SEQ ID NO: 20 or 22, and wherein the placeholder sequence comprises SEQ ID NO: 21 or 23 or a sequence having at least 80% identity to SEQ ID NO: 21 or 23; (v) the miRNA biomarker is miR-302a, wherein the nucleic acid probe comprises SEQ ID NOS: 30, or a sequence having at least 80% identity to SEQ ID NO: 30, and wherein the placeholder sequence comprises SEQ ID NO: 31 or a sequence having at least 80% identity to SEQ ID NO: 31; (vi) the miRNA biomarker is miR-155, wherein the nucleic acid probe comprises any one of SEQ ID NOS: 37-40, or a sequence having at least 80% identity to SEQ ID NO: 37-40, and wherein the placeholder sequence comprises SEQ ID NO: 41 or a sequence having at least 80% identity to SEQ ID NO: 41; or (vii) the miRNA biomarker is miR-124, wherein the nucleic acid probe comprises any one of SEQ ID NOS: 42-45, or a sequence having at least 80% identity to SEQ ID NO: 42-45, and wherein the placeholder sequence comprises SEQ ID NO: 46 or a sequence having at least 80% identity to SEQ ID NO: 46.

[0012] In some embodiments of the foregoing or related aspects, the plasmonic-active nanoparticle is a nanosphere, a nanoshell, a nanorod, or a nanostar. In some embodiments, the plasmonic-active nanoparticle comprises a single metal or more than one metal, and wherein the metal is selected from the group consisting of gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iron (Fe), copper (Cu), aluminum (Al), and zinc (Zn). In some embodiments, the plasmonic-active nanoparticle is a silver nanosphere, gold nanosphere, silver nanoshell, gold nanoshell, silver nanorod, gold nanorod, silver nanostar, or gold nanostar. In someembodiments, the plasmonic-active nanoparticle is a gold nanostar. In some embodiments, the plasmonic-active nanoparticle is embedded in a silica shell. In some embodiments, the plasmonic-active nanoparticle further comprises a component attached to a surface of the nanoparticle. In some embodiments, the component comprises a synthetic polymer, a cell penetrating peptide, a membrane anchor, a bioreceptor, a synthetic peptide, or a combination thereof. In some embodiments, the component comprises the synthetic polymer, and wherein the synthetic polymer is selected from a PEG, a N-isopropyl acrylamide (NIP AM), or a combination thereof. In some embodiments, the component comprises the cell penetrating peptide, and wherein the cell penetrating peptide comprises a polycationic peptide, a human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription (TAT) peptide, a HIV-1 cysteine-terminated TAT (cTAT) peptide, or a combination thereof. In some embodiments, the component comprises the membrane anchor, and wherein the membrane anchor is selected from a cationic polymer, a lipid, cholesterol or a derivative thereof, porphyrin, tocopherol, or a combination thereof. In some embodiments, the cationic polymer comprises poly-lysine. In some embodiments, the component comprises the bioreceptor, and wherein the bioreceptor comprises an antibody or antigen-binding domain thereof. In some embodiments, the component comprises PEG. In some embodiments, the component comprises poly-lysine. In some embodiments, the component comprises PEG and polylysine.

[0013] In some embodiments of the foregoing or related aspects, the sequence that forms a stem loop, the placeholder sequence, or both comprise at least one modified nucleoside. In some embodiments, the at least one modified nucleoside comprises a 2'-modification or a locked nucleic acid. In some embodiments, the 2'-modification is selected from 2'- aminoethyl, 2'-fluoro, 2'-O-methyl, and 2'-O-methoxyethyl. In some embodiments, the sequence that forms a stem loop, the placeholder sequence, or both comprise at least one modified internucleoside linkage. In some embodiments, the optical label comprises a Raman label, a positively-charged hydrophobic near infrared (NIR) dye, a fluorescence label, or an absorbance label. In some embodiments, the second optical label is linked to a surface of the plasmonic active nanoparticle by a linker. In some embodiments, the second optical label comprises a Raman label, a positively-charged hydrophobic NIR dye, a fluorescence label, or an absorbance label.

[0014] In some aspects, the disclosure provides a cell comprising a nanoprobe described herein. In some embodiments, the cell further comprises at least one second nanoprobe fordetecting a second target nucleic acid. In some embodiments, the cell is a stem cell-derived cell. In some embodiments, the cell is an insulin producing cell.

[0015] In some aspects, the disclosure provides a method of monitoring an engineered cell in a subject for inflammation and / or hypoxia, comprising administering the engineered cell to a transplant site of the subject, wherein the engineered cell comprises a nanoprobe described herein for detecting a target nucleic acid (e.g., miRNA) biomarker for hypoxia and / or inflammation, and detecting an optical signal from the optical label upon exposing the transplant site to electromagnetic radiation, wherein an increase in the optical signal indicates the presence of the target nucleic acid (e.g., miRNA) biomarker of inflammation and / or hypoxia, thereby providing for monitoring of the engineered cell. In some embodiments, the engineered cell comprises a stem cell-derived cell. In some embodiments, the engineered cell comprises an insulin producing cell.

[0016] In some aspects, the disclosure provides a method of monitoring an engineered cell in a subject for inflammation and / or hypoxia, comprising administering the engineered cell to a transplant site of the subject, wherein the engineered cell comprises a nanoprobe described herein for detecting a target nucleic acid (e.g., miRNA) biomarker for hypoxia and / or inflammation, and detecting the signal at the first Raman shift and the signal at the second, different Raman shift upon exposing the treatment site to electromagnetic radiation, wherein an increase in a ratio of the signal at the first Raman shift to the signal at the second, different Raman shift indicates the presence of the target nucleic acid, thereby providing for monitoring of the engineered cell. In some embodiments, the engineered cell comprises a stem cell-derived cell. In some embodiments, the engineered cell comprises an insulin producing cell.

[0017] In some aspects, the disclosure provides a method of characterizing an engineered cell at a transplant site in a subject, comprising administering the engineered cell to the subject, wherein the engineered cell comprises a nanoprobe comprising (a) a plasmonic-active nanoparticle, (b) a nucleic acid probe comprising a sequence that forms a stem loop, wherein the stem loop has an open and a closed configuration, (b) a first end attached to the nanoparticle, and (c) a second end attached to an optical label, wherein the optical label emits a signal at a first Raman shift (cm'1) that is increased in the closed configuration relative to the open configuration, (c) a placeholder sequence configured to maintain the stem loop in the open configuration in the absence of a target nucleic acid, and (d) a second optical label operably linked to the nanoparticle, wherein the second optical label emits a signal at a second, different Raman shift (cm'1) that is substantially similar in the open and closedconfiguration, and detecting the signal and the second signal from the transplant site, wherein an increase in the signal as compared to the second signal indicates presence of the target nucleic acid, thereby characterizing the engineered cell.

[0018] In some embodiments of the foregoing or related aspects, the target nucleic acid is a biomarker of proliferation, cell death, inflammation, hypoxia, nutrient deprivation, differentiation, de-differentiation, teratoma formation, endoplasmic reticulum (ER) stress, metabolic stress, endoplasmic reticulum stress, oxidative stress, and / or activation of a cytokine signaling pathway. In some embodiments, the target nucleic acid is a biomarker of inflammation or hypoxia. In some embodiments, the target nucleic acid is a biomarker of teratoma formation and / or proliferation. In some embodiments, the target nucleic acid is a biomarker of de-differentiation. In some embodiments, the target nucleic acid is a biomarker of metabolic stress. In some embodiments, the target nucleic acid is a biomarker of ER stress.

[0019] In some embodiments of the foregoing or related aspects, the method further comprises administering to the subject at least one immunosuppressant to the subject (e.g., wherein the target nucleic acid is a biomarker of inflammation). In some embodiments, the method further comprises illuminating a three-dimensional volume comprising the transplant site, or portion thereof, with a photothermal wavelength (e.g., wherein the target nucleic acid is a biomarker of proliferation or teratoma formation). In some embodiments, illuminating the three-dimensional volume, or a portion thereof, with the photothermal wavelength induces thermal damage to the engineered cells. In some embodiments, the method further comprises administering a repeat dose of the engineered cell (e.g., wherein the target nucleic acid is a biomarker of dedifferentiation, metabolic stress, or ER stress). In some embodiments, the method further comprises administering a therapeutic modulator of ER stress (e.g., wherein the target nucleic acid is a biomarker of ER stress). In some embodiments, the detecting is by surface enhanced Raman spectroscopy (SERS) or surface enhanced resonance Raman spectroscopy (SERRS). In some embodiments, the electromagnetic radiation comprises a wavelength in the NIR region. In some embodiments, the engineered cell comprises at least one additional nanoprobe, and wherein the method comprises detecting one or more signals from the at least one additional nanoprobe. In some embodiments, the transplant site is subcutaneous or intramuscular. In some embodiments, the engineered cell is a stem-cell derived cell. In some embodiments, engineered cell is an insulin producing cell. In some embodiments, the subject has type I diabetes.

[0020] In some aspects, the disclosure provides a composition comprising (i) an insulin producing cell comprising a nanoprobe, and (ii) one or more factors selected from aproangiogenic factor, a parathyroid gland (PTG) factor, an anti-inflammatory factor, and a combination thereof.

[0021] In some aspects, the disclosure provides a composition comprising a population comprising insulin producing cells, wherein a plurality of insulin producing cells of the population comprise a nanoprobe, and wherein the population comprises at least one function of human islets selected from the group consisting of: (i) glucose-responsive secretion of C- peptide; (ii) glucose-responsive secretion of insulin; (iii) insulin granule biogenesis, trafficking, and / or exocytosis; and (iv) a combination of (i)-(iii). In some embodiments, the plurality of insulin producing cells are present in cell clusters.

[0022] In some aspects, the disclosure provides a method for improving performance of a population comprising insulin producing cells, comprising contacting a population comprising dissociated insulin producing cells with a nanoprobe to induce uptake, and reaggregating to form a population comprising nanoprobe-loaded insulin producing cells, wherein the population comprising nanoprobe-loaded insulin producing cells is characterized by at least one function of human islets selected from the group consisting of: (i) glucoseresponsive secretion of C-peptide; (ii) glucose-responsive secretion of insulin; (iii) insulin granule biogenesis, trafficking, and / or exocytosis; and (iv) a combination of (i)-(iii). In some embodiments, the population comprising nanoprobe-loaded insulin producing cells are present in cell clusters following the reaggregating.

[0023] In some embodiments of the foregoing or related aspects, the at least one function is improved as compared to a control population lacking the nanoprobe. In some embodiments, the population exhibits the at least one function in a manner comparable to human islets. In some embodiments, the at least one function is retained under hypoxic and / or nutrient poor conditions. In some embodiments, the population comprising insulin producing cells secrete an increased level of insulin in response to an increased level of glucose. In some embodiments, the population comprising insulin producing cells is characterized by a glucose stimulated insulin secretion (GSIS) response. In some embodiments, the GSIS response is in vitro and / or in vivo. In some embodiments, the insulin producing cell secretes an increased level of C-peptide in response to an increased level of glucose.

[0024] In some embodiments, the cell clusters comprise a longest diameter of about 50 pm to 350 pm. In some embodiments, the cell clusters comprise a longest diameter of about 100 pm to about 200 pm. In some embodiments, the cell clusters comprise about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 cells. In some embodiments, the population comprising insulinproducing cells are present in a liquid suspension. In some embodiments, the population comprising insulin producing cells is encapsulated in a degradable material. In some embodiments, the population comprises stem cell-derived islets. In some embodiments, the population comprises primary donor islets.

[0025] In some aspects, the disclosure provides a method of controlling stem cell-derived cells at a transplant site in a subject, comprising administering to the subject a population comprising stem cell -derived cells, wherein a plurality of stem cell -derived cells comprise a nanoprobe configured to emit an optical signal upon electromagnetic excitation, and measuring the optical signal across a three-dimensional volume comprising the transplant site at a first time point and at least one additional time point using spectroscopy. In some embodiments, the spectroscopy comprises SERS or SERRS. In some embodiments, the spectroscopy comprises illuminating the three-dimensional volume with electromagnetic excitation, wherein the electromagnetic excitation comprises a wavelength in the NIR region. In some embodiments, the wavelength is about 650 nm to about 900 nm. In some embodiments, the spectroscopy is performed with a laser configured to produce the wavelength, optionally a continuous wave laser. In some embodiments, the laser produces the wavelength at a laser power of about 5 mW to about 20 mW. In some embodiments, the laser produces the wavelength with a power density of about 5 to about 100 milliwatts / cm2. In some embodiments, the spectroscopy is performed with an acquisition time of about 1 second to about 20 seconds. In some embodiments, the wavelength penetrates at a tissue depth of about 1 mm to about 20 mm. In some embodiments, the method further comprises measuring a predetermined change in the optical signal at the first time point as compared to the at least one additional time point. In some embodiments, the method the method further comprises illuminating the three-dimensional volume, or a portion thereof, with a photothermal wavelength upon measuring the predetermined change. In some embodiments, the photothermal wavelength induces thermal damage to the population comprising stem cell- derived cells.

[0026] In some aspects, the disclosure provides a method of controlling stem cell-derived cells at a transplant site in a subject, comprising administering to the subject a population comprising stem cell -derived cells, wherein a plurality of stem cell -derived cells comprise a nanoprobe configured to emit an optical signal upon electromagnetic excitation, and measuring the optical signal across a three-dimensional volume comprising the transplant site at a first time point and at least one additional time point upon illuminating the three- dimensional volume with electromagnetic excitation, and illuminating the three-dimensionalvolume, or a portion thereof, with a photothermal wavelength upon measuring a predetermined change in the optical signal at the first time point as compared to the at least one additional time point.

[0027] In some embodiments of the foregoing or related aspects, the electromagnetic excitation comprises a wavelength in the NIR region. In some embodiments, the wavelength is about 650 nm to about 900 nm.

[0028] In some embodiments of the foregoing or related aspects, the first time point is immediately following the administering or within about 1-2 weeks following the administering. In some embodiments, the at least one additional time point is about is about 1 day to about 30 days following the first time point. In some embodiments, the at least one additional time point is a cycle of once per every about 1 to about 180 days following the first time point. In some embodiments, the at least one additional time point is a cycle of once per every about 5 minutes to about 24 hours following the first time point. In some embodiments, the predetermined change is determined by a machine learning algorithm trained on a dataset of transplant images. In some embodiments, the predetermined change is a marker of teratoma formation or proliferation. In some embodiments, illuminating the three- dimensional volume, or a portion thereof, with the photothermal wavelength induces thermal damage to the population comprising stem cell-derived cells.

[0029] In mbodiments of the foregoing or related aspects, the photothermal wavelength for inducing thermal damage is about 650 nm to about 900 nm. In some embodiments, the illuminating is performed with a laser configured to produce the photothermal wavelength for inducing thermal damage, optionally wherein the laser is a continuous wave laser. In some embodiments, the laser produces the photothermal wavelength for inducing thermal damage at a laser power of about 20 mW to about 500 mW. In some embodiments, the laser produces the photothermal wavelength for inducing thermal damage at a power density of about 0.1 W / cm2to about 2 W / cm2. In some embodiments, the illuminating is performed with an irradiation time of about 30 seconds to about 30 minutes. In some embodiments, the photothermal wavelength for inducing thermal damage penetrates at a tissue depth of about 1 mm to about 20 mm.

[0030] In some aspects, the disclosure provides a method of treating a disease in a subject, comprising administering to an extrahepatic site of the subject a composition comprising an insulin producing cell, wherein the insulin producing cell comprises a nanoprobe, and wherein the insulin producing cell is characterized by reduced sensitivity to immune killing.

[0031] In some embodiments of the foregoing or related aspects, the extrahepatic site is subcutaneous or intramuscular. In some embodiments, immune killing comprises an anti -beta cell immune response. In some embodiments, the immune killing comprises a host versus graft immune response. In some embodiments, the insulin producing cell comprises a gene modification, wherein the insulin producing cell is characterized by reduced sensitivity to immune killing as compared to a control cell lacking the gene modification.

[0032] In some embodiments of the foregoing or related aspects, the gene modification comprises a disruption, inactivation, and / or loss of function of one or more genes. In some embodiments, the one or more genes encodes a major histocompatibility complex (MHC) class I molecule and / or a regulator of transcription of an MHC class I molecule. In some embodiments, the MHC class I molecule comprises a human leukocyte antigen (HLA) class I molecule. In some embodiments, the HLA class I molecule is selected from the group consisting of beta-2 microglobulin (B2M), HLA- A, HLA-B, HLA-C, and a combination thereof. In some embodiments, the regulator of transcription of the MHC class I molecule is selected from the group consisting of RFX5, RFXAP, RFXANK, X2BP, NFY, NFY-A, NFY-B, NFY-C, IRF1, NLRC5, and a combination thereof. In some embodiments, the one or more genes encodes a MHC class II molecule and / or a regulator of transcription of an MHC class II molecule. In some embodiments, the MHC class II molecule comprises an HLA class II molecule. In some embodiments, the HLA class II molecule is selected from the group consisting of HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, and a combination thereof. In some embodiments, the regulator of transcription of the MHC class II molecule is CIITA. In some embodiments, the one or more genes encodes a survival factor selected from the group consisting of: TXNIP; ZNF143; FOXOI; JNK and a combination thereof. In some embodiments, the one or more genes encodes tet methylcytosine dioxygenase 2 (Tet2). In some embodiments, the gene modification comprises an insertion and / or amplification of one or more genes. In some embodiments, the one or more genes encodes a protein selected from the group consisting of CD47; programmed death -ligand 1 (PDL1); A20; HLA-E; HLA-G; CTLA-4; mesencephalic astrocyte derived neurotrophic factor (MANF); and a combination thereof. In some embodiments, the composition comprises one or more tolerogenic factors.

[0033] In some embodiments of the foregoing or related aspects, the composition comprises a proangiogenic factor, a parathyroid gland (PTG) factor, an anti-inflammatory factor, or a combination thereof.

[0034] In some embodiments of the foregoing or related aspects, the one or more factors is selected from vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, platelet derived growth factor (PDGF) AA, PDGF-BB, gamma-aminobutyric acid (GABA), leptin, serotonin, parathyroid hormone (PTH), hormone growth factor (HGF), osteopontin, parathyroid hormone-related protein (PTHrP), and a combination thereof. In some embodiments, the one or more factors comprises or consists of VEGF, angiopoietin-1, and HGF. In some embodiments, the one or more factors comprises or consists of osteopontin, PTH, and PTHrP. In some embodiments, the one or more factors comprises (i) a concentration of VEGF, wherein the concentration is about 0.1 pg / mL to about 10 pg / mL; (ii) a concentration of angiopoietin-1, wherein the concentration is about 10 pg / mL to about 1,000 pg / mL; (iii) a concentration of angiopoietin-2, wherein the concentration is about 10 pg / mL to about 1,000 pg / mL; (iv) a concentration of PDGF-AA, wherein the concentration is about 0.1 pg / mL to about 10 pg / mL; (v) a concentration of PDGF-BB, wherein the concentration is about 10 pg / mL to about 1,000 pg / mL; (vi) a concentration of GABA, wherein the concentration is about 0.1 pM to about 50 pm; (vii) a concentration of leptin, wherein the concentration is about 1 pM to about 500 pm; (viii) a concentration of serotonin, wherein the concentration is about 1 pM to about 500 pm; (ix) a concentration of PTH, wherein the concentration is about 0.1 pg / mL to about 10 pg / mL; (x) a concentration of HGF, wherein the concentraiton is about 10 ng / mL to about 1,000 ng / mL; (xi) a concentration of osteopontin, wherein the concentration is about 10 ng / mL to about 1,000 ng / mL; (xii) a concentration of PTHrP, wherein the concentration is about 1 ng / mL to about 500 ng / mL; or (xiii) any combination of (i)-(xii).

[0035] In some embodiments of the foregoing or related aspects, the one or more factors are operably linked to a surface of the insulin producing cell. In some embodiments, the composition further comprises a polymer matrix, wherein the insulin producing cell and the one or more factors are encapsulated in the polymer matrix. In some embodiments, the composition comprises the one or more factors and insulin producing cell in a liquid suspension. In some embodiments, the insulin producing cell is differentiated from a stem cell. In some embodiments, the stem cell is a pluripotent stem cell. In some embodiments, the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell. In some embodiments, the insulin producing cell is generated from a PDX1+ / NKX6.1+ progenitor cell, and wherein the PDX1+ / NKX6.1+ progenitor cell is generated from the stem cell.

[0036] In some embodiments of the foregoing or related aspects, a plurality of insulin producing cells of the population are present in cell clusters. In some embodiments, the cellclusters comprise a longest diameter of about 50 pm to 350 pm or about 100 pm to about 200 pm. In some embodiments, the cell clusters comprise about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 cells. In some embodiments, the population comprising insulin producing cells are present in a liquid suspension. In some embodiments, the population comprising insulin producing cells are encapsulated in a degradable material. In some embodiments, the population comprising insulin producing cells is differentiated from a population comprising stem cells. In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells. In some embodiments, the population comprising insulin producing cells is generated from PDX1+ / NKX6.1+ progenitor cells, and wherein the PDX1+ / NKX6.1+ progenitor cells are generated from the population comprising stem cells.

[0037] In some aspects, the disclosure provides a pharmaceutical composition comprising a nanoprobe described herein, and a pharmaceutically acceptable carrier.

[0038] In some aspects, the disclosure provides a kit comprising a nanoprobe described herein or a pharmaceutical composition comprising the nanoprobe described herein, and instructions for contacting a cell with the nanoprobe or the pharmaceutical composition.

[0039] In some aspects, the disclosure provides a pharmaceutical composition comprising a cell described herein, and a pharmaceutically acceptable carrier.

[0040] In some aspects, the disclosure provides a kit comprising a cell described herein, or a pharmaceutical composition comprising the cell, and instructions for monitoring the cell following administration of the cell or the pharmaceutical composition to a subject.

[0041] In some aspects, the disclosure provides a pharmaceutical composition comprising a cell composition described herein, and a pharmaceutically acceptable carrier.

[0042] In some aspects, the disclosure provides a kit comprising a cell composition described herein, or a pharmaceutical composition comprising the cell composition, and instructions for administering the composition or the pharmaceutical composition to a subject.

[0043] In some aspects, the disclosure provides for use of a composition comprising an insulin producing cell for treating a disease or disorder in a subject, wherein the insulin producing cell comprises a nanoprobe, and wherein the insulin producing cell is characterized by reduced sensitivity to immune killing.

[0044] In some aspects, the disclosure provides for use of a composition comprising an insulin producing cell in the manufacture of a medicament for treating a disease or disorder ina subject,, wherein the insulin producing cell comprises a nanoprobe, and wherein the insulin producing cell is characterized by reduced sensitivity to immune killing.

[0045] In some embodiments of the foregoing or related aspects, the subject received a daily infusion of insulin prior to administration of the composition. In some embodiments, the subject had a medical history of severe hypoglycemic events. In some embodiments, the subject is characterized by no residual endogenous islet cell function prior. In some embodiments, the subject has undetectable blood C-peptide level when measured using a mixed meal tolerance test prior to the administration. In some embodiments, the disease is characterized by high blood sugar levels for a prolonged period of time. In some embodiments, the disease is diabetes. In some embodiments, the disease is Type 1 diabetes. In some embodiments, the disease is Type 2 diabetes.BRIEF DESCRIPTION OF THE FIGURES

[0046] FIG. 1 is a schematic showing stages of differentiation of human pluripotent stem cells to insulin producing mature beta cells (“stem cell-derived islets” or “sc-islets”) using an exemplary differentiation protocol described herein. Cell markers expressed at each stage of differentiation and day of culture following initial seeding of the human pluripotent stem cells is indicated. Clusters of cells generated at approximately day 20 or later of the protocol that contain mature beta cells are referred to as “stem-cell derived islets” or “sc-islets.”

[0047] FIG. 2A includes a top panel showing the percentage of cells at approximately day 3- 4 of the differentiation protocol of FIG. 1 expressing markers of definitive endoderm (FOXA2 and SOX17). The bottom panel shows a brightfield image of clusters formed by the cells (scale bar = 100 pm).

[0048] FIG. 2B includes a top panel showing the percentage of cells at approximately day 10-11 of the differentiation protocol of FIG. 1 expressing markers of pancreatic progenitors (PDX1 and NKX6.1). The bottom panel shows a brightfield image of clusters formed by the cells (scale bar = 100 pm).

[0049] FIG. 2C includes a top panel showing the percentage of cells at approximately day 20+ of the differentiation protocol of FIG. 1 expressing markers of hormone producing beta cells (C-peptide and NKX6.1). The bottom panel shows a brightfield image of clusters formed by the cells (scale bar = 100 pm).

[0050] FIG. 3A is a graph showing percentage of sc-islets (left panel) or human donor islets (right panel) expressing c-peptide (“CPEP”) above a background level as measured by flow cytometry.

[0051] FIG. 3B is a graph showing c-peptide expression by sc-islets following low glucose stimulation, high glucose stimulation, or high glucose stimulation combined with KC1.

[0052] FIG. 4 is a graph showing blood glucose levels (mg / dL) over time in diabetic mice that received a sc-islet transplant under the kidney capsule. Diabetes was induced by streptozotocin (“STZ”) treatment on day 0 and transplants were administered on day 5. Diabetes was defined by a blood glucose level >400 mg / dL and diabetes reversal by blood glucose <250 mg / dL.

[0053] FIGs. 5A-5B are graphs showing c-peptide levels in serum (pg / mL) under fasting or glucose-stimulated conditions for mice that received a subcutaneous transplant of sc-islets alone (FIG. 5A) or sc-islets combined with PTG-secreted soluble factors (FIG. 5B). The c- peptide levels are shown for the indicated weeks following transplant.

[0054] FIG. 6 is a graph showing c-peptide levels in serum (pg / mL) under fasting or glucose-stimulated conditions for mice that received a subcutaneous transplant of sc-islets alone (left panel) or sc-islets combined with a reduced set of PTG-secreted soluble factors (right panel). The c-peptide levels are shown for the indicated weeks following transplant.

[0055] FIG. 7A is a Raman spectrum obtained from pig skin following injection of IS-Cy7- miR-21 gold nanostars (AuNSs) with measurements performed at the indicated laser power.

[0056] FIG. 7B is a Raman spectrum obtained from pig skin following injection of IS-Cy7- miR-21 AuNSs with measurements performed at the indicated laser power and tissue depth.

[0057] FIG. 8A depicts an image of an in vivo transplant of sc-islets loaded with AuNS containing a Raman label generated by stitching together SERS signals collected across a plane of the transplant.

[0058] FIG. 8B depicts a series of planar images of a sc-islet transplant obtained over time of the transplant of FIG. 8A.

[0059] FIGs. 9A-9C are graphs showing integrated SERS signal of in vivo transplants containing the indicated number of sc-islet clusters (loaded with AuNSs containing a Raman label) at 8 or 14 days following transplantation.

[0060] FIG. 10A is a schematic showing an exemplary nanoprobe of the disclosure for detecting a target nucleic acid. The nanoprobe has a stem -loop strand linked to a AuNS at one end and to a Raman label at the other end. The nanoprobe further includes a placeholder strand having a first region complementary to a portion of the stem-loop strand and a secondregion complementary to the target nucleic acid, with the first region and second region overlapping. The stem-loop strand is maintained in an open configuration when bound to a placeholder strand, thereby producing a Raman signal comparable to background in the absence of the target nucleic acid (“OFF probe”). The placeholder strand is displaced in the presence of the target nucleic acid, thereby producing a Raman signal higher than background (“ON” probe).

[0061] FIG. 10B is a schematic showing synthesis of the exemplary nanoprobe according to FIG. 10A with further functionalization to include a surface-appended Raman label (“internal standard”). According to the design, the intensity of the Raman signal from the internal standard remains substantially the same in the presence or absence of the placeholder strand, while the intensity of the Raman label appended to the stem-loop strand (“target label”) is altered.

[0062] FIG. 11A is a Raman spectrum of exemplary nanoprobes according to the design of FIG. 10B having a placeholder strand with complementarity to miR21 (referred to as “MM_A700-miR21_BHQ3” nanoprobes). Shown is Raman signal for the target label (ATTO 700) and internal standard (BHQ3) for MM_A700-miR21_BHQ3 nanoprobes in the presence of the placeholder sequence or the placeholder sequence and the miR21 target sequence.

[0063] FIG. 11B is an image of AuNS nanoprobes present in the cytoplasm of a mouse embryonic fibroblast (MEF) obtained using transmission electron microscopy.

[0064] FIG. 11C is a graph showing normalized SERS signal measured for MM A700- miR21_BHQ3 nanoprobe in MCF-7 cells transfected with miR21. Control cells received no miR21. Normalized SERS signal is the ratio of target label signal to internal standard signal.

[0065] FIG. 12A is a graph showing expression of miR-146a in insulin producing cells or MEFs following exposure to a mixture of cytokines. Control cells were untreated. The insulin producing cells were from the rat insulinoma cell line (Insl), human donor islets, or day 20 sc-islets.

[0066] FIG. 12B is a graph showing expression of miR-224 in sc-islets following exposure to a mixture of cytokines or interferon. Control cells were untreated.

[0067] FIG. 12C are graphs showing expression of miR-210 in insulin producing cells or MEFs following culture under hypoxic conditions. Control cells were cultured under normoxic conditions. Insulin producing cells were from the Insl cell line, human donor islets, or sc-islets.

[0068] FIG. 13 is a schematic showing an exemplary method of the disclosure for loading islets with nanoprobes. The islets are obtained as clusters of human donor derived islets or sc-islets. The method includes the steps of dissociating the islets, introducing the nanoprobes (e.g., via electroporation), and reaggregating the islets to form clusters.

[0069] FIG. 14A includes a graph showing percentage of total nanoprobe-loaded islets in clusters at 7 days following the reaggregation step of FIG. 13. Control cells received no nanoprobe. The left panel provides brightfield images of clusters for nanoprobe-loaded and control islets.

[0070] FIG. 14B is a graph showing percentage of live nanoprobe-loaded islets in clusters at 7 days following the reaggregation step of FIG. 13. Control cells received no nanoprobe.

[0071] FIG. 15 is a graph showing relative abundance of mRNA for the indicated islet identity genes in sc-islets loaded with IS-Cy7-miR-21 nanoprobe. Control cells received no nanoprobe. mRNA expression is shown as fold-change compared to control.

[0072] FIG. 16A is a graph showing percentage of cells expressing C-peptide following glucose stimulation for human donor islets loaded with IS-Cy7-miR-21 nanoprobe. Control cells received no nanoprobe.

[0073] FIG. 16B includes a graph (left panel) showing percentage of cells expressing C- peptide following glucose stimulation for sc-islets loaded with IS-Cy7-miR-21 nanoprobe. Control cells received no nanoprobe. The right panel is a graph showing the ratio at high to low glucose stimulation (“stimulation index”) for nanoprobe-loaded and control cells.

[0074] FIGs. 17A-17B are representative immunofluorescent images of tissue harvested from a transplant of sc-islets loaded with IS-Cy7-miR-21 nanoprobe in an always-on configuration. Control was transplant of sc-islets containing no nanoprobe. FIG. 17A shows staining for insulin (INS), glucagon (GCG), and nuclear DNA. FIG. 17B shows staining for INS, Ki67 (proliferation marker), and nuclear DNA.

[0075] FIG. 17C is a representative immunohistochemistry image of tissue harvested from a transplant of sc-islets loaded with IS-Cy7-miR-21. Control was transplant of sc-islets containing no nanoprobe. The tissue was stained for cleaved caspase 3, with arrows indicating positive cells.

[0076] FIG. 17D is a graph showing percentage of Ki67-positive cells in tissue harvested from a transplant of sc-islets loaded with IS-Cy7-miR-21. Control was transplant of sc-islets containing no nanoprobe.

[0077] FIGs. 18A-18D are representative images of GFP-sc-islets clusters (expressing GFP in conjunction with insulin) loaded with polylysine-coated PEGylated gold nanoprobes (“pK GP”) following exposure to photothermal irradiation (FIG. 18D) or no photothermal irradiation (FIG. 18C); and of control sc-islets clusters without pK GP and exposed tophotothermal irradiation (FIG. 18B) or no photothermal irradiation (FIG. 18A). For each, the left panel shows a brightfield image and the right panel shows GFP fluorescence measured under identical imaging conditions for each figure.

[0078] FIGs. 19A-19D are representative images of GFP-sc-islets clusters loaded with PEGylated gold nanoprobes (“GP”) or polylysine-coated PEGylated gold nanoprobes (“pK GP”) following exposure to photothermal irradiation (FIGs. 19B and 19D respectively) or no photothermal irradiation (FIGs. 19A and 19C respectively). For each, the left panel shows a brightfield image and the right panel shows GFP fluorescence measured under identical imaging conditions for each figure.

[0079] FIGs. 20A-20B are graphs showing viability of sc-islets containing pK GP and exposed to photothermal irradiation. Control cells were sc-islets with or without pK GP and not exposed to photothermal irradiation, and sc-isclets with pK GP and not exposed to photothermal irradiation. Viability is shown as percentage of total cells (FIG. 20A) or absolute counts of viable cells (FIG. 20B).

[0080] FIG. 21 is a graph showing Raman signal for sc-islets containing PEGylated gold nanoprobes (“Nanoprobe”) or PEGylated gold nanoprobes coated with poly lysine (“Polylysine Nanoprobe”).DETAILED DESCRIPTION

[0081] The present disclosure relates, at least in part, to cell compositions comprising SCD cells and methods of use thereof. In some embodiments, the SCD cells are differentiated from pluripotent stem cells. Pluripotent stem cells, under certain culture conditions, can be differentiated into numerous cell types, including neurons, hepatocytes, cardiomyocytes, and endocrine cells (e.g., insulin producing endocrine cells). A therapeutic approach of the disclosure is to administer a cell composition comprising SCD cells (e.g., SCD cells differentiated from pluripotent stem cells) to a subject for the purpose of restoring a normal organ or tissue function that is impaired or lost due to a disease or disorder.

[0082] The success of the SCD cell transplant depends, at least in part, on the ability of the cells to engraft at the transplant site, maintain differentiation state, evade immune detection / killing, and function as effector cells. There are numerous challenges to achieving these criteria. First, it is considered desirable to administer the SCD cell transplant to sites that are readily accessible (e.g., subcutaneous or intramuscular transplant sites), such that a subject can receive the transplant in a simple and substantially non-invasive procedure, butsuch sites can be detrimental to survival of SCD cells due to poor nutrient conditions, inflammatory insult, and / or hypoxia. Second, SCD cell transplants are associated with the risk of emerging neoplastic cells that drive teratoma formation. Such cells may be residual pluripotent stem cells, progenitor cells not fully differentiated, and / or SCD cells that dedifferentiate to a pluripotent state following in vivo administration. Third, the SCD cell transplant is susceptible to immune killing, either resulting from the subject’s immune system recognizing the SCD cell transplant as foreign and activating an immune response to eliminate the transplant and / or an autoimmune response to the SCD cells associated with the subject’s disease or disorder (i.e., an autoimmune response that resulted in destruction of endogenous cells for which the SCD cell transplant is intended to replace). Thus, there is a need for approaches that promote survival, function, and safety of SCD cells under conditions of physiological stress and immune attack associated with in vivo transplantation. The methods and compositions of the disclosure contribute to addressing this need, and in particular the need for SCD cell transplants comprising insulin producing cells for treatment of diabetes (e.g., type 1 diabetes).

[0083] In some aspects, the disclosure provides methods and compositions for monitoring the status of the SCD cell transplant following administration to the subject.

[0084] In some embodiments, the disclosure provides nanoprobes configured for optical detection that when combined with the SCD cell transplant as described herein, are used to visualize the SCD cell transplant following in vivo administration. In some embodiments, visual images of the SCD cell transplant are collected over time following in vivo administration and analyzed (e.g., using an artificial intelligence algorithm) to detect changes that indicate desired homeostasis of the SCD cell transplant or informs the need for a clinical intervention. For example, detecting a change indicating uncontrolled growth of the SCD cells informs the need to eliminate the SCD cell to prevent teratoma formation. In some embodiments, eliminating the SCD cell transplant is performed by illuminating the transplant site with photothermal radiation that is absorbed by the nanoprobes and converted to heat energy for ablating surrounding cells. As another example, detecting a change indicating a decline in survival of the SCD cells informs a clinician to “re-dose” the transplant by administering a second composition of SCD cells. As a further example, detecting a change indicating increased inflammation indicates the subject would benefit from receiving an immunosuppressant in order to promote longevity of the transplanted cells.

[0085] In some embodiments, the disclosure provides nanoprobes configured for detection of a target nucleic acid, wherein the target nucleic acid is a biomarker of a cellular phenotypeand / or differentiation state. In some embodiments, the biomarker comprises an altered (e.g., increased or decreased) expression level in a cell described herein upon a change in a cellular phenotype and / or differentiation state. In some embodiments, the disclosure provides target nucleic acids that are biomarkers for inflammation, wherein the target nucleic acid comprises an altered (e.g., increased or decreased) expression level upon exposure to inflammatory conditions. In some embodiments, the disclosure provides target nucleic acids that are biomarkers for hypoxia, wherein the target nucleic acid comprises an altered (e.g., increased or decreased) expression level upon exposure to hypoxic conditions. In some embodiments, the disclosure provides methods for monitoring a SCD cell transplant described herein comprising administering to a subject a SCD cell transplant comprising the nanoprobes configured for detection of a target nucleic acid, and measuring a signal from the nanoprobe following in vivo administration, wherein detection of a signal indicates the presence of the target nucleic acid. In some embodiments, detecting the presence of the target nucleic acid is used to inform a clinical intervention. In some embodiments, detecting a target nucleic acid identified herein as a biomarker of inflammation indicates the subject would benefit from receiving an immunosuppressant or an anti-inflammatory medication. In some embodiments, detecting a target nucleic acid identified herein as a biomarker of hypoxia indicates the subject would benefit from receiving supplemental oxygen (e.g., via hyperbaric oxygen therapy).

[0086] In some aspects, the disclosure provides compositions comprising SCD cells engineered to evade immune detection according to a method described herein. In some embodiments, the stem cell-derived cells comprise genomic modifications described herein that reduce or mitigate recognition by a subject’s immune system following transplantation in vivo.

[0087] In some aspects, the disclosure provides compositions comprising SCD cells and a support factor, wherein the support factor promotes survival and function of the SCD cells following in vivo transplantation. In some embodiments, the support factors promote vascularization of the composition. In some embodiments, the support factors increase resilience of the composition to hypoxic and / or nutrient deprived conditions. In some embodiments, the support factors maintain the SCD cells in a differentiated state.

[0088] The methods and compositions of the disclosure are applicable to SCD cell transplants for treating numerous human diseases. In particular aspects, the disclosure provides methods for treating diabetes in a subject, comprising administering a composition comprising insulin producing cells (e.g., SCD P cells) described herein.

[0089] The disclosure further provides pharmaceutical compositions comprising the cell compositions described herein and delivery devices comprising the cell compositions or delivery devices. The disclosure further provides kits comprising the cell compositions, pharmaceutical compositions, or delivery devices and instructions for administering for treating a disease in a subject and / or monitoring the cell composition following in vivo administration. In some aspects, the disclosure provides kits comprising the nanoprobes described herein and instructions for contacting a cell with the nanoprobe to induce uptake.Definitions

[0090] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0091] As used herein, the term “stem cell-derived cell” or “SCD cell” are interchangeably used to refer to a cell differentiated from a stem cell (such as a pluripotent stem cell described herein (e.g., embryonic stem cells, induced pluripotent stem cells (iPSC)) a multipotent cell, or an unipotent cell), a precursor cell differentiated from a stem cell, a partially reprogrammed somatic cell (i.e., a somatic cell which has been partially reprogrammed to an intermediated state between an iPSC and the somatic cell from which it was derived). Generally, the term refers to cells differentiated from a stem cell ex vivo, rather than cells differentiated from stem cells in a living organism. The term encompasses cells differentiated from stem cells for administering to a subject as a stem cell therapy.

[0092] As used herein, the term “stem cell” refers to cells of the body characterized by the potential to differentiate into a cell having a more specialized or differentiated phenotype and the potential to proliferate without substantially differentiating. For example, in some embodiments, a stem cell refers to an undifferentiated mother cell whose descendants (progeny) specialize by differentiation, e.g., by acquiring one or more functional properties. Stem cells are classified, at least in part, based upon their development potential. A stem cell that is “totipotent” refers to one having the capacity to differentiate into all embryonic and extraembryonic cell types. A stem cell that is “pluripotent” refers to one having the capacity to differentiate to each of the three germ cell layers. A stem cell that is “multipotent” refers to one having the capacity to differentiate into all cell types within one particular lineage (e.g., hematopoietic stem cells are multipotent stem cells having capacity to differentiate into allblood cell types). A stem cell that is “unipotent” refers to one having the capacity to differentiate into a single cell lineage (e.g., spermatogenic stem cells).

[0093] As used herein, the term “differentiate” or “differentiation” refers to the process in which a stem cell progresses from a less specialized state to a more specialized state.

[0094] As used herein, the term “pluripotent cell” refers to a cell characterized by the capacity to self-renew and proliferate while remaining in an undifferentiated state and that can, under proper conditions, be induced to differentiate into a more specialized cell type.

[0095] As used herein, the term “pluripotent stem cell” refers to a pluripotent cell having the potential to differentiate into any of the three germ layers: endoderm (e.g., the stomach lining, gastrointestinal tract, lungs, etc.), mesoderm (e.g., blood, bone, muscle, urogenital tissue, etc), or ectoderm (e.g., epidermal tissue and nervous system tissue).

[0096] As used herein, the terms “progenitor” and “precursor” cell are used interchangeably herein and refer to cells that have a cellular phenotype that is more primitive (e.g., is at an earlier step along a developmental pathway or progression than is a fully differentiated cell) relative to a cell which it can give rise to by differentiation. In some embodiments, a progenitor cell gives rise to multiple distinct differentiated cell types or to a single differentiated cell type by exposure to an environment and set of conditions that promote differentiation along a certain developmental pathway.

[0097] As used herein, the term “endoderm” refers to a cell of one of the three primary germ cell layers in the early embryo (the other two germ cell layers are mesoderm and ectoderm). An endoderm cell differentiates to give rise to the embryonic gut and then to linings of the respiratory tract, the digestive tract, the liver, and the pancreas. The term encompasses endogenous endoderm cells in a subject and SCD endoderm cells (e.g., endoderm cells differentiated from a pluripotent stem cell, such as by the methods described herein).

[0098] A “cell of endoderm origin” refers to one having differentiated from an endoderm cell. Exemplary cells of endoderm origin include, but are not limited to cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. In some embodiments, the cell of endoderm origin is a definitive endoderm cell.

[0099] As used herein, the term “definitive endoderm” refers to a cell differentiated from an endoderm cell and which retains capability to be differentiated into cells of the liver, lung, pancreas, thymus, intestine, stomach and / or thyroid. In some embodiments, the definitive endoderm cell is characterized by expression of one or more cell markers. In some embodiments, the one or more cell markers comprises SOX17. In some embodiments, the one or more cell markers comprises SOX17, CXCR4, FOXA2, and CD 177. In someembodiments, the definitive endoderm cells do not substantially express PDX1 (i.e., PDX1 negative). In some embodiments, the definitive endoderm cells do not substantially express pluripotency markers such as OCT3 / 4, SOX2, TRA-1-60, and NANOG (i.e., negative for these proteins.) The expression of SOX17 and other cell markers of definitive endoderm may be assessed by any method known by the skilled person such as immunochemistry or flow cytometry, e.g., using marker-specific antibody, or quantitative RT-PCR.

[0100] As used herein, the term “posterior foreguf ’ refers to a cell differentiated from a definitive endoderm cell and which retains capability to be differentiated into cells of the lung, liver, pancreas, stomach, and intestine. In some embodiments, the posterior foregut cell is characterized by expression of one or more cell markers. In some embodiments, the one or more cell markers comprises CDX2, FOXA2, HNF6, SOX17, HNF4A, HNF1B, FOXA1, and c-KIT. In some embodiments, the posterior foregut cell is FOXA-2 positive. In some embodiments, the posterior foregut cell expresses SOX2.

[0101] A cell of the “pancreatic endoderm” refers to a cell differentiated from definitive endoderm that retains the capability to differentiate into multiple pancreatic lineages, but no longer has the capacity to differentiate into non-pancreatic lineages accessible to definitive endoderm cells. The term encompasses endogenous pancreatic endoderm cells in a subject and pancreatic endoderm cells differentiated from a stem cell (e.g., a pluripotent stem cell), such as by the methods described herein.

[0102] As used herein, the term “pancreatic progenitor,” “pancreatic endocrine progenitor,” “pancreatic precursor,” pancreatic endocrine precursor” are interchangeably used to refer to a cell of endoderm origin that has the capacity to differentiate into a pancreatic endocrine cell, pancreatic exocrine cell, or a pancreatic duct cell. Such cells are committed to differentiating to at least one type of pancreatic cell, e.g., a pancreatic a cell, P cell, 5 cell, PP cell, or 8 cell, acinar cell, duct cell etc. The term encompasses endogenous pancreatic progenitor cells in a subject and pancreatic progenitor cells differentiated from a stem cell (e.g., a pluripotent stem cell), such as by the methods described herein. Pancreatic progenitor cells are characterized by expression of at least one of the following cell markers: NGN3, NKX2.2, NKX6.1, NEUROD, ISL-1, PAX4, PAX6, SOX9, HNF6, FOXA2, PDX1, and ARX. The expression of cell markers of pancreatic progenitor cell may be assessed by any method known by the skilled person such as immunochemistry or flow cytometry, e.g., using marker-specific antibody, or quantitative RT-PCR.

[0103] As used herein, the term “PDX-1 -positive pancreatic progenitor” refers to a pancreatic endoderm cell having capacity to differentiate into a pancreatic P cell or P-like cell. A PDX-1positive cell is characterized by expression of pancreatic and duodenal homeobox 1 (PDX-1). In some embodiments, the PDX-1 positive cell is further characterized by expression of at least one of the following cell markers: PTF1A, HNF6, NKX2.2, HNF4A, HNF1B, F0XA1, F0XA2, SOX9, NGN3, and NKX6.1. In some embodiments, the PDX-1 positive cell is further characterized by expression of NKX6.1 (referred to herein as a “PDX-1, NKX6.1- positive pancreatic progenitor”).

[0104] As used herein, the term “insulin-producing cell” refers to any cell that produces, stores, and / or secretes detectable amounts of insulin. In some embodiments, an insulin producing cell has the capacity to produce and secrete insulin similar to that produced and secreted by a beta cell of the islets of Langerhans. The term encompasses pancreatic P cells (e.g., human donor pancreatic P cells), pancreatic-like P cells, and SCD p cells that synthesize insulin (i.e., transcribe the insulin gene and translate the mRNA product thereof to generate insulin), express insulin (e.g., package and store insulin into secretory granules), and secrete insulin (e.g., release insulin into the extracellular space) in a constitutive or inducible manner. In some embodiments, a population of insulin producing cells is characterized by a glucose stimulated insulin secretion (GSIS) response that resembles that exhibited by a pancreatic P cell. In some embodiments, the population of insulin producing cells comprises non-insulin producing cells.

[0105] As used herein, the term “glucose stimulated insulin secretion response” or “GSIS response,” used interchangeably herein, refers to secretion of insulin by an insulin producing cell upon contact with glucose. According to the canonical model, the GSIS response results from the uptake of glucose (through a glucose transporter) by the insulin producing cell, which in turn increases production of adenosine triphosphate (ATP). And increase in the ATP / ADP ratio results in closing of potassium channels of the cell, resulting in depolarization of the cell and opening of calcium channels. This results in insulin secretion. In some embodiments, the GSIS response is measured in vitro by contacting the insulin producing cell with glucose at a concentration of about 1 mM to about 30 mM and measuring extracellular secretion of insulin (or c-peptide as an insulin surrogate) using, e.g., an ELISA. Functional assessment of purified human pancreatic islets: glucose stimulated insulin release by ELISA - A Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium CellR4 2014; 2(2):e900 for methods to measure GSIS in vitro. In some embodiments, an increase in extracellular secretion of insulin as compared to an insulin producing cell not contacted with glucose indicates a GSIS response. In some embodiments, the extracellular secretion of insulin is at least about 5%, about 10%, about 20%, about 30%,about 40%, about 50% of the total insulin stored by the insulin producing cell following contacting with glucose (e.g., at a concentration of about 1 mM to about 30 mM). In some embodiments, the extracellular secretion of insulin is about 1% to about 20%, of the total insulin stored by the insulin producing cell following contacting with glucose (e.g., at a concentration of about 1 mM to about 30 mM). In some embodiments, the GSIS response is measured in vivo by administering a glucose solution to a subject following a period of fasting and measuring blood insulin levels. In some embodiments, an increase in blood insulin levels following the administering as compared to prior to the administering indicates a GSIS response.

[0106] As used herein, the term “SCD p cell” or “stem cell-derived P cell” are interchangeably used to refer to an SCD cell that display at least one pancreatic P cell marker, express insulin, and display a glucose stimulated insulin secretion (GSIS) response mimicking an endogenous mature pancreatic P cell.

[0107] As used herein, the term “pancreatic P cell marker” refers to polypeptides, nucleic acids, metabolites, or analytes expressed by pancreatic p. In some embodiments, the pancreatic P cell marker is selected from: NKX6.1, PDX1, insulin, c-peptide, amylin, E- cadherin, Hnf3p, PCI / 3, GLUT2, PC2, ZnT-8, ISL1, Pax6, Pax4, NeuroD, Hnflb, Hnf-6,MAFA, MAFB, NKX2.2, PH3, UCN3, ENTPD3, and a combination thereof. In some embodiments, the pancreatic P cell markers comprise NKX6.1 and c-peptide. In some embodiments, the pancreatic P cell markers comprise NKX6.1 and insulin. In some embodiments, the pancreatic P cell markers comprise C-peptide, PDX1, NKX6.1, MAFA,MAFB, UCN3, and ENTPD3.

[0108] As used herein, the term “SCD a cell” or “stem cell-derived a cell” are interchangeably used to refer to an SCD cell that displays at least one marker of a pancreatic a cell (e.g., GCG, PDX1, IRX, PC2), does not express a pancreatic P cell marker (e.g., PCI, NKX6-1), a pancreatic 5 cell marker (e.g., somatostatin), a pancreatic y cell marker (e.g., pancreatic polypeptide), or a pancreatic a cell marker (e.g., ghrelin), and express and secrete glucagon. In some embodiments, a SCD a does not express a pancreatic hormone other than glucagon. In some embodiments, a SCD a cell does not express insulin and / or somatostatin.

[0109] As used herein, the term “SCD 5 cell” or “stem cell-derived 5 cell” are interchangeably used to refer to an SCD cell that displays at least one marker of a pancreatic 5 cell and express and secrete somatostatin. In some embodiments, a SCD 5 cell does not express a pancreatic hormone other than somatostatin. In some embodiments, a SCD 5 cell does not express insulin, glucagon, pancreatic polypeptide, ghrelin, or a combination thereof.

[0110] As used herein, the term “SCD y cell” or “stem cell-derived y cell” are interchangeably used to refer to an SCD cell that displays at least one marker of a pancreatic y cell and express and secrete pancreatic polypeptide. In some embodiments, a SCD y cell does not express a pancreatic hormone other than pancreatic polypeptide. In some embodiments, a SCD y cell does not express insulin, glucagon, somatostatin, Ghrelin, or a combination thereof.[OHl] As used herein, the term “SCD a cell” or “stem cell-derived a cell” are interchangeably used to refer to an SCD cell that displays at least one marker of a pancreatic a cell and express and secrete ghrelin. In some embodiments, a SCD a cell does not express a pancreatic hormone other than ghrelin. In some embodiments, a SCD a cell does not express insulin, glucagon, somatostatin, pancreatic polypeptide, or a combination thereof.

[0112] As used herein, the term “cell cluster,” “cluster,” “cell aggregate,” or “aggregate” are interchangeably used to refer to a group of cells having direct cell-to-cell contact. For example, in some embodiments, a plurality of cells in a cell cluster are in directed contact with a least one additional cell present in the cluster.

[0113] As used herein, the term “longest diameter” in reference to a cell cluster described herein refers to the distance between the two farthest points present on the surface of the cell cluster.

[0114] As used herein, the term “ancillary materials” refers to components, reagents, and materials used during the manufacture of a cell therapy product that are not intended to be part of the final product. Exemplary ancillary materials include, but are not limited to, cell isolation reagents, culture and cryopreservation media, and disposables such as plasticware or bioprocessing bags.

[0115] As used herein, the term “allogeneic” refers to a cell, a cell population, or a biological sample comprising the cells that are obtained from a donor that is different than the subject that receives the cell, cell population, or biological sample, or a downstream product thereof. For example, the term encompasses stem cells or progenitor cells that are derived from a donor and differentiated into SCD cells that are administered to a subject that is different than the donor. A transplant comprising the allogeneic cells is referred to as an “allogeneic transplant.” A subject’s immune response against an allogeneic transplant is referred to as an “allogeneic immune response.”

[0116] As used herein, the term “autologous” refers to a cell, a cell population, or a biological sample comprising the cells that are obtained from an individual that also receives the cell, cell population, or biological sample, or a downstream product thereof. For example,the term encompasses adult somatic cells derived from an individual that are reprogrammed (e.g. reprogrammed to iPSCs) ex vivo, differentiated to SCD cells, and administered to the same individual. A transplant comprising the autologous cells is referred to as an “autologous transplant.”

[0117] As used herein, the term “Major histocompatibility complex class I” or “MHC-I” generally refer to a class of biomolecules that are found on the cell surface of all nucleated cells in vertebrates, including mammals, e.g, humans; and function to display peptides of non-self or foreign antigens, e.g, proteins, from within the cell (i.e. cytosolic) to cytotoxic T cells, e.g, CD8+ T cells, in order to stimulate an immune response. In some embodiments, a MHC-I biomolecule is HLA-A (NCBI Gene ID No: 3105), HLA-B (NCBI Gene ID No: 3106), HLA-C (NCBI Gene ID No: 3107), or B2M (NCBI Gene ID No: 567).

[0118] As used herein, the term “Major histocompatibility complex class II” or “MHC-II” generally refer to a class of biomolecules that are typically found on the cell surface of antigen-presenting cells in vertebrates, including mammals, e.g., humans; and function to display peptides of non-self or foreign antigens, e.g., proteins, from outside of the cell (extracellular) to cytotoxic T cells, e.g. , CD8+ T cells, in order to stimulate an immune response. In some embodiments, a MHC-II biomolecule is HLA-DPA (NCBI Gene ID No: 3113), HLA-DPB (NCBI Gene ID No: 3115), HLA-DMA (NCBI Gene ID No: 3108), HLA- DMB (NCBI Gene ID No: 3109),HLA-DOA (NCBI Gene ID No: 3111), HLA- DOB (NCBI Gene ID No: 3112), HLA-DQA (NCBI Gene ID No: 3117), HLA-DQB (NCBI Gene ID No: 3119), HLA-DRA (NCBI Gene ID No: 3122), or HLA-DRB (NCBI Gene ID No: 3123).

[0119] As used herein, the term “transcriptional regulator of MHC-I or MHC-II” generally refers to a biomolecule that modulates, e.g, increases or decreases, the expression of a MHC-I and / or MHC-II human leukocyte antigen. In some embodiments, the transcriptional regulator is CIITA (NCBI Gene ID No: 4261) or NLRC5 (NCBI Gene ID No: 84166).

[0120] As used herein, the term “survival factor” generally refers to a protein (e.g., expressed by a polynucleotide as described herein) that, when increased or decreased in a cell, enables the cell, e.g., a SCD cell or insulin producing cell (e.g., a SCD p cell) described herein, to survive after transplantation or engraftment into a host subject at higher survival rates relative to an unmodified cell. In some embodiments, the survival factor is ZNF143 (NCBI Gene ID No: 7702), TXNIP (NCBI Gene ID No: 10628), FOXO1 (NCBI Gene ID No: 2308), JNK (NCBI Gene ID No: 5599), or MANF (NCBI Gene ID No: 7873).

[0121] As used herein, the term “tolerogenic factor” refers to a protein (e.g., expressed by a polynucleotide) that, when increased or decreased in a cell, enables the cell, e.g., a SCD cellor insulin producing cell (e.g., a SCD P cell) described herein, to inhibit or evade immune rejection after transplantation or engraftment into a host subject at higher rates relative to an unmodified cell. In some embodiments, a tolerogenic factor is a human tolerogenic factor. In some embodiments, a tolerogenic factor is HLA-E (NCBI Gene ID No: 3133), HLA-G (NCBI Gene ID No: 3135), CTLA-4 (NCBI Gene ID No: 1493), CD47 (NCBI Gene ID No: 961), or PD-L1 (NCBI Gene ID No: 29126).

[0122] As used herein, the term “unmodified cell” refers to a cell that has not been subjected to a genetic modification involving a polynucleotide or gene that encodes a MHC-I, MHC-I, transcriptional regulator of MHC-I or MHC-II, survival factor, and / or tolerogenic factor.

[0123] As used herein, the term “within or near a gene” refers to a site or region of genomic DNA that is an intronic or extronic component of a said gene or is located proximal to said gene.

[0124] As used herein, the term “support factor” refers to any component that when combined with a cell composition of the disclosure (e.g., a cell composition comprising a SCD cell or an insulin producing cell), promotes a desired function of the cell composition compared to a cell composition lacking the support factor. The term encompasses, but is not limited to, components that are a molecule (e.g., a nucleic acid, protein, hormone, carbohydrate, lipid, steroid, a pharmacological agent), a nanoprobe, a nanoparticle, or a cell (e.g., a CD34+ cell or a cell derived from PTG tissue). The desired functions encompass, but are not limited to, improved survival (e.g., in vitro or in vivo), insulin production (for an insulin producing cell), and / or tolerance to hypoxic and / or nutrient deficient conditions.

[0125] As used herein, the term “diabetes” or “diabetic disorder” or “diabetes mellitus,” are used interchangeably to refer to a disease which is marked by elevated levels of sugar (glucose) in the blood. Diabetes can be caused by too little insulin (a protein produced by the pancreas to regulate blood sugar), resistance to insulin, or both. Diabetes mellitus includes, without limitation, type 1 diabetes, type 2 diabetes, or surgical diabetes.

[0126] As used herein, the term “type 1 diabetes,” as used herein, refers to a chronic disease that occurs when the pancreas produces too little insulin to regulate blood sugar levels appropriately. Type 1 diabetes is also interchangeably referred to as “insulin-dependent diabetes mellitus,” “IDMM,” “juvenile onset diabetes,” “autoimmune diabetes,” and “diabetes — type 1.” Type 1 diabetes is the result of a progressive autoimmune destruction of pancreatic P cells with subsequent insulin deficiency.

[0127] As used herein, the term "type 2 diabetes" (also referred to as “non-insulin-dependent diabetes mellitus” or “adult-onset diabetes”) refers to a metabolic disorder in individuals whoexhibit insulin resistance and who usually exhibit relative, rather than absolute, insulin deficiency. Illustrative, but non limiting criteria for determining whether an individual has type 2 diabetes, include one or more of the following: (1) a confirmed fasting plasma glucose value of greater than or equal to 126 milligrams / deciliter (mg / dL), (2) in the presence of symptoms of diabetes, a confirmed non-fasting plasma glucose value of greater than or equal to 200 mg / dL, and (3) with an oral glucose tolerance test (by administering 75 grams of anhydrous glucose dissolved in water, in accordance with World Health Organization standards, and then measuring the plasma glucose concentration 2 hours later), a confirmed glucose value of greater than or equal to 200 mg / dL.

[0128] As used herein, the term “surgically induced diabetes” or “surgical diabetes” refers to diabetes cause by some surgical procedure, such as when surgery on the pancreas impacts its ability to produce insulin either permanently or temporarily.

[0129] As used herein, the term "a cell derived from a parathyroid gland" means a cell isolated from the tissue of a parathyroid gland. In some embodiments, a cell derived from a parathyroid gland is a CD34+ cell. In other embodiments, the cell derived from parathyroid gland are CD45 CD34+. In another embodiment, the cell derived from parathyroid gland secrets one or more pro-angiogenic substances such as, but not limited to, VEGF, PDGF, and angiopoietin. In another embodiment, the cell derived from parathyroid gland secrets one or more parathyroid hormones (such as, but not limited to, GABA, leptin, serotonin, PTH and PTHrP).

[0130] As used herein, the term “CD34+ cell” refers to a cell that expresses the progenitor cell antigen CD34, also known as CD34 antigen, which is a protein that in humans is encoded by the CD34 gene (OMIM: 142230; NM_001025109; NP_001020280). In some embodiments of the disclosure, the CD34+ cell is derived from a parathyroid gland. In some embodiments, the CD34+ cell is a SCD cell. In some embodiments, the CD34+ cell is differentiated from a pluripotent stem cell (e.g., ESC or iPSC).

[0131] As used herein, the “first region of complementarity” in reference to a placeholder strand described herein refers to the nucleotide sequence present in the placeholder strand having sufficient complementarity to a contiguous nucleotide sequence present in a stem loop strand described herein to hybridize with, associate, with, and / or bind to the stem loop strand.

[0132] As used herein, the term “duplex region” refers to the double-stranded oligonucleotide resulting from hybridization, association, and / or binding of a placeholder strand described herein with a stem loop strand described herein.

[0133] As used herein, the “second region of complementarity” in reference to a placeholder strand described herein refers to the nucleotide sequence present in the placeholder strand having sufficient complementarity to a contiguous nucleotide sequence present a target nucleic acid to hybridize with, associate with, and / or bind to the target nucleic acid.

[0134] As used herein, the “target sequence” in a target nucleic acid refers to a contiguous nucleotide sequence present in the target nucleic acid that hybridizes to the region of complementarity present in the placeholder strand. In some embodiments, the binding site spans the entire length of the target nucleic acid. In some embodiments, the binding site spans a portion of the target nucleic acid.

[0135] As used herein, a "SERS reporter molecule" refers to any molecule or chemical compound that is capable of producing a Raman spectrum when it is illuminated with radiation of an appropriate wavelength. The term "SERS reporter molecule" is used interchangeably herein with "Raman-active molecule" or "SERS- active molecule."

[0136] As used herein, the term “transplanting” refers to placement of cells or cell clusters of the cell composition into a subject by a method or route of administration that results in at least partial localization of the administered cell composition at the transplant site.

[0137] As used herein, the term “treating” and “treatment” refer to administering to the subject an effective amount of the cell composition such that the subject experiences a reduction in at least one symptom of the disease and / or an improvement in a clinical outcome associated with the disease. In some embodiments, the clinical outcome comprises alleviation of one or more symptoms, stabilized (e.g., not worsened) state of disease, delayed or slowed progression of the disease, ameliorated or palliation of the disease state, and remission (e.g., partial or total) of the disease. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Treatment can further refer to an improvement in a clinical outcome without further therapeutic intervention.

[0138] As used herein, the terms “subject,” “patient,” or “individual” are used interchangeably herein, and refer to an animal, for example, a human from whom cells can be obtained and / or to whom treatment, including prophylactic treatment, with the cell compositions as described herein, is provided. The terms “non-human animals” and “nonhuman mammals” as used interchangeably herein, includes mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term “subject” also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish. “Patient in need thereof or “subject in need thereof is referred to herein as a patient diagnosed with or suspected of having a disease or disorder (e.g., diabetes).

[0139] As used herein, the term “parathyroid gland factor” or “PTG factor” are interchangeably used to refer to soluble factors present in parathyroid gland (PTG) tissue. Methods to identify soluble factors present in PTG tissue are known in the art (see, e.g., US Pat No 11,951,136). It is to be understood the term need not be limited to soluble factors harvested from PTG tissue and encompasses the soluble factors when obtained through other means (e.g., chemical synthesis, recombinant protein expression). Further, the term encompasses derivatives or variants of soluble present in PTG tissue. In some embodiments, the PTG factor comprises a proangiogenic factor. In some embodiments, the PTG factor comprises a hormone. Exemplary PTG factors of the disclosure are identified in Table 10.

[0140] As used herein, the term “pro-angiogenic factor” refers to a molecule (e.g., a polypeptide, small molecule, nucleic acid, etc) that triggers angiogenic signaling pathways to induce formation of new blood vessels. Exemplary pro-angiogenic factors of the disclosure include, but are not limited to, fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), NRP-1, angiopoietin (Ang) 1, Ang2, platelet derived growth factor (PDGF), PDGFR receptor (PDGFR), TGFP, endoglin, TGFP receptor, CCL2, VE-cadherin, CD31, ephrin, plasminogen activators, AC133, 1D1, semaphorins, and Nogo-A.Cell Compositions

[0141] In some embodiments, the disclosure provides compositions comprising a cell described herein.

[0142] In some embodiments, the disclosure provides compositions comprising a population of cells, wherein the cells or a plurality of cells are SCD cells described herein. In some embodiments, the population of cells of the composition is substantially homogeneous (i.e., limited to cells of a single differentiation and / or phenotypic state). For example, in some embodiments, at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of cells of the population are characterized by a common differentiation and / or phenotypic state. In some embodiments, the population of cells is heterogenous (i.e., composed of plurality of subpopulations of cells, each characterized by a distinct differentiation and / or phenotypic state). For example, in some embodiments, about 1% to about 50% of cells of the population are characterized by a common differentiation and / or phenotypic state and the remainder of cells in the population are characterized by a different differentiation and / or phenotypic state. Exemplarydifferentiation and / or phenotypic states are characterized by expression of a cell marker, expression and / or secretion of a hormone or soluble factor, proliferation rate, cell division, cellular response (e.g., to altered extracellular conditions), and metabolic activity. In some embodiments, the population of cells is substantially free of progenitor or precursor cells from which the SCD was derived. In some embodiments, the progenitor or precursor cells account for not more than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% of total cells within the population.

[0143] In some embodiments, the disclosure provides compositions comprising a population of cells, wherein the cells or a plurality of cells of the population are insulin producing cells. In some embodiments, the insulin producing cells are primary P cells. In some embodiments, the insulin producing cells are SCD p cells described herein. In some embodiments, the cells or the plurality of cells are formed into an organoid. In some embodiments, the cells or the plurality of cells are formed into clusters. In some embodiments, the cells or the plurality of cells are formed into spherical or substantially spherical clusters. In some embodiments, the clusters comprise a longest diameter of about 50 pm to about 1000 pm. In some embodiments, the clusters comprise a longest diameter of about 100 pm to about 350 pm. In some embodiments, the clusters comprise a longest diameter of about 100 pm to about 200 pm. In some embodiments, the population of cells further comprises at least one additional cell type of endoderm origin.

[0144] In some embodiments, the composition further comprises a nanoprobe described herein. In some embodiments, the nanoprobe is internalized by a plurality of cells in the population. In some embodiments, the nanoprobe is attached to the surface of a plurality of cells of the population. In some embodiments, the nanoprobe is configured to emit an optical signal for detection of the cell. In some embodiments, the nanoprobe is configured to induce thermal damage to cells or a plurality of cells of the population upon excitation with a photothermal wavelength. Nanoprobes configured to emit an optical signal and / or to induce thermal damage are further described as “Nanoprobes for SERS Detection and / or photothermal irradiation” herein. In some embodiments, the nanoprobe for SERS detection comprises a metallic nanoparticle (e.g., a gold nanoparticle, e.g., a gold nanostar), an optical reporter (e.g., an optical reporter operably linked to a surface of the metallic nanoparticle), and optionally a component operably linked to the surface of the optical reporter. In some embodiments, the nanoprobe is configured to detect a target nucleic acid, wherein the target nucleic acid is a biomarker expressed by cells of the population. Nanoprobes configured todetect a target nucleic acid are further described as “Nanoprobes for Target Acid Detection” herein.

[0145] In some embodiments, the composition comprises at least about 10 to about 100, about 10 to about 500, about 100 to about 500, about 100 to about IxlO3, about 500 to about 5xl03, about IxlO3to about 5xl03, about 2x103to about 5x103, or about 3x103to about 5x103nanoprobes per cell of the population of cells. In some embodiments, the nanoprobe is configured to emit an optical signal for detection by spectroscopy (e.g., SERS or SERRS) upon illumination with a wavelength and / or to induce thermal damage to cells or a plurality of cells of the population upon excitation with a photothermal wavelength, wherein the composition comprises at least about IxlO3to about 5xl03, about 2xl03to about 5xl03, or about 3xl03to about 5xl03nanoprobes per cell of the population of cells. Methods to quantify the number of nanoprobes per volume are known in the art, and include, for example, Inductively Coupled Plasma Mass Spectrometry (ICP-MS), microscopy, tunable resistive pulse sensing. In some embodiments, the number of nanoprobes per cell is determined by quantifying the number of cells per volume, and multiplying the number of nanoprobes per volume by the reverse of the number of cells per volume.

[0146] In some embodiments, the composition comprises a first nanoprobe and at least one second nanoprobe. In some embodiments, the first nanoprobe and / or the at least one second nanoprobe are each independently (i) internalized by a plurality of cells in the population, or (ii) attached to a surface of a plurality of cells of the population. In some embodiments, the first nanoprobe and the at least one second nanoprobe are each configured to detect a target nucleic acid, wherein the target nucleic acid is a biomarker expressed by cells of the population, and wherein the target nucleic acid detected by the first nanoprobe and by the at least one second nanoprobe are different. In some embodiments, the composition independently comprises at least about 10 to about 100, about 10 to about 500, about 100 to about 500, about 100 to about IxlO3, about 500 to about 5xl03of each of the first nanoprobe and of at least one second nanoprobe.

[0147] In some embodiments, the first nanoprobe is configured to detect the target nucleic acid, and the at least one second nanoprobe is configured for detection by spectroscopy (e.g., SERS or SERRS) upon illumination with a wavelength. In some embodiments, the at least one second nanoprobe induces thermal damage to cells or a plurality of cells of the population upon excitation with a photothermal wavelength. In some embodiments, the composition independently comprises at least about 10 to about 100, about 10 to about 500, about 100 to about 500, about 100 to about IxlO3, about 500 to about 5xl03of the firstnanoprobe. In some embodiments, the composition comprises at least about lxl03to about 5xl03, about 2xl03to about 5xl03, or about 3xl03to about 5xl03of the at least one second nanoprobe per cell of the population of cells.

[0148] In some embodiments, the wavelength and the photothermal wavelength are each generated by a laser, but using a different laser power, power density, and / or illumination time. For example, at least one of the laser power, power density and / or illumination time is higher for generating the photothermal wavelength for inducing thermal damage of the cells or a plurality of cells of the population as compared to the wavelength for detection.

[0149] In some embodiments, the biomarker is an indicator of a cellular phenotype, process, and / or response. In some embodiments, the biomarker is an indicator of a stress response. In some embodiments, the biomarker is an indicator of an inflammatory response. In some embodiments, the biomarker is an indicator of cell growth. In some embodiments, the biomarker is an indicator of apoptosis. In some embodiments, the biomarker is an indicator of DNA damage. In some embodiments, the biomarker is an indicator of hypoxia.

[0150] In some embodiments, the cells or a plurality of cells of the population comprising the nanoprobe are characterized by an improved property. In some embodiments, the cells or the plurality of cells are insulin producing cells, wherein the improved property is expression, storage, and / or secretion of insulin.

[0151] In some embodiments, the composition further comprises a support factor described herein. In some embodiments, the cells or a plurality of cells of the population are characterized by an improved property in the presence of the support factor. In some embodiments, the improved property comprises increased survival, e.g., under hypoxic, inflamed, and / or nutrient deprived conditions, and / or reduced sensitivity to immune killing following administration to a subject.

[0152] In some embodiments, the support factor is a cell derived from a parathyroid gland (PTG) tissue described herein. In some embodiments, the population of cells comprising the cell derived from PTG tissue is characterized by increased viability following in vivo administration, e.g., as compared to a population of cells lacking the cell derived from PTG tissue.

[0153] In some embodiments, the support factor is a CD34+ cell described herein. In some embodiments, the population of cells comprising the CD34+ cell is characterized by increased viability following in vivo administration, e.g., as compared to a population of cells lacking the CD34+ cell.

[0154] In some embodiments, the support factor is a PTG hormone, a proangiogenic factor, and / or a cytokine. In some embodiments, the population of cells comprising the PTG hormone, proangiogenic factor, and / or cytokine is characterized by increased viability following in vivo administration, e.g., as compared to a population of cells lacking the PTG hormone, proangiogenic factor, and / or cytokine.

[0155] In some embodiments, the support factor is a tolerogenic factor. In some embodiments, the population of cells comprising the tolerogenic factor is characterized by a reduced sensitivity to immune killing following in vivo administration, e.g., as compared to a population of cells lacking the tolerogenic factor.

[0156] In some embodiments, the cells or the plurality of cells comprise a genetic modification described herein for immune evasion. In some embodiments, the genetic modification comprises a gene knockout, wherein the gene knockout reduces an autoimmune or allogeneic immune response to the cells or the plurality of cells following in vivo administration. In some embodiments, the genetic modification comprises a gene insertion of a tolerogenic factor, wherein the gene insertion reduces an autoimmune or allogeneic immune response to the cells or the plurality of cells following in vivo administration.Stem Cell Derived Cells

[0157] In some embodiments, a SCD cell of the disclosure is differentiated from a totipotent stem cell. In some embodiments, a SCD cell of the disclosure is differentiated from a pluripotent stem cell. In some embodiments, a SCD cell of the disclosure is differentiated from a multipotent stem cell. In some embodiments, a SCD cell of the disclosure is differentiated from a unipotent stem cell. In some embodiments, a SCD cell of the disclosure is differentiated from an adult stem cell. Adult stem cells are undifferentiated lineage- committed cells found in various bodily tissues that have the capacity for self-renewal and can be differentiated into certain specialized cell types based upon their tissue of origin. In some embodiments, a SCD cell of the disclosure is differentiated from a partially reprogrammed somatic cell (e.g., a somatic cell which has been partially reprogrammed to an intermediate state between an iPSC and the somatic cell from which it was derived). In some embodiments, a SCD cell of the disclosure is differentiated from a progenitor cell.

[0158] The ability of a stem cell to give rise to cells of greater developmental potential may occur naturally or upon culture with various factors or conditions, as further described herein.

[0159] In some embodiments, the SCD cell comprises a differentiation phenotype characterized by one or more functional properties. In some embodiments, the one or more functional properties comprises the ability to further differentiate into specialized cells, e.g., in cell culture and / or subsequent to in vivo administration to a subject. In some embodiments, the one or more functional properties are characteristic of a terminal differentiation state. In some embodiments, the one or more functional properties comprises the ability to remain viable for an extended duration, e.g., in cell culture and / or subsequent to in vivo administration to a subject.

[0160] In some embodiments, the SCD cell is a cell of any tissue or organ. In some embodiments, the SCD cell is a skin cell, parathyroid cell, intestinal cell, endocrine cell, cardiac cell, brain cell, kidney cell, liver cell, digestive tract cell, salivary gland cell, adrenal gland cell, prostate cell, lung cell, pancreatic cell, bone cell, immune cell, hematopoietic cell, vascular cell, ocular cell, connective tissue cell, musculoskeletal cell. In some embodiments, the SCD cell is a cell of adipose tissue, skin, heart, vascular system, musculoskeletal system, bone, eye, cornea, immune system, connective tissue, lung, kidney, liver, pancreas, heart, stomach, or intestine.

[0161] In some embodiments, the SCD cell is an allogeneic cell. In some embodiments, the SCD cell is an autologous cell. In some embodiments, the SCD cell is a hepatocyte. In some embodiments, the SCD cell is a cardiomyocyte. In some embodiments, the SCD cell is a neural cell. In some embodiments, the SCD cell is a mesenchymal cell. In some embodiments, the SCD cell is a pancreatic beta cell. In some embodiments, the SCD cell is of the human pancreatic lineage.Pluripotent Stem Cells

[0162] In some embodiments, the SCD cells of the disclosure are generated from pluripotent stem cells (e.g., human pluripotent stem cells). Pluripotent stem cells have the ability to differentiate into cells of all three germ layers (i.e., ectoderm, mesoderm, and endoderm) and have the characteristics of self-renewability.

[0163] As understood by the skilled artisan, sources of pluripotent stem include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). The generation of pluripotent stem cells (e.g., human pluripotent stem cells) is generally known in the art.

[0164] In some embodiments, a SCD cell of the disclosure is differentiated from an ESC. In some embodiments, a SCD cell of the disclosure is differentiated from a human ESC. ESCsare pluripotent cells isolated from the inner cell mass of the early mammalian embryo that implants into the uterus that have capacity for self-renewal and can be differentiated to multiple types of adult cells in vitro (Thomson, et al (1998) Science 282: 1145-1147). Methods for maintaining ESCs in culture are known in the art. Exemplary methods are described in US10 / 486,408; US11 / 021,618; US11 / 165,305; US11 / 573,662; US12 / 729,084; US12 / 093,590; US11 / 993,399; US11588,693; US11 / 681,687; US11 / 807,223; US11 / 773,944; US12 / 099759; US12 / 107020; US12 / 618649; US12 / 765714; US11 / 838054; US12 / 264760; US14 / 201630; US14 / 106330; and PCT / US2016 / 061442, each of which are hereby incorporated by reference.

[0165] In some embodiments, a SCD cell of the disclosure is differentiated from an ESC cell line. In some embodiments, the ESC cell line is established from cells of blastocytes / unfertilized embryos. In some embodiments, the ESC cell line is established from cells that received a somatic cell nuclear transfer (e.g., ooplasm or nuclear transfer). In some embodiments, the ESC cell line is established from germ cell lineage cells (e.g., oocyte or testicular cells).

[0166] Exemplary ESC cell lines are known in the art and include, but are not limited to the H9, CyT49, CyT25, CyT203, CyT212, MEL-1 ESC cell lines. In some embodiments, the ESC cell line is one made available through the NIH Embryonic Stem Cell Registry or the HHMI HUES collection. In some embodiments, the ESC cell line is the MEL-1 ESC cell line (NIH registration number: 0139) (Micallef et al (2011) Diabetologia 55:694). In some embodiments, the ESC cell line is one made available through the California Institute for Regenerative Medicine (CIRM) (word wide web: cirm.ca.gov / cirm-information-applicants- clinically-compatible-hpsc-lines / ).

[0167] In some embodiments, a SCD cell of the disclosure is differentiated from an ESC cell line established under current Good Manufacturing Practices (cGMP). Clinical ESC cell lines refer to ESC cell lines cultured in the absence of animal -derived components and under cGMP conditions. Exemplary ESC cell lines established under cGMP include, but are not limited to, those made commercially available by WiCell (see world wide web: wicell.org / home / stem-cells / catalog-of-stem-cell-lines / collections / cgmp-cell-banks.cmsx).

[0168] In some embodiments, a SCD cell of the disclosure is differentiated from an iPSC. In some embodiments, a SCD cell of the disclosure is differentiated from a human iPSC. As used herein, the terms “iPS cell” and “induced pluripotent stem cell” are used interchangeably and refer to a pluripotent stem cell artificially derived (e.g., induced or by complete reversal) from a non-pluripotent cell, typically an adult somatic cell, for example,by inducing a forced expression of one or more genes. iPSCs are reprogrammed from somatic cells into the embryonic-like pluripotent state by overexpression of key reprogramming genes and have a similar capacity for self-renewal and differentiation (Takahashi, et al (2007) Cell 131 :861-72; Yu, et al (2007) Science! 18: 1917-20; Seki, et al (2015) World J. Stem Cells'! : 116; Lakshmipathy &Vermuri Ed. Methods in Molecular Biology: Pluripotent Stem Celsl, Methods and Protocols Springer 2013; each of which are herein incorporated by reference). Generally, iPSCs are generated from somatic cells (e.g., blood cells, fibroblasts) by transient expression of a reprogramming factor using, e.g., an episomal vector. Once the cells are reprogrammed due to expression of the reprogramming factors, they become pluripotent and express the factors from endogenous genes. Exemplary reprogramming factors include, but are not limited to, OCT4, KLF4, SOX2, c-Myc, SOKMNLT, NANOG, LIN28, and SV40L T antigen.

[0169] In some embodiments, an SCD cell of the disclosure is differentiated from an iPSC generated from a somatic cell (e.g., a blood cell, a fibroblast) induced to express a reprogramming factor selected from OCT4, KLF4, SOX2, c-Myc, SOKMNLT, NANOG, LIN28, SV40L T antigen, and a combination thereof. In some embodiments, the reprogramming factor comprises OCT4. In some embodiments, the reprogramming factor comprises OCT4 and SOX2. In some embodiments, the reprogramming factor comprises OCT4, SOX2, and KLF4. In some embodiments, the reprogramming factor comprises OCT4, SOX2, KLF4, and c-Myc. In some embodiments, the reprogramming factor further comprises one or more of SOKMNLT, NANOG, LIN28, and SV40L T antigen. In some embodiments, the somatic cell is obtained from a donor. In some embodiments, the somatic cell is obtained from the subject intended to receive a transplant comprising the SCD cell.

[0170] Methods for establishing or determining pluripotency of a pluripotent stem cell described herein are known in the art. In some embodiments, the method comprises assaying for expression of a pluripotency-specific factor using, e.g., flow cytometry, immunohistochemistry, Western blot, and / or ELISA. In some embodiments, the pluripotency-specific factor comprises NANOG, OCT4, SOX2, ESRRB, TRA-1-60, TRA-1- 81, SSEA4, or a combination thereof. In some embodiments, the method comprises differentiating the pluripotent stem cell, wherein successful differentiation is an indication of pluripotency.Insulin-Producing Cells

[0171] In some embodiments, the disclosure provides a population comprising insulin producing cells for use in the compositions and methods described herein.

[0172] Pancreatic islets (also known as “islets of Langerhans”) are regions of the pancreas that contain endocrine cells (e.g., hormone-producing cells, such as, but not limited to, insulin-producing cells). During embryonic development, pancreatic islets are derived from definitive endoderm during gastrulation (see Jin, et al (2022) Cell Regeneration 11 :24). The endoderm folds to form two buds that fuse to form the pancreatic endoderm. The pancreatic endothelium, which comprises multipotent progenitor cells, undergoes morphological changes and forms exocrine and endocrine components. The endocrine cells comprise insulin (INS) producing pancreatic P cells, glucagon (GCG)-producing a cells, somatostain (SST)- producing 5 cells, pancreatic polypeptide (PP)-producing cells, and ghrelin-producing s-cells.

[0173] The P cells in islets sense increases in blood glucose concentrations and release an appropriate amount of insulin in response, thereby enabling sugar uptake by the liver and peripheral tissues (see Nair, et al (2020) Nat Rev Endocrinol 16:506). Once glucose levels in the blood are lowered, P cells respond by terminating insulin secretion (see Nair (2020); Blum, et al (2012) Nat Biotech 30:261). Islet a cells release glucagon to stimulate hepatic gluconeogenesis and glycogenolysis, which in turn raises blood glucose levels (Id.).Inhibition of both insulin and glucagon secretion is mediated by somatostatin, which is released by islet 5 cells (see van der Meulen, et al (2015) Nat Med 21 :769). The interaction between release of insulin, glucagon, and somatostatin maintains glucose concentrations below about 5.6 mM in a healthy human adult, which in turn is needed to avoid hypoglycemia (low blood sugar) and hyperglycemia (high blood sugar). In subjects with type 1 diabetes mellitus (T1DM), insulin producing p cells are destroyed due to an autoimmune response. In subjects with type 2 diabetes mellitus (T2DM), p cells are unable to produce sufficient insulin to meet physiological demand. There is currently no cure for T1DM or T2DM, and subjects require exogenous insulin administration. The downside of insulin delivery via subcutaneous injection is that it does not mimic the temporal glucose control provided by endogenous insulin producing p cells and other pancreatic islet cells. Thus, there is a need for improved therapies for treatment of diabetes.

[0174] Accordingly, the present disclosure provides compositions comprising insulin producing cells for administering to a subject to treat conditions associated with defective P cell function (e.g., T1DM and / or T2DM).

[0175] In some embodiments, the insulin producing cell is a human donor pancreatic P cell. Sources of human donor pancreatic P cells are known in the art and include, e.g., the Networkfor Pancreatic Organ Donors with Diabetes (see word wide web: jdrfn-pod.org), Prodo Laboratories (see prodolabs.com), and the Alberta Diabetes Institute IsletCore (see word wide web: ualberta.ca / alberta-diabetes / core-services / isletcore.html). In some embodiments, the insulin producing cell comprises a primary pancreatic P cell obtained from a human donor. In some embodiments, the insulin producing cell comprises a primary pancreatic P cell obtained from a human cadaver.

[0176] In some embodiments, the insulin producing cell is a cell of a human P cell line. Sources of human p cell lines are known in the art (see, e.g., Scharfmann, et al (2019) JCI 129:3511).

[0177] In some embodiments, the insulin producing cell is a non-human insulin producing cell. In some embodiments, the insulin producing cell is a porcine pancreatic P cell. In some embodiments, the insulin producing cell is derived from a porcine pancreatic islet. In some embodiments, the insulin producing cell is a murine pancreatic P cell. In some embodiments, the insulin producing cell is derived from a mouse or rat pancreatic islet.

[0178] In some embodiments, the insulin producing cell is derived from an insulinoma cell line. In some embodiments, the insulinoma cell line is a mouse, rat, or human insulinoma cell line. Sources of insulinoma cell lines are known in the art (see, e.g., Labriola et al (2009) BMC Cell Biol. 10:49; Efrat et al (1993) Diabetes 42(6): 901-907; Knaack et al (1994) Diabetes 43(12): 1413-1417).

[0179] In some embodiments, the insulin producing cell is a SCD P cell. In some embodiments, the SCD p cell is differentiated from a pluripotent stem cell according to a method described herein. In some embodiments, the SCD p cell is differentiated from an endoderm cell. In some embodiments, the insulin producing cell is differentiated from a cell of endoderm origin. In some embodiments, the SCD P cell is differentiated from a definitive endoderm cell. In some embodiments, the SCD p cell is differentiated from a pancreatic endoderm cell. In some embodiments, the SCD p cell is differentiated from a pancreatic progenitor cell, or precursor thereof. In some embodiments, the SCD P cell is differentiated from a PDX-1 positive pancreatic progenitor. In some embodiments, the SCD P cell is differentiated from a PDX-1, NKX6.1- positive pancreatic progenitor.Methods of Generating Populations Comprising Stem Cell Derived Insulin ProducingPancreatic Cells

[0180] Provided herein are methods for generating cell compositions comprising SCD P cells. In some embodiments, the cell composition is generated from a population comprising pluripotent stem cells described herein. In some embodiments, the cell composition further comprises at least one additional endocrine cell type. In some embodiments, the at least one additional endocrine cell type is selected from an SCD a cell, a SCD 5 cell, a SCD y cell, and a SCD 8 cell. In some embodiments, the cell composition comprises not more than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% pancreatic progenitor cells.

[0181] Differentiation of stem cells into specialized cell types often involve providing signal modulators at specific time points in a manner that mimics signals and development pathways from the embryo (Irion, et al (2008) Cold Spring Harb Symp Quant Biol 73: 101- 110). For example, strategies developed for generating insulin producing cells from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are based upon approaches that mimic embryonic pancreatic development. Without wishing to be limited to theory, a pluripotent stem cell in the course of normal ontogeny can differentiate first to an endoderm cell that is capable of forming pancreas cells and other endoderm cell types. Further differentiation of an endoderm cell leads to the pancreatic pathway, where, in some embodiments, -98% of the cells become exocrine, ductular, or matrix cells, and -2% become endocrine cells. Endoderm cells can also be differentiated into other cells of endodermal origin, e.g. lung, liver, intestine, thymus etc. Early endocrine cells are islet progenitors, which can then differentiate further into functional endocrine cells which secrete insulin, glucagon, somatostatin, or pancreatic polypeptide. As appreciated by the skilled artisan, directed differentiation is performed by application of cytokines (e.g., epidermal growth factor, bFGF) and signaling modulators (e.g., bone morphogenetic proteins, y- secretase inhibitors) at each stage of development to activate or inhibit specific signaling pathways involved in the generation of adult pancreatic P cells (D’ Amour, et al (2006) Nat Biotech 24: 1392-1401; Rezania, et al (2014) Nat. Biotechnol 32: 1121-33; Zhang, et al (2009) Cell Res 19:429-38). Exemplary methods for differentiating a pluripotent stem cell to generate a SCD p cell of the disclosure are further described in PCT / US2016 / 028963, PCT / US2021 / 044080, and PCT / US2021 / 013735, each of which is hereby incorporated by reference. In some embodiments, the method is described in Parent, et al (2022) Stem Cell Reports, 17:979; Nair, et al (2019) Protoc Exchange doi.org / 10.1038 / protex.2018.140; and Nair et al (2019) Nat Cell Biol 21 :263, each of which are herein incorporated by reference.

[0182] In some embodiments, the SCD p cells of the disclosure are generated by differentiating pluripotent stem cells (e.g., ESCs or iPSCs) into a committed pancreatic cell lineage. In some embodiments, the method comprises stepwise differentiation of pluripotent stem cells (e.g., an ESC or an iPSC) to definitive endoderm cells, cells of the pancreatic lineage, and then to pancreatic P cells. In some embodiments, the population comprising pluripotent stem cells (e.g., human ESCs) are characterized by expression of OCT4, NANOG, and SOX2. In some embodiments, the pluripotent stem cells of the population are human ESCs. In some embodiments, the pluripotent stem cells of the population are a human ESC cell line described herein or known in the art. In some embodiments, the pluripotent stem cells of the population are a cGMP human ESC cell line described herein or known in the art.

[0183] In vivo, the definitive endoderm is generated by the process of gastrulation of embryogenesis, in which epiblast cells differentiate to form the three germ layers. Definitive endoderm cells then give rise to cells of various tissues, including pancreatic, liver, lung, thyroid, thymus, and epithelial lining cells (“endoderm lineage” cells). The definitive endoderm forms the primitive gut tube, which becomes the pharynx, esophagus, stomach, duodenum, small and large intestine, and associated organs (e.g., pancreas, lung, thyroid, thymus, parathyroid, and liver). The pancreas, liver, and duodenum differentiate from cells of the posterior gut tube, which are characterized by expression of haematopoietically expressed homeobox (HHEX), pancreatic and duodenal homeobox 1 (PDX1), one cut homeobox 1 (ONECUT1 or HNF6), and hepatocyte nuclear factor 4 alpha (HNF4A).

[0184] In some embodiments, the directed differentiation of a population comprising pluripotent stem cells to a population comprising definitive endoderm cells is the first stage of generating a population comprising SCD P cells. In some embodiments, the first stage is performed by contacting the population comprising pluripotent stem cells with culture conditions described herein. In some embodiments, the contacting results in a population comprising definitive endoderm cells following a duration of about 1 day, about 2 days, about 3 days, or about 4 days. In some embodiments, the contacting results in a population comprising definitive endoderm cells following a duration of about 3 days or about 4 days.

[0185] In some embodiments, the directed differentiation of a population comprising definitive endoderm cells to a population comprising posterior foregut is the second stage of generating a population comprising SCD p cells. In some embodiments, the second stage is performed by contacting the population comprising definitive endoderm cells with a culture condition described herein. In some embodiments, the contacting results in a populationcomprising posterior foregut following a duration of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days. In some embodiments, the contacting results in a population comprising primitive gut tube following a duration of about 2 days, about 3 days, or about 4 days. In some embodiments, the contacting results in a population comprising posterior foregut following a duration of about 2 days, about 3 days, or about 4 days.

[0186] In some embodiments, the directed differentiation of a population comprising posterior gut tube to a population comprising pancreatic progenitor cells is the third stage of generating a population comprising SCD P cells. In some embodiments, the third stage is performed by contacting the population comprising posterior foregut with a culture condition described herein. In some embodiments, the contacting results in a population comprising pancreatic progenitor cells following a duration of about 1 day, about 2 days, about 3 days, or about 4 days.

[0187] In some embodiments, the directed differentiation of a population comprising pancreatic progenitor cells to a population comprising insulin producing pancreatic cells is the fourth stage of generating a population comprising SCD P cells. In some embodiments, the fourth stage is performed by contacting the population comprising pancreatic progenitor cells with a culture condition described herein. In some embodiments, the contacting results in a population comprising SCD P cells following a duration of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or longer. In some embodiments, the contacting results in a population comprising endocrine progenitor cells following a duration of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days. In some embodiments, the contacting results in a population comprising immature islets following a duration of about 8 days, about 9 days, about 10 days, or about 11 days. In some embodiments, the contacting results in a population comprising mature islets following a duration of about 15 days, about 16 days, about 17 days, about 18 days, or longer.

[0188] In some embodiments, contacting a cell population described herein with a culture medium is performed according to any cell culture modality known in the art. For example, in some embodiments, the contacting is performed using 2D culture (see, e.g., Marek- Trzonkowska, et al (2012) Diabetes Care 35: 1817; Schulz, et al (2015) Stem Cell TranslMed 4:927, each of which are hereby incorporated by reference). In some embodiments, the contacting is performed in multi -well plates under orbital stirring (see, e.g., Nair, et al (2019) Nat Cell Biol 21 :263; Schulz, et al (2012) PLoS One 7:e37004, each of which are hereby incorporated by reference). In some embodiments, the contacting is performed using rollerbottles (see, e.g., Schulz, et al (2015) Stem Cell Transl Med 4:927). In some embodiments, the contacting is performed using a bioreactor, such as a vertical-wheel bioreactor (e.g., a PBS Vertical -Wheel bioreactor), or wave-agitation bioreactor (see, e.g., Somerville, et al (2012) J. Transl Med 10:69; Fraser, et al (2018) Mol Ther Methods Clin Dev 8: 198, each of which are hereby incorporated by reference). In some embodiments, the contacting is performed using a stirred suspension bioreactor (see, e.g., Krawetz, et al (2010) Tissue Eng Part C. Methods 16:573; Kempf, et al (2014) Stem Cell Rep 3: 1132, each of which are hereby incorporated by reference). In some embodiments, the contacting is performed in the presence of a microcarrier (e.g., polystyrene, glass, alginate, or dextran beads) (see, e.g., Lock, et al (2009) Tissue Eng Part A 15:2051; Bardy, et al (2013) Tissue Eng Part C Methods 19:166, each of which are hereby incorporated by reference). In some embodiments, the culturing described herein is performed under stirring or shaking. In some embodiments, the population comprising cells are suspended in a container, wherein the suspension is stirred or shaken.

[0189] In some embodiments, the culture medium is xeno-free. In some embodiments, the culture medium does not comprise animal products derived from a non-human.(i) Directed Differentiation to Definitive Endoderm Cells

[0190] In some embodiments, the method comprises a first stage of differentiation, wherein the first stage of differentiation comprises differentiating the population comprising pluripotent stem cells (e.g., human ESC or iPSC cells) to a second population comprising definitive endoderm cells. In some embodiments, the differentiating comprises contacting the population comprising pluripotent stem cells with (i) a first culture medium comprising a ROCK inhibitor, a TGFP superfamily growth factor described herein (e.g., activin A), and an epidermal growth factor family polypeptide (e.g., Heregulin P-1), (ii) a second culture medium comprising a TGFP superfamily growth factor described herein (e.g., activin A) and a WNT activator described herein (e.g., CHIR99021), and (iii) a third culture medium comprising a TGFP superfamily growth factor described herein (e.g., activin A).

[0191] In some embodiments, the first stage of differentiation comprises contacting the population comprising pluripotent stem cells with a first culture medium comprising (i) a ROCK inhibitor; (ii) Heregulin P-1; and (iii) activin A. In some embodiments, the contacting is performed for a duration of at least 1 day. In some embodiments, the contacting is performed for about 1 to about 5 days. In some embodiments, the contacting is performed forabout 1 day. In some embodiments, the first culture medium comprises a concentration of ROCK inhibitor, wherein the concentration is at least about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, or about 10 pM. In some embodiments, the concentration is about 5 pM to about 50 pM. In some embodiments, the concentration of ROCK inhibitor is about 5 pM to about 20 pM. In some embodiments, the concentration of ROCK inhibitor is about 10 pM. In some embodiments, the first culture medium comprises a concentration of Heregulin P-1, wherein the concentration is at least about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, or about 10 ng / ml. In some embodiments, the concentration of Heregulin P-1 is about 5 ng / ml to about 50 ng / ml. In some embodiments, the concentration of Heregulin P-1 is about 5 ng / ml to about 20 ng / ml. In some embodiments, the concentration of Heregulin P-1 is about 10 ng / ml. In some embodiments, the first culture medium comprises a concentration of activin A, wherein the concentration is at least about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, or about 10 ng / ml. In some embodiments, the concentration is about 5 ng / ml to about 50 ng / ml. In some embodiments, the concentration of activin A is about 5 ng / ml to about 20 ng / ml. In some embodiments, the concentration of activin A is about 10 ng / ml.

[0192] In some embodiments, the first stage of differentiation further comprises contacting the population with a second culture medium, wherein the second culture medium comprises a TGFP superfamily growth factor described herein (e.g., activin A), and a WNT activator described herein (e.g., CHIR99021). In some embodiments, the population of cells contacted with the first culture medium, or a portion thereof, are transferred to the second culture medium.

[0193] In some embodiments, the second culture medium comprises (i) a concentration of activin A, wherein the concentration is at least about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL; and (ii) a concentration of the CHIR99021, wherein the concentration is at least about 0.1 pM, 0.5 pM, 1.0 pM, 1.5 pM, 2.0 pM, 2.5 pM, or 3 pM. In some embodiments, the concentration of activin A is about 10 ng / mL to about 200 ng / mL. In some embodiments, the concentration of the CHIR99021 is about 0.1 pM to about 10 pM.

[0194] In some embodiments, the second culture medium further comprises a concentration of vitamin C, wherein the concentration is at least about 0.05 mM, about 0.1 mM, about 0.15 mM, about 0.2 mM, or about 0.25 mM. In some embodiments, the concentration of vitamin Cis about 0.05 mM to about 2 mM. In some embodiments, the concentration of vitamin C is about 0.05 mM to about 0.25 mM.

[0195] In some embodiments, the second culture medium further comprises an ITS supplement (e.g., an Gibco ITS supplement). In some embodiments, the ITS supplement comprises insulin, transferrin, and selenium.

[0196] In some embodiments, the second culture medium further comprises fetal bovine serum (FBS). In some embodiments, the second culture medium further comprises a concentration of FBS, wherein the concentration of FBS is at least about 0.05%, 0.1%, 0.15%, or 0.2%. In some embodiments, the concentration of FBS is about 0.05% to about 10%. In some embodiments, the concentration of FBS is about 0.2%.

[0197] In some embodiments, contacting with the second culture medium is performed for a duration of at least about 1 day. In some embodiments, the duration is about 1 day to about 10 days. In some embodiments, the duration is about 1 day.

[0198] In some embodiments, the first stage of differentiation further comprises contacting the population with a third culture medium, wherein the third culture medium a TGFP superfamily growth factor described herein (e.g., activin A). In some embodiments, the population of cells contacted with the second culture medium, or a portion thereof, are transferred to the third culture medium.

[0199] In some embodiments, the third culture medium comprises a concentration of activin A, wherein the concentration is at least about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL. In some embodiments, the concentration of activin A is about 10 ng / mL to about 200 ng / mL.

[0200] In some embodiments, the third culture medium further comprises a concentration of vitamin C, wherein the concentration is at least about 0.05 mM, about 0.1 mM, about 0.15 mM, about 0.2 mM, or about 0.25 mM. In some embodiments, the concentration of vitamin C is about 0.05 mM to about 2 mM. In some embodiments, the concentration of vitamin C is about 0.05 mM to about 0.25 mM.

[0201] In some embodiments, the third culture medium comprises an ITS supplement (e.g., Gibco ITS supplement). In some embodiments, the ITS supplement comprises insulin, transferrin, and selenium.

[0202] In some embodiments, the third culture medium further comprises fetal bovine serum (FBS). In some embodiments, the third culture medium further comprises a concentration of FBS, wherein the concentration of FBS is at least about 0.05%, 0.1%, 0.15%, or 0.2%. Insome embodiments, the concentration of FBS is about 0.05% to about 10%. In some embodiments, the concentration of FBS is about 0.2%.

[0203] In some embodiments, contacting with the third culture medium is performed for a duration of at least about 1 day. In some embodiments, the duration is about 1 day to about 10 days. In some embodiments, the duration is about 1 day.

[0204] In some embodiments, the first culture medium, the second culture medium, and / or the third culture medium each comprise a base media, wherein the base media is any defined media for human pluripotent stem cell culture known in the art. In some embodiments, the defined media is free of ancillary materials that are animal -derived (e.g., non-human animal or human-derived). In some embodiments, the defined media is StemFit Basisc03 complete cell medium.

[0205] In some embodiments, a plurality of cells of the second population are characterized by expression of a cell marker. In some embodiments, expression of the cell marker is increased in a plurality of cells of the second population as compared to the population comprising pluripotent stem cells from which it was derived. In some embodiments, a plurality of cells of the second population is characterized by expression of CXCR4, SOX17, and / or FOXA2, but do not substantially express PDX1. In some embodiments, at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% of cells of the second population are characterized by expression of CXCR4, SOX17, and FOXA2, but do not substantially express PDX1. In some embodiments, a plurality of cells of the second population are definitive endoderm cells. In some embodiments, at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% of cells of the second population are definitive endoderm cells.(ii) Directed Differentiation to Posterior Foregut Cells

[0206] In some embodiments, the methods described herein for generating a population comprising SCD P cells further comprise a second stage of differentiation, wherein the second stage of differentiation comprises differentiating the second population comprising definitive endoderm cells to a third population comprising posterior foregut cells. In some embodiments, a plurality of cells of the second population are definitive endoderm cells, wherein the second stage of differentiation comprises differentiating the definitive endodermcells to posterior foregut cells. In some embodiments, the differentiating comprises contacting the second population with a first culture medium comprising a growth factor of the fibroblast growth factor (FGF) family described herein (e.g., KGF). In some embodiments, the differentiating further comprising contacting the second population with a second culture medium comprising an activator of the retinoic acid signaling pathway described herein (e.g., TTNPB), an activator of protein kinase C (PKC) described herein (e.g., PMA), an inhibitor of bone morphogenetic protein (BMP) signaling described herein (e.g., LDN193189), and / or an inhibitor of sonic hedgehog (SHH) signaling described herein (e.g., SANT1). In some embodiments, the population comprising definitive endoderm contacted with the first culture medium, or a portion thereof, are then contacted with the second culture medium.

[0207] In some embodiments, the second stage of differentiation comprises contacting the second population comprising definitive endoderm cells with a first culture medium, wherein the first culture medium comprises the fibroblast growth factor (FGF) family (e.g., KGF).

[0208] In some embodiments, the first culture medium comprises a concentration of KGF, wherein the concentration is at least about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, or about 50 ng / mL. In some embodiments, the concentration of KGF is about 5 ng / mL to about 200 ng / mL. In some embodiments, the concentration of KGF is about 50 ng / mL.

[0209] In some embodiments, the first culture medium further comprises vitamin C. In some embodiments, the first culture medium further comprises a concentration of vitamin C, wherein the concentration is about at least about 0.1 mM, about 0.15 mM, about 0.2 mM, or about 0.25 mM. In some embodiments, the concentration of vitamin C is about 0.1 mM to about 3 mM.

[0210] In some embodiments, the first culture medium comprises an ITS supplement (e.g., Gibco ITS supplement). In some embodiments, the ITS supplement comprises insulin, transferrin, and selenium.

[0211] In some embodiments, the first culture medium further comprises FBS. In some embodiments, the first culture medium further comprises a concentration of FBS, wherein the concentration of FBS is at least about 0.5%, about 1%, about 1.5%, or about 2%. In some embodiments, the concentration of FBS is about 0.5% to about 10%. In some embodiments, the concentration of FBS is about 2%.

[0212] In some embodiments, the first culture medium comprises a base media, wherein the base media is any defined media for human pluripotent stem cell culture known in the art. Insome embodiments, the defined media is free of ancillary materials that are animal -derived (e.g., non-human animal or human-derived). In some embodiments, the defined media is StemFit Basisc03 complete cell medium.

[0213] In some embodiments, contacting with the first culture medium is performed for a duration of at least about 1 day, about 2 days, or about 3 days. In some embodiments, the contacting is performed for a duration of about 1 day to about 5 days. In some embodiments, the contacting is performed for a duration of about 1 day, about 2 days, or about 3 days.

[0214] In some embodiments, the second culture medium comprises a retinoic pathway activator described herein (e.g., TTNPB), a SHH signaling inhibitor described herein (e.g., Sant-1), a PKC activator described herein (e.g., PMA), and / or a BMP signaling inhibitor described herein (e.g., LDN193189). In some embodiments, the second culture medium comprises the retinoic pathway activator, the SHH signaling inhibitor, the PKC activator, and the BMP signaling inhibitor. In some embodiments, the second culture medium comprises TTNPB, Sant-1, PMA, and LDN193189.

[0215] In some embodiments the second culture medium comprises (i) a concentration of TTNPB, wherein the concentration is at least about 0.5 nM, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, or about 3 nM; (ii) a concentration of Sant-1, wherein the concentration is at least about 50 nM, about 100 nM, about 150 nM, about 200 nM, or about 250 nM; (iii) a concentration of PMA, wherein the concentration is at least about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, or about 30 nM; and (iv) a concentration of LDN193189, wherein the concentration is at least about 50 nM, about 100 nM, about 150 nM, about 200 nM, or about 250 nM. In some embodiments the concentration of TTNPB is about 0.5 nM to about 10 nM. In some embodiments the concentration of TTNPB is about 3 nM. In some embodiments, the concentration of Sant-1 is about 50 nM to about 500 nM. In some embodiments, the concentration of Sant-1 is about 250 nM. In some embodiments, the concentration of PMA is about 5 nM to about 100 nM. In some embodiments, the concentration of PMA is about 30 nM. In some embodiments, the concentration of LDN193189 is about 50 nM to about 500 nM. In some embodiments, the concentration of LDN193189 is about 250 nM.

[0216] In some embodiments, the second culture medium further comprises non-essential amino acids, sodium pyruvate, and neurocult.

[0217] In some embodiments, the second culture medium further comprises a concentration of vitamin C, wherein the concentration is about at least about 0.1 mM, about 0.15 mM,about 0.2 mM, or about 0.25 mM. In some embodiments, the concentration of vitamin C is about 0.1 mM to about 3 mM.

[0218] In some embodiments, contacting with the second culture medium is performed for a duration of at least about 1 day, about 2 days, or about 3 days. In some embodiments, the contacting is performed for a duration of about 1 day to about 5 days. In some embodiments, the contacting is performed for a duration of about 1 day, about 2 days, or about 3 days.

[0219] In some embodiments, a plurality of cells of the third population are characterized by expression of a cell marker. In some embodiments, expression of the cell marker is increased in a plurality of cells of the third population as compared to the population comprising definitive endoderm cells from which it was derived. In some embodiments, at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% of cells of the third population are characterized by expression of PDX1, HNF6, FOXA2, HNF4A, HNF1B, and / or FOXA1. In some embodiments, a plurality of cells of the third population are posterior foregut cells. In some embodiments, at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% of cells of the third population are posterior foregut cells.(iii) Directed Differentiation to PDX1, NKX6.1 -Positive Pancreatic Progenitor Cells

[0220] In some embodiments, the methods described herein for generating a population comprising SCD P cells further comprise a third stage of differentiation, wherein the third stage of differentiation comprises differentiating a third population comprising posterior foregut cells to a fourth population comprising pancreatic progenitor cells (e.g., PDX1, NKX6.1 -positive pancreatic progenitor cells). In some embodiments, the differentiating comprises contacting the population comprising posterior foregut cells with (i) a first culture medium culture medium comprising epidermal growth factor (EGF) and / or a retinoic pathway activator described herein (e.g., TTNPB); and (ii) a second culture medium comprising EGF and / or a growth factor of the fibroblast growth factor (FGF) family described herein (e.g., KGF). In some embodiments, the population comprising posterior foregut cells contacted with the first culture medium, or a portion thereof, are then contacted with the second culture medium. In some embodiments, the population contacted with the second culture medium is then contacted with the third culture medium.

[0221] In some embodiments, the third stage of differentiation comprises contacting the population comprising posterior foregut cells with a first culture medium, wherein the first culture medium comprises EGF. In some embodiments, the first culture medium comprises a concentration of EGF, wherein the concentration is at least about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL. In some embodiments, the concentration of EGF is about 10 ng / mL to about 1,000 ng / mL. In some embodiments, the concentration of EGF is about 100 ng / mL.

[0222] In some embodiments, the first culture medium further comprises a retinoic pathway activator described herein (e.g., TTNPB). In some embodiments, the first culture medium further comprises a concentration of TTNPB, wherein the concentration is at least about 0.5 nM, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, or about 3 nM. In some embodiments the concentration of TTNPB is about 0.5 nM to about 10 nM. In some embodiments the concentration of TTNPB is about 3 nM.

[0223] In some embodiments, the first culture medium further comprises non-essential amino acids, sodium pyruvate, and neurocult.

[0224] In some embodiments, the first culture medium further comprises a concentration of vitamin C, wherein the concentration is about at least about 0.1 mM, about 0.15 mM, about 0.2 mM, or about 0.25 mM. In some embodiments, the concentration of vitamin C is about 0.1 mM to about 3 mM.

[0225] In some embodiments, contacting with the first culture medium is performed for a duration of at least about 1 day, about 2 days, or about 3 days. In some embodiments, the contacting is performed for a duration of about 1 day to about 5 days. In some embodiments, the contacting is performed for a duration of about 1 day, about 2 days, or about 3 days.

[0226] In some embodiments, the population of cells is further contacted with a second culture medium, wherein the second culture medium comprises EGF. In some embodiments, the second culture medium comprises a concentration of EGF, wherein the concentration is at least about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL. In some embodiments, the concentration of EGF is about 10 ng / mL to about 1,000 ng / mL. In some embodiments, the concentration of EGF is about 100 ng / mL.

[0227] In some embodiments, the second culture medium further comprises a concentration of KGF, wherein the concentration is at least about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL,or about 50 ng / mL. In some embodiments, the concentration of KGF is about 5 ng / mL to about 200 ng / mL. In some embodiments, the concentration of KGF is about 50 ng / mL.

[0228] In some embodiments, the second culture medium further comprises non-essential amino acids, sodium pyruvate, and neurocult.

[0229] In some embodiments, the second culture medium further comprises a concentration of vitamin C, wherein the concentration is about at least about 0.1 mM, about 0.15 mM, about 0.2 mM, or about 0.25 mM. In some embodiments, the concentration of vitamin C is about 0.1 mM to about 3 mM.

[0230] In some embodiments, the first culture medium and the second culture medium each comprises a base media, wherein the base media is any defined media for human pluripotent stem cell culture known in the art. In some embodiments, the defined media is free of ancillary materials that are animal-derived (e.g., non-human animal or human-derived). In some embodiments, the defined media is StemFit Basisc03 complete cell medium.

[0231] In some embodiments, a plurality of cells of the fourth population are characterized by expression of PDX1 (e.g., as measured by immunohistochemistry, ELISA, or flow cytometry). In some embodiments, the plurality is further characterized by expression of HNF4A, HNF1B, FOXA1, FOXA2, SOX9, NKX6-1, NGN3, and / or PTF1 A (e g., as measured by immunohistochemistry, qPCR, ELISA, or flow cytometry). In some embodiments, a plurality of cells of the fourth population are characterized by expression of PDX1 and NKX6-1 (e.g., as measured by immunohistochemistry, qPCR, ELISA, or flow cytometry). In some embodiments, a plurality of cells of the fourth population are characterized by expression of PDX1, HNF4A, HNF1B, FOXA1, FOXA2, SOX9, NKX6-1, NGN3, and PTF1A (e.g., as measured by immunohistochemistry, qPCR, ELISA, or flow cytometry).

[0232] In some embodiments, at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% of cells of the fourth population are characterized by expression of PDX1 (e.g., as measured by immunohistochemistry or flow cytometry). In some embodiments, at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% of cells of the fourth population are characterized by expression of PDX1 and NKX6-1 (e.g., as measured by immunohistochemistry or flow cytometry). In some embodiments, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% of cells ofthe fourth population are characterized by expression of PDX1 and NKX6-1 (e.g., as measured by immunohistochemistry or flow cytometry).

[0233] In some embodiments, at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% of cells of the fourth population are pancreatic progenitor cells (e.g., PDX1+, NKX6-1+ pancreatic progenitor cells). In some embodiments, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% of cells of the fourth population are pancreatic progenitor cells (e.g., PDX1+, NKX6-1+ pancreatic progenitor cells).(iv) Insulin Producing Endocrine Cells

[0234] In some embodiments, the method further comprises a fourth stage of differentiation, wherein the fourth stage of differentiation comprises differentiating the fourth population comprising pancreatic progenitor cells (e.g., PDX1, NKX6.1 -positive pancreatic progenitor cells) to a fifth population comprising insulin producing endocrine cells (e.g., SCD P cells). In some embodiments, a plurality of cells of the fourth population are PDX1+, NKX6-1+ pancreatic progenitor cells, wherein the fourth stage of differentiation comprises differentiating the PDX1+, NKX6-1+ pancreatic progenitor cells to insulin producing endocrine cells (e.g., SCD p cells). In some embodiments, the differentiation comprises contacting the population comprising PDX1+, NKX6-1+ pancreatic progenitor cells with (i) a first culture medium comprising an inhibitor of TGFP type I receptor (Alk5) described herein (e.g., Alk5i II), a thyroid hormone described herein (e.g., triiodothyronine), a BMP signaling inhibitor described herein (e.g., LDN193189), and / or an inhibitor of gamma secretase (Notch signaling) described herein (e.g., XXi); and (ii) a second culture medium comprising an inhibitor of Alk5 described herein (e.g., Alk5i II), a thyroid hormone described herein (e.g., triiodothyronine), a BMP signaling inhibitor described herein (e.g., LDN193189), and / or an inhibitor of gamma secretase (Notch signaling) described herein (e.g., XXi). In some embodiments, the population comprising PDX1+, NKX6-1+ pancreatic progenitor cells is first contacted with the first culture medium, whereupon the population or a portion thereof, is then contacted with the second culture medium.

[0235] In some embodiments, the first culture medium comprises an inhibitor of TGFP type I receptor (Alk5) described herein (e.g., Alk5i II), a thyroid hormone described herein (e.g., triiodothyronine), a BMP signaling inhibitor described herein (e.g., LDN193189), and / or aninhibitor of gamma secretase (Notch signaling) described herein (e.g., XXi). In some embodiments, the first culture medium comprises the inhibitor of TGFP type I receptor (Alk5), the thyroid hormone, the BMP signaling inhibitor, and the inhibitor of gamma secretase (Notch signaling).

[0236] In some embodiments, the first culture medium comprises (i) a concentration of Alk5i II, wherein the concentration is at least about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, or about 10 pM; (ii) a concentration of triiodothyronine, wherein the concentration is at least about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, or about 1 pM; (iii) a concentration of LDN193189, wherein the concentration is at least about 0.01 pM, about 0.05 pM, about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, or about 0.5 pM; and (iv) a concentration of XXi, wherein the concentration is at least about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, or about 1 pM.

[0237] In some embodiments, the first culture medium comprises a concentration of Alk5i II of about 1 pM to about 20 pM. In some embodiments, the first culture medium comprises a concentration of Alk5i II of about 10 pM.

[0238] In some embodiments, the first culture medium comprises a concentration of triiodothyronine of about 0.1 pM to about 10 pM. In some embodiments, the first culture medium comprises a concentration of triiodothyronine of 1 pM.

[0239] In some embodiments, the first culture medium comprises a concentration of LDN193189 of about 0.01 pM to about 5 pM. In some embodiments, the first culture medium comprises a concentration of LDN193189 of about 0.5 pM.

[0240] In some embodiments, the first culture medium comprises a concentration of XXi of about 0.1 pM to about 10 pM. In some embodiments, the first culture medium comprises a concentration of XXi of about 1 pM.

[0241] In some embodiments, the first culture medium further comprises non-essential amino acids, sodium pyruvate, insulin, selenium, transferrin, zinc, cysteine, vitamin C, and bovine serum albumin (BSA).

[0242] In some embodiments, the first culture medium comprises a concentration of zinc, wherein the concentration is at least about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, or about 10 pM. In some embodiments, the concentration of zinc is about 10 pM.

[0243] In some embodiments, the first culture medium comprises a concentration of vitamin C, wherein the concentration is at least about 10 pM, about 50 pM, about 100 pM, or about 150 pM. In some embodiments, the concentration of vitamin C is about 155 pM.

[0244] In some embodiments, the first culture medium comprises a concentration of BSA, wherein the concentration is at least about 0.1%, about 0.5%, about 1%, about 1.5%, or about 2%. In some embodiments, the concentration of BSA is about 2%.

[0245] In some embodiments, contacting with the first culture medium is performed for a duration of at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In some embodiments, the contacting is performed for a duration of about 3 days to about 10 days. In some embodiments, the contacting is performed for a duration of about 6 days, about 7 days, or about 8 days.

[0246] In some embodiments, the second culture medium comprises an inhibitor of Alk5 described herein (e.g., Alk5i II), a thyroid hormone described herein (e.g., triiodothyronine), a BMP signaling inhibitor described herein (e.g., LDN193189), and / or an inhibitor of gamma secretase (Notch signaling) described herein (e.g., XXi). In some embodiments, the second culture medium comprises the inhibitor of TGFP type I receptor (Alk5), the thyroid hormone, the BMP signaling inhibitor, and the inhibitor of gamma secretase (Notch signaling).

[0247] In some embodiments, the second culture medium comprises (i) a concentration of Alk5i II, wherein the concentration is at least about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, or about 10 pM; (ii) a concentration of triiodothyronine, wherein the concentration is at least about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, or about 1 pM; (iii) a concentration of LDN193189, wherein the concentration is at least about 0.01 pM, about 0.05 pM, about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, or about 0.5 pM; and (iv) a concentration of XXi, wherein the concentration is at least about 0.1 pM, about 0.2 pM, about 0.3 pM, about 0.4 pM, about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, or about 1 pM.

[0248] In some embodiments, the second culture medium comprises a concentration of Alk5i II of about 1 pM to about 20 pM. In some embodiments, the second culture medium comprises a concentration of Alk5i II of about 10 pM.

[0249] In some embodiments, the second culture medium comprises a concentration of triiodothyronine of about 0.1 pM to about 10 pM. In some embodiments, the second culture medium comprises a concentration of triiodothyronine of 1 pM.

[0250] In some embodiments, the second culture medium comprises a concentration of LDN193189 of about 0.01 pM to about 5 pM. In some embodiments, the second culture medium comprises a concentration of LDN193189 of about 0.5 pM.

[0251] In some embodiments, the second culture medium comprises a concentration of XXi of about 0.1 pM to about 10 pM. In some embodiments, the second culture medium comprises a concentration of XXi of about 1 pM.

[0252] In some embodiments, the second culture medium further comprises non-essential amino acids, sodium pyruvate, zinc, cysteine, vitamin C, heparin, glutamax, and BSA.

[0253] In some embodiments, the first second medium comprises a concentration of zinc, wherein the concentration is at least about 1 pM, about 2 pM, about 3 pM, about 4 pM, about5 pM, about 6 pM, about 7 pM, about 8 pM, about 9 pM, or about 10 pM. In some embodiments, the concentration of zinc is about 10 pM.

[0254] In some embodiments, the second culture medium further comprises a concentration of vitamin C, wherein the concentration is at least about 10 pM, about 50 pM, about 100 pM, or about 150 pM. In some embodiments, the concentration of vitamin C is about 155 pM.

[0255] In some embodiments, the second culture medium comprises a concentration of BSA, wherein the concentration is at least about 0.1%, about 0.5%, about 1%, about 1.5%, or about 2%. In some embodiments, the concentration of BSA is about 2%. In some embodiments, the second culture medium comprises a concentration of BSA, wherein the concentration is at least about 1 pg / mL, about 5 pg / mL, or about 10 pg / mL. In some embodiments, the concentration of BSA is about 10 pg / mL.

[0256] In some embodiments, contacting with the second culture medium is performed for a duration of at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about6 days, or about 7 days. In some embodiments, the contacting is performed for a duration of about 3 days to about 10 days. In some embodiments, the contacting is performed for a duration of about 6 days, about 7 days, or about 8 days.

[0257] In some embodiments, a plurality of cells of the fifth population are characterized by expression of a marker (e.g., as measured by immunohistochemistry, ELISA, or flow cytometry). In some embodiments, a plurality of cells of the fifth population are characterized by expression of NKX6-1 and C-peptide (e.g., as measured by immunohistochemistry, ELISA, or flow cytometry). In some embodiments, a plurality of cells of the fifth population are characterized by expression of NKX6-1 and INS (e.g., as measured by immunohistochemistry, ELISA, qPCR, or flow cytometry). In some embodiments, the plurality of cells of the fifth population are further characterized by expression of PDX1,CHGA, SIX2, MAFA, NKX2.2, NEUROD, ISL1, UCN3, ENTPD3, and / or MAFB (e.g., as measured by immunohistochemistry, ELISA, qPCR, or flow cytometry). In some embodiments, at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% of the fifth population are characterized by expression of NKX6-1 and c-peptide (e.g., as measured by immunohistochemistry or flow cytometry). In some embodiments, about 5% to about 50% of the fifth population are characterized by expression of NKX6-1 and c-peptide (e.g., as measured by immunohistochemistry or flow cytometry). In some embodiments, at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of cells of the fifth population are pancreatic P cells. In some embodiments, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of cells of the fifth population are SCD p cells. In some embodiments, about 5% to about 50% of cells of the fifth population are SCD p cells.

[0258] In some embodiments, the fifth population comprises clusters of cells, wherein the clusters of cells comprise the SCD p cells. In some embodiments, the cluster of cells further comprises SCD a cells and / or SCD 5 cells. In some embodiments, the cluster of cells comprises at least about 10, about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, about IxlO3, about 1.5xl03, about 2xl03, about 3xl03, about 4xl03, about 5xl03, or about IxlO4. In some embodiments, the cell cluster comprises not more than about IxlO5, 50xl04, 40xl04, 30xl04, 20xl04, 10xl04, 5xl04, IxlO4, 50xl03, 40xl03, 30xl03, 20xl03, 10xl03, or 5xl03cells.

[0259] In some embodiments, the cell cluster is substantially spherical. In some embodiments, the cell cluster is not spherical. In some embodiments, the cell cluster comprises a longest diameter of about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, about 100 pm, about 110 pm, about 120 pm, about 130 pm, about 140 pm, about 150 pm, about 160 pm, about 170 pm, about 180 pm, about 190 pm, about 200 pm, about 210 pm, about 220 pm, about 230 pm, about 240 pm, about 250 pm, about 260 pm, about 270 pm, about 280 pm, about 290 pm, about 300 pm, about 310 pm, about 320 pm, about 330 pm, about 340 pm, about 350 pm, about 360 pm, about 370 pm, about 380 pm, about 390 pm, about 400 pm, about 410 pm, about 420 pm, about 430 pm, about 440 pm, about 450 pm, about 460 pm, about 470 pm, about 480 pm, about 490 pm, or about 500 pm. In some embodiments, the cell cluster comprises a longest diameter of about 100 pm toabout 300 pm. In some embodiments, the cell cluster comprises a longest diameter of about 100 pm to about 200 pm.

[0260] In some embodiments, the cell compositions comprising insulin-producing cells described herein are cultured under conditions and supplemented with nutrients to improve survival rates. It has been shown in vitro that nutrient deprivation and hypoxia can independently kill mature insulin producing cells such as human islets. Combination of nutrient deprivation and hypoxia that occurs during ischemia act additively to kill insulin producing cells. SCD pancreatic precursor cells and immature insulin producing cells are more resistant to nutrient deprivation and hypoxia alone, but these two factors act synergistically to kill pancreatic precursor cells and immature insulin producing cells in vitro. However, generating pancreatic precursor cells and immature insulin producing cells under physiological oxygen tension of 5% can confer hypoxia resistance without affecting differentiation or function. As such, in some embodiments, insulin-producing cells are cultured prior to transplantation in an atmosphere having any of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% oxygen concentration. In another embodiment, the transplanted insulin producing cells are supplemented during and / or after transplantation with one or more amino acids. In some non-limiting embodiments, the amino acids are alanine and glutamine. Further information regarding pre-culturing insulin-producing cells under low oxygen conditions prior to transplant and nutritional supplementation of these cells can be found in Faleo, et al ., Stem Cell Reports, 2017, 9(3): 807- 19, the disclosure of which is incorporated by reference herein in its entirety.Differentiation Factors

[0261] The present disclosure provides factors for differentiating a population comprising pluripotent stem cells described herein (e.g., a population comprising ESCs or a population comprising iPSCs) to a population comprising insulin producing endocrine cells (e.g., insulin producing SCD p cells). In some embodiments, the factors are used in the methods described herein to differentiate the population comprising pluripotent stem cells to a population comprising definitive endoderm cells. In some embodiments, the factors are used in the methods described herein to differentiate a population comprising definitive endoderm cells to a population comprising posterior foregut cells. In some embodiments, the factors are used in the methods described herein to differentiate a population comprising posterior foregut cells to a population comprising PDX1, NKX6.1 -positive pancreatic progenitor cells to apopulation comprising insulin producing endocrine cells (e.g., insulin producing SCD P cells).(i) Rho Kinase (ROCK) Signaling Pathway

[0262] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use inhibitors of the ROCK signaling pathway. ROCK is a serine / threonine kinase downstream of Ras homolog gene family, member A (RhoA), a small GTPase protein in the Rho family. RhoA is converted between an inactive and active state by binding to GTP. Once activated, RhoA associates with ROCK to mediate downstream signaling.

[0263] In some embodiments, the ROCK inhibitor comprises a compound set forth in Table 1Table 1: Exemplary ROCK inhibitors of the Disclosure(ii) Transforming Growth Factor P (TGFP) Superfamily

[0264] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use growth factors of the TGFP superfamily. As used herein, the term “TGFP superfamily” refers to polypeptides that interact with TGFP receptors. In some embodiments, the TFGP superfamily comprises polypeptides of the activin subfamily (e.g., activin A, activin B, and activin AB), polypeptides of the inhibin subfamily (e.g., inhibin A and inhibin B), bone morphogenetic proteins, polypeptides of the TGFP subfamily (e.g., TGFpi, TGFP2, TGFP3), polypeptides of the growth and differentiation factor (GDF) family (e.g., GDF1, GDF2, GDF3, GDF5, GDF6, GDF8, GDF9, GDF 10, GDF11, and GDF 15), and polypeptides of the left-right dermination factor family (e.g., lefty-1).

[0265] In some embodiments, the growth factor of the TGFP superfamily is activin A. In some embodiments, the growth factor of the TGFP superfamily is human activin A. The amino acid sequence for human activin A is publicly available through the Uniprot database using reference number A0A1B0GXA9.(iii) WNT Signaling Pathway Activators

[0266] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use activators of the WNT signaling pathway. Wnt proteins are secreted morphogens that play roles in stem cell proliferation and self-renewal (see, e.g., Bonnet et al (2021) RSC Chem Biol 2: 1144). In some embodiments, the activator is a small molecule that activates the Wnt signaling pathway. In some embodiments, the activator is a small molecule inhibitor of GSK-3p. Exemplary activator include, but are not limited to, TWS119 (Selleckchem Cat No SI 590),IQ-1 (StemCell Technologies Cat No 72774), SB216763 (Tocris Cat No 1616), SB415286 (Tocris Cat No S2729), CHIR9902 (Selleckchem Cat No S1263), and L807mts (see Licht- Murava, et al (2016) Sci Signal 9:ral 10). In some embodiments, the activator is CHIR99021. In some embodiments, the activator is a salt of CHIR99021.(iv) Fibroblast Growth Factor (FGF) Family

[0267] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use a polypeptide of the FGF family. In some embodiments, the polypeptide of the FGF family comprises keratinocyte growth factor (KGF). In some embodiments, the polypeptide of the FGF family is human KGF. Human KGF is also referred to as fibroblast growth factor 7. Sequence information for KGF is known in the art (see, e.g., UniProt Ref No P21781).

[0268] In some embodiments, the polypeptide of the FGF family comprises FGF2. Sequence information for FGF2 is known in the art (see, e.g., UniProt Ref No P09038).

[0269] In some embodiments, the polypeptide of the FGF family comprises FGF8. Sequence information for FGF8 is known in the art (see, e.g., UniProt Ref No P55075).

[0270] In some embodiments, the polypeptide of the FGF family comprises FGF10. Sequence information for FGF10 is known in the art (see, e.g., UniProt Ref No 015520).

[0271] In some embodiments, the polypeptide of the FGF family comprises FGF21. Sequence information for FGF21 is known in the art (see, e.g., UniProt Ref No Q9NSA1).(v) Retinoic Acid Signaling Pathway Modulation

[0272] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use a modulator of retinoic acid signaling. Retinoic acid (RA) is a metabolite of retinol (vitamin A) that functions as a ligand of nuclear RA receptors (RARs), which include RARa, RARP, and RARy. RARs bind to DNA and recruit nuclear receptor coactivators or nuclear receptor corepressors to activate or repress transcription.

[0273] In some embodiments, the modulator is an activator (agonist) of RA signaling, such as any activator of RA signaling known in the art. In some embodiments, the modulator is an agonist of RARa, RARP, and / or RARy. In some embodiments, the modulator is an agonist of RARa, RARP, and RARy. In some embodiments, the modulator is a selective RARP agonist. In some embodiments, the modulator is a selective RARa agonist. In some embodiments, themodulator is a selective RARy agonist. In some embodiments, the activator comprises retinoic acid. In some embodiments, the activator comprises an analog of retinoic acid. In some embodiments, the activator is a compound set forth in Table 2. In some embodiments, the activator is TTNPB.Table 2: Exemplary RA Signaling Pathway Activators of the Disclosure(vi) Protein Kinase C (PKC) Activators

[0274] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use an activator of one or more PKCs. PKCs are serine / threonine kinases that function in multiple cellular signaling pathways. There are multiple PKC isoforms, including conventional PKCs (a, pi), alternatively spliced PCKs (PII and y), novel PCKs (5, 9, a, r|), and atypical PCKs (S, and 1 / ). Canonical PKC signaling is activated by phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4, 5 -biphosphate (PIP2) to diacylglycerol (DAG), which in turn activates PKC and inositol triphosphate (IP3) to mobilize intracellular calcium. Many PKCs are activated by phorbol esters, e.g., phorbol 12-myristate 13-acetate (PMA), that anchor PCKs in their active conformation to membranes. In some embodiments, the activator is bryostatin- 1. In some embodiments, the activator is diacylglycerol. In some embodiments, the activator is PMA. In some embodiments, the activator is prostatin (13-O-acetyl-12-deoxyphorbol). In some embodiments, the activator is Phorbol 12, 13 -dibutyrate. See Kawano, et al (2021)Pharmaceutics 13: 1748 (herein incorporated by reference) for exemplary PKC activators for use in the methods of the disclosure.(vii) Bone Morphogenetic Protein (BMP) Signaling Pathway Inhibitors

[0275] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use inhibitors of the BMP signaling pathway. The BMP signaling family is a subset of the TGFP superfamily. In humans, the family contains at least 20 family members (see Bragdon, et al (2011) Cell Signal 23:609). For example, the family includes multiple TGFp / GDF / activin type I receptors (ALK2, ALK3, ALK1, and ALK6) and type II receptors (BMPRII, ActRIIa, and ActRIIb). Endogenously, dimeric ligands facilitate assembly of receptor heterodimers, such that the constitutively active type II receptor phosphorylates a type I receptor. Activated type I receptors phosphorylate SMAD effectors (SMADs 1, 5, and 8) to facilitate nuclear translocation in complex with SMAD4.

[0276] In some embodiments, the BMP signaling pathway inhibitor comprises a compound set forth in Table 3. In some embodiments, the BMP signaling pathway inhibitor is a compound described in Dinter, et al (2019) Methods Mol Biol 1891 :221, herein incorporated by reference.

[0277] In some embodiments, the BMP signaling pathway inhibitor comprises a compound described in Hao, et al (2010) ACS Chem Biol 5:245, herein incorporated by reference.

[0278] In some embodiments, the BMP signaling pathway inhibitor comprises LDN-193189. LDN-193189 is a selective inhibitor of ALK1, ALK2, ALK3, and ALK6 (IC50 = 0.8, 0.8, 5.3, and 16.7 nM respectively; see Sanvitale, et al (2013) PLOS One 8:e62721). In some embodiments, the BMP signaling pathway inhibitor comprises any salt of LDN-193189. In some embodiments, the BMP signaling pathway inhibitor comprises the dihydrochloride salt of LDN-193189.Table 3: Exemplary BMP Signaling Pathway Inhibitors of the Disclosure(viii) Sonic Hedgehog (SHH) Signaling Pathway Inhibitors

[0279] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) uses an inhibitor of the SHH pathway. The hedgehog signaling pathway is initiated by SHH, Indian hedgehog, and Desert hdedgehog, with SHH being the most widely expressed and potent of the three ligands. The receptor for SHH is Patchedl (PITCHI). Binding of SHH to PITCHI relieves repressed of Smoothed (SMO) by PITCHI, which activates SMO signaling activity. SMO signaling activates GLI transcription factors, resulting in changes in gene expression. See Carpenter (2019) Drug Saf 42:263.

[0280] In some embodiments, the SHH signaling pathway inhibitor is a compound set forth in Table 4. In some embodiments, the SHH signaling pathway inhibitor is SANT1. In some embodiments, the SHH signaling pathway inhibitor is a salt of SANT2.Table 4: Exemplary SHH signaling pathway inhibitors of the disclosure(ix) Epidermal Growth Factor (EGF) Family

[0281] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use polypeptides of the EGF family. In some embodiments, the polypeptide of the EGF family is human wild-type EGF polypeptide. Sequence information for human wild-type EGF polypeptide is accessible via UniProt database reference Q6QBS2. In some embodiments, the polypeptide is a variant of human wild-type EGF polypeptide. In some embodiments, the variant comprises at least 80%, 85%, 90%, or 95% identity to human wild-type EGF polypeptide. In someembodiments, the polypeptide is a truncation of human wild-type EGF polypeptide, wherein the truncation retains binding to the EGF receptor.(x) TGF-P Signaling Pathway Inhibitors

[0282] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use inhibitors of the TGFP signaling pathway. In some embodiments, the inhibitor of the TGFP signaling pathway comprises an inhibitor of TGFP receptor (also referred to as “ALK5”).

[0283] In some embodiments, the inhibitor is a compound identified in Table 5.

[0284] In some embodiments, the TGFP signaling pathway comprises a ALK5 inhibitor described in US Pat Pub 20100267731, US20090186076, US20070142376, and US20230092449, each of which are herein incorporated by reference.Table 5: Exemplary TGFp Signaling Pathway Inhibitors of the Disclosure(xi) Thyroid Hormone Signaling Pathway Activators

[0285] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use activators of the thyroid hormone signaling pathway. In some embodiments, the activator comprises a compound set forth in Table 6. In some embodiments, the activator comprises triiodothyronine (T3).Table 6: Exemplary Thyroid Hormone Signaling Pathway Activators of the Disclosure(xii) y-Secretase Inhibitors

[0286] As described herein, the methods of the disclosure for generating populations comprising SCD insulin producing pancreatic cells (e.g., SCD P cells) use inhibitors of y- secretase.

[0287] In some embodiments, the inhibitor of y-secretase comprises a compound set forth in Table 7. In some embodiments, the inhibitor comprises XXI. In some embodiments, the inhibitor comprises DAPT.Table 7: Exemplary Inhibitors of y-Secretase of the DisclosureFunctional Characteristics of Insulin Producing Cells

[0288] In some embodiments, provided herein are cell compositions comprising insulin producing cells. In some embodiments, the insulin producing cells comprise SCD P cells. In some embodiments, the cell composition further comprises SCD a cells, SCD 5 cells, SCD y, or SCD 8 cells. In some embodiments, endocrine progenitor cells account for not more than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% of total cells in the cell composition.

[0289] In some embodiments, the insulin producing cells comprise one or more functional properties comparable to primary human beta cells. Suitable methods for measuring the functionality of insulin producing cells are known in the art, see e.g., Nair, et al (2019) Nat Cell Biol 21 :263-274; Velazco-Cruz et al, (2019) Stem Cell Reports 12:351-65; Hogrebe et al., (2020), Nat Biotech, 38:460-70; Rezania et al., (2014) Nature Biotech 32: 1121-33; Russ et al., (2015), EMBO Journal 34: 1759-72; and Pagliuca et al., (2014), Cell, 159:428-39.

[0290] In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by the capacity to regulate insulin secretion in a manner similar to in situ pancreatic beta cells. For example, in some embodiments, the insulin producing cells secrete insulin in response to an increase in glucose in the extracellular environment.

[0291] In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by a response to a glucose challenge. In some embodiments, the response is substantially comparable to the response of endogenous islets to a glucose challenge.

[0292] In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by a total insulin content that is substantially comparable to primary human islet beta cells.

[0293] In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by an expression of one or more cell-specific markers (e.g., C-peptide, NKX6.1, and PDX1) that is substantially comparable to that of primary human islet beta cells, e.g., as measured by flow cytometry.

[0294] In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by an expression of mRNAs encoding prohormone convertase enzymes that are substantially comparable to that of primary human islet beta cells, e.g., as measured by quantifying thelevels of PCI and PC2 mRNAs. In some embodiments, PCI and PC2 mRNA levels are measured by qPCR, RNA-seq, or any other method of measuring gene expression levels that is known in the art.

[0295] In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by a metabolic state that is substantially comparable to primary human islet beta cells, e.g., as measured by a Seahorse analyzer.

[0296] In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by increased calcium signaling (e.g., as measured by microscopy or flow cytometry) in response to a secretagogue. In some embodiments, the insulin producing cells (e.g., SCD p cells) is characterized by increased calcium signaling (e.g., as measured by microscopy or flow cytometry) in response to glucose. In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by insulin secretion that is enhanced in response to a secretagogue.

[0297] In some embodiments, the insulin producing cells (e.g., SCD P cells) packages insulin into secretory granules (e.g., as measured by electron microscopy). In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by a quantity of insulin secretory granules that is substantially comparable to that present in primary human islet beta cells (e.g., as measured by electron microscopy). In some embodiments, the insulin producing cells (e.g., SCD p cells) is characterized by the presence of crystalline insulin granules (e.g., as measured by electron microscopy).

[0298] In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by a level of mitochondrial energization that is substantially comparable to that of primary human islet beta cells.

[0299] In some embodiments, the insulin producing cells (e.g., SCD p cells) secrete a level of c-peptide following administration in vivo that is substantially comparable to that produced following administration of primary human islet beta cells.

[0300] In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by a morphology substantially similar to the morphology of endogenous pancreatic islet P cells.

[0301] In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by an in vitro GSIS response that is substantially similar to the GSIS response of endogenous pancreatic islet P cells. In some embodiments, the insulin producing cells (e.g., SCD p cells) exhibit a response to multiple glucose challenges (e.g, at least one, at least two, or at least three or more sequential glucose challenges), e.g., in vitro. In some embodiments, theresponse resembles the response of endogenous pancreatic islet P cells to multiple glucose challenges.

[0302] In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by an in vivo GSIS response that is substantially similar to the GSIS response of endogenous pancreatic islet P cells. In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by an in vitro and an in vivo GSIS response that is substantially similar to the GSIS response of endogenous pancreatic islet P cells. In some embodiments, the insulin producing cells (e.g., SCD P cells) exhibit a GSIS response within about 2 weeks of in vivo transplantation. In some embodiments, the insulin producing cells (e.g., SCD p cells) exhibit a GSIS response within about 3 weeks of in vivo transplantation. In some embodiments, the insulin producing cells (e.g., SCD p cells) exhibit a GSIS response within about 3 weeks of in vivo transplantation. In some embodiments, the insulin producing cells (e.g., SCD p cells) exhibit a GSIS response within about 4 weeks of in vivo transplantation. In some embodiments, the insulin producing cells (e.g., SCD P cells) exhibit a GSIS response within about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks of in vivo transplantation. In some embodiments, the insulin producing cells (e.g., SCD p cells) exhibit a GSIS response within about 12 weeks of in vivo transplantation.

[0303] In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by a stimulation index (i.e., ratio of insulin produced at high to low glucose concentration) of greater than 1. In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by a stimulation index of greater than 1.1. In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by a stimulation index of greater than 2. See Functional assessment of purified human pancreatic islets: glucose stimulated insulin release by ELISA - A Standard Operating Procedure of the NIH Clinical Islet Transplantation Consortium CellR4 2014; 2(2):e900 for methods to determine the stimulation index.

[0304] In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized as monohormonal. In some embodiments, the insulin producing cells (e.g., SCD P cells) express insulin, but does not substantially express another hormone (e.g., glucagon, somatostatin, PP).

[0305] In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by apoptosis in response to cytokines.

[0306] In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by an ability to reverse diabetes in an animal model of diabetes. Animal models of diabetes are known in the art, see, e.g., Kottaisamy et al (2021) Laboratory animal research, 37(1), 23; and Singh et al (2024) Front. Endocrinol. 15, 1359685. In some embodiments, the animal model of diabetes uses NOD-SCID Gamma mice, NOD mice, B6 mice, or Ball / c mice. In some embodiments, the animal model of diabetes uses immunodeficient mice. In some embodiments, diabetes is induced in the mice by injecting with streptozotocin. In some embodiments, the insulin producing cells (e.g., SCD p cells) are characterized by lowering blood glucose levels in an animal model of diabetes to a greater extent than animals that are not treated with insulin producing cells. In some embodiments, the insulin producing cells (e.g., SCD P cells) are characterized by an ability to lower blood glucose levels in the animals to blood glucose levels of less than about 250 mg / dL, about 200 mg / dL, or about 150 mg / dL. An exemplary animal model of diabetes is described in Example 2.Genetic Modifications

[0307] In some embodiments, a plurality of cells (e.g., a plurality of SCD cells or a plurality of insulin producing cells) of a cell composition described herein comprise a genetic modification to reduce immunogenicity following administration to a subject.

[0308] In some embodiments, the cell composition is characterized by reduced susceptibility to an allogeneic immune response following administration to a subject as compared to a cell composition lacking the genetic modification. For example, in some embodiments, the cell composition comprises SCD cells differentiated from pluripotent stem cells not obtained from the subject, wherein the SCD cells comprise the genetic modification, and wherein the cell composition is characterized by a reduced allogenic immune response as compared to the cell composition lacking the genetic modification. In some embodiments, the genetic modification renders the cell composition less susceptible to a host-versus-graft disease following administration to the subject.

[0309] In some embodiments, the cell composition is characterized by reduced susceptibility to an autologous immune response following administration to a subject as compared to a cell composition lacking the genetic modification. For example, in some embodiments, the cell composition comprises insulin producing cells comprising the genetic modification, wherein the cell composition is administered to a subject having type 1 diabetes, and wherein the cell composition is characterized by reduced susceptibility to the subject’s autoimmune responseas compared to a cell composition lacking the genetic modification. In some embodiments, the subject’s autoimmune response is characterized by a T cell mediated immune response against pancreatic beta cells.

[0310] In some embodiments, the genetic modification comprises a genomic disruption in at least one gene. In some embodiments, the genomic disruption reduces or eliminates expression of a gene product encoded by the at least one gene. In some embodiments, the genomic disruption results in expression of non-functional or inactive gene product.

[0311] In some embodiments, the genetic modification comprises a genomic disruption of a gene encoding a major histocompatibility class (MHC) molecule. In some embodiments, a cell composition comprising a genomic disruption in a gene encoding an MHC molecule is characterized by reduced susceptibility to a T cell-mediated immune response following in vivo administration. In some embodiments, the gene encodes HL A- A, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, B2M, NLRC5, CUT A, RFX5, RFXAP, or RFXANK.

[0312] In some embodiments, the gene encodes an MHC class I molecule. In some embodiments, the MHC class I molecule comprises is a human leukocyte antigen (HLA) class I molecule. In some embodiments, the gene encodes beta-2 microglobulin. In some embodiments, the gene encodes HL A- A. In some embodiments, the gene encodes HLA-B. In some embodiments, the gene encodes HLA-C.

[0313] In some embodiments, the gene encodes a regulator of transcription of an MHC Class I molecule. In some embodiments, the gene encodes regulatory factor X (RFX). In some embodiments, the gene encodes RFX-5. In some embodiments, the gene encodes RFXANK. In some embodiments, the gene encodes RFXAP. In some embodiments, the gene encodes X2 box-binding protein (X2BP). In some embodiments, the gene encodes nuclear factor Y (NFY). In some embodiments, the gene encodes NFY-A. In some embodiments, the gene encodes NFY-B. In some embodiments, the gene encodes NFY-C. In some embodiments, the gene encodes IFN regulatory factor 1 (IRF1). In some embodiments, the gene encodes NOD-, LRR-and CARD-containing 5 (NLRC5).

[0314] n some embodiments, the gene encodes an MHC class II molecule. In some embodiments, the MHC class II molecule is an HLA class II molecule. In some embodiments, the gene encodes HLA-DP. In some embodiments, the gene encodes HLA- DM. In some embodiments, the gene encodes HLA-DOA. In some embodiments, the gene encodes HLA-DOB. In some embodiments, the gene encodes HLA-DQ. In some embodiments, the gene encodes HLA-DR.

[0315] In some embodiments, the gene encodes a regulator of transcription of an MHC Class II molecule. In some embodiments, the gene encodes CIITA.

[0316] In some embodiments, the gene encodes an epigenetic modulator. In some embodiments, the gene encodes tet methylcytosine dioxygenase 2 (Tet2).

[0317] In some embodiments, the genomic disruption is induced using a gene editing technology (e.g., CRISPR / Cas, TALENs, zinc finger nuclease, meganuclease, or homing endonuclease systems).

[0318] In some embodiments, the genetic modification comprises an insertion of a transgene encoding a tolerogenic factor at a target genomic locus. In some embodiments, the tolerogenic factor comprises a checkpoint protein. In some embodiments, the checkpoint protein is CD47. In some embodiments, the checkpoint protein comprises CTLA4. In some embodiments, the checkpoint protein comprises PDL1. In some embodiments, the checkpoint protein comprises PD1. In some embodiments, the tolerogenic factor is PDL1, PD1, HLA-E, HLA-G, CTLA-4, and / or CD47.

[0319] In some embodiments, the insertion is induced using a gene editing system comprising a site-directed endonuclease (e.g., CRISPR / Cas, TALENs, zinc finger nuclease, meganuclease, or homing endonuclease systems) and a donor polynucleotide comprising the transgene. In some embodiments, the target genomic locus is a safe harbor locus. In some embodiments, the target genomic locus is within or near a gene encoding an MHC molecule described herein. In some embodiments, the target genomic locus is within or near a gene encoding an activator of transcription of an MHC molecule described herein.

[0320] In some embodiments, the genomic modification (e.g., genetic disruption or gene insertion) comprises a gene encoding a survival factor described herein. In some embodiments, the survival factor is a human survival factor. In some embodiments, the survival factor is a member of a pathway involved in cell survival. In some embodiments, the pathway involved in cell survival is activated by hypoxia, reactive oxygen species, nutrient deprivation, and / or oxidative stress. In some embodiments, the genetic modification of at least one survival factor enables a SCD or insulin producing cell comprising the genetic modification to survive for a longer time period, e.g,, at least 1.05, at least 1.1, at least 1.25, at least 1.5, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, or at least 50 times longer time period, than an unmodified cell following engraftment. In some embodiments, the survival factor is inserted into a cell. In some embodiments, a survival factor is deleted from a cell, e.g, a universal donor cell. In some embodiments, the gene encodes thioredoxin interacting protein (TXNIP). In some embodiments, the gene encodesforkhead box, class O (FOXOI). In some embodiments, the gene encodes zinc finger protein 143 (ZNF143). In some embodiments, the gene encodes c-Jun N-terminal kinase (JNK). In some embodiments, the gene encodes mesencephalic astrocyte derived neurotrophic factor (MANF). In some embodiments, the genomic modification is a disruption of the gene encoding the survival factor. In some embodiments, the genomic modification is an insertion of the gene encoding the survival factor.

[0321] In some embodiments, the genetic modification is one described in WO2023133568, WO2021044377, WO2021044379, WO2022269393, and / or WO2022144856, each of which is herein incorporated by reference.

[0322] In some embodiments, the cell composition comprises SCD cells, wherein the genetic modification is introduced prior to differentiation of the SCD cells. In some embodiments, the genetic modification is introduced subsequent to differentiation of the SCD cells. In some embodiments, the cell composition comprises SCD p cells derived from a population comprising pluripotent stem cells, wherein the genetic modification is introduced prior to differentiating the population comprising pluripotent stem cells. In some embodiments, the genetic modification is introduced at any stage of differentiating the population comprising pluripotent stem cells to the cell composition comprising SCD P cells.Support Factors

[0323] In some embodiments, the cell compositions of the disclosure comprise cells described herein (e.g., SCD cells and / or insulin producing cells) and a support factor. In some embodiments, the cells and the support factor are combined in a suspension. In some embodiments, the support factor is operably linked to the surface of a plurality of the cells. In some embodiments, the cells and the support factor are encapsulated in a delivery device described herein. In some embodiments, the support factor is a gene product described herein, wherein a plurality of cells of the composition are engineered to express the gene product. In some embodiments, the cells of the composition are transfected with the support factor, such that the support factor is internalized by a plurality of the cells.

[0324] In some embodiments, the cell composition comprising the support factor exhibits improved survival compared to a cell composition lacking the support factor. In some embodiments, the survival is improved in vitro. In some embodiments, the survival is improved following administration in vivo. For example, in some embodiments, the cell composition comprising the support factor exhibits improved survival for duration of at leastabout 2 weeks, about 4 weeks, about 8 weeks, about 12 weeks, about 24 weeks, or longer compared to a cell composition lacking the support factors. In some embodiments, the cell composition comprising the support factor exhibits improved angiogenesis compared to a cell composition lacking the support factor. In some embodiments, the angiogenesis is improved following administration in vivo, e.g., within about 2 weeks, about 4 weeks, about 8 weeks, or about 12 weeks following administration in vivo.

[0325] In some embodiments, the cell composition comprises an insulin producing cell described herein (e.g., a SCD p cell) and a support factor, wherein the cell composition exhibits an improved GSIS response compared to a cell composition lacking the support factor. In some embodiments, the improved GSIS response is an in vitro or in vivo GSIS response. In some embodiments, the cell composition exhibits increased insulin expression compared to a cell composition lacking the support factor. In some embodiments, the cell composition exhibits increased c-peptide expression compared to a cell composition lacking the support factor

[0326] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a support factor, wherein the support factor comprises a cell capable of secreting a proangiogenic factor, a PTG factor, or both. In some embodiments, the cell composition comprises a first cell capable of secreting a PTG hormone and a second cell capable of secreting a proangiogenic factor. In some embodiments, the cell composition comprises a cell capable of secreting both a proangiogenic factor and PTG hormone. In some embodiments, the cell is one derived from a PTG tissue. In some embodiments, the cell is a CD34+ cell. In some embodiments, the cell is a hematopoietic progenitor cell. Methods for co-transplanting an insulin producing cell and a cell derived from a PTG tissue or a CD34+ cell are further described in US Pat No. 11,951,136 (herein incorporated by reference).

[0327] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a support factor, wherein the support factor comprises a PTG factor (e.g., a PTG hormone), a proangiogenic factor, a cytokine, an anti-inflammatory factor, or a combination thereof. In some embodiments, the PTG factor is selected from gamma- aminobutyric acid (GABA), leptin, serotonin, parathyroid hormone (PTH), hormone growth factor (HGF), osteopontin, parathyroid hormone-related protein (PTHrP), vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, platelet derived growth factor (PDGF) AA, PDGF-BB, and a combination thereof. In some embodiments, the PTG factor is selected from GABA, leptin, serotonin, PTH, HGF, osteopontin, PTHrP, and a combination thereof. In some embodiments, the proangiogenic factor and / or the PTG factor isselected from VEGF, angiopoietin-1, angiopoietin-2, PDGF-AA, PDGF-BB, and a combination thereof. In some embodiments, the cytokine comprises IL-6.

[0328] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a support factor selected from GABA, leptin, serotonin, PTH, HGF, osteopontin, PTHrP, VEGF, angiopoietin-1, angiopoietin-2, PDGF-AA, PDGF-BB, and a combination thereof.

[0329] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a support factor selected from GABA, leptin, serotonin, PTH, HGF, osteopontin, PTHrP, VEGF, angiopoietin-1, angiopoietin-2, PDGF-AA, PDGF-BB, IL-6, and a combination thereof.

[0330] In some embodiments, the cell composition comprises an insulin producing cell describe herein and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 support factors selected from GABA, leptin, serotonin, PTH, HGF, osteopontin, PTHrP, VEGF, angiopoietin-1, angiopoietin-2, PDGF-AA, PDGF-BB, and IL-6.

[0331] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a concentration of VEGF, wherein the concentration is at least about 0.1 pg / mL, 0.3 pg / mL, 0.5 pg / mL, 1 pg / mL, 1.5 pg / mL, 2 pg / mL, or 3 pg / mL. In some embodiments, the concentration of VEGF is about 0.1 pg / mL to about 10 pg / mL. In some embodiments, the concentration of VEGF is about 3 pg / mL.

[0332] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a concentration of angiopoietin-1, wherein the concentration is at least about 10 pg / mL, 30 pg / mL, 50 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, or 300 pg / mL. In some embodiments, the concentration of angiopoietin-1 is about 10 pg / mL to about 1,000 pg / mL. In some embodiments, the concentration of angiopoietin-1 is about 300 pg / mL.

[0333] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a concentration of angiopoietin-2, wherein the concentration is at least about 10 pg / mL, 30 pg / mL, 50 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, or 300 pg / mL. In some embodiments, the concentration of angiopoietin-2 is about 10 pg / mL to about 1,000 pg / mL. In some embodiments, the concentration of angiopoietin-2 is about 300 pg / mL.

[0334] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a concentration of PDGF-AA, wherein the concentration is at least about 0.1 pg / mL, 0.3 pg / mL, 0.5 pg / mL, 1 pg / mL, 1.5 pg / mL, 2 pg / mL, or 3 pg / mL. In some embodiments, the concentration of PDGF-AA is about 0.1 pg / mL to about 10 pg / mL. In some embodiments, the concentration of PDGF-AA is about 3 pg / mL.

[0335] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a concentration of PDGF-BB, wherein the concentration is at least about 10 pg / mL, 30 pg / mL, 50 pg / mL, 100 pg / mL, 150 pg / mL, 200 pg / mL, or 300 pg / mL. In some embodiments, the concentration of PDGF-BB is about 10 pg / mL to about 1,000 pg / mL. In some embodiments, the concentration of PDGF-BB is about 300 pg / mL.

[0336] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a concentration of GABA, wherein the concentration is at least about 0.1 pM, 0.5pM, IpM, 1.5pM, 2 pM, 3pM, 4pM, 5pM, 6pM, 7pM, 8pM, 9pM, or 10 pM. In some embodiments, the concentration of GABA is about 0.1 pM to about 50 pm. In some embodiments, the concentration of GABA is about 10 pM.

[0337] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a concentration of leptin, wherein the concentration is at least about 1 pM, 5 pM, 10 pM, 15 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, or 100 pM. In some embodiments, the concentration of leptin is about 1 pM to about 500 pm. In some embodiments, the concentration of leptin is about 500 pM.

[0338] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a concentration of serotonin, wherein the concentration is at least about 1 pM, 5 pM, 10 pM, 15 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, or 100 pM. In some embodiments, the concentration of serotonin is about 1 pM to about 500 pm. In some embodiments, the concentration of serotonin is about 500 pM.

[0339] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a concentration of PTH, wherein the concentration is at least about 0.1 pg / mL, 0.3 pg / mL, 0.5 pg / mL, 1 pg / mL, 1.5 pg / mL, 2 pg / mL, 3 pg / mL, or 4 pg / mL. In some embodiments, the concentration of PTH is about 0.1 pg / mL to about 10 pg / mL. In some embodiments, the concentration of PTH is about 4 pg / mL.

[0340] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a concentration of HGF, wherein the concentration is at least about 10 ng / mL, 30 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, or 300 ng / mL. In some embodiments, the concentration of HGF is about 10 ng / mL to about 1,000 ng / mL. In some embodiments, the concentration of HGF is about 300 ng / mL.

[0341] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a concentration of osteopontin, wherein the concentration is at least about 10 ng / mL, 30 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 300 ng / mL, or 400ng / mL. In some embodiments, the concentration of osteopontin is about 10 ng / mL to about 1,000 ng / mL. In some embodiments, the concentration of osteopontin is about 400 ng / mL.

[0342] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a concentration of PTHrP, wherein the concentration is at least about 1 ng / mL, 5 ng / mL 10 ng / mL, 30 ng / mL, 50 ng / mL, or 100 ng / mL. In some embodiments, the concentration of PTHrP is about 1 ng / mL to about 500 ng / mL. In some embodiments, the concentration of PTHrP is about 100 ng / mL.

[0343] In some embodiments, the cell composition comprises an insulin producing cell describe herein and a support factor, wherein the support factor comprises a nanoprobe described herein. In some embodiments, the cell composition comprising the nanoprobe exhibits an improved GSIS response compared to a cell composition lacking the nanoprobe. In some embodiments, the GSIS response is an in vitro GSIS response. In some embodiments, the GSIS response is an in vivo GSIS response. In some embodiments, the cell composition comprising the nanoprobe exhibits increased insulin production as compared to a cell composition lacking the nanoprobe. In some embodiments, the nanoprobe comprises a metallic nanoparticle described herein. In some embodiments, and without being bound by theory, an insulin producing cell comprising an internalized nanoprobe are capable of improved storage, transport, and secretion of insulin-containing granules compared to an insulin producing cell lacking the nanoprobe.Nanoprobes

[0344] Various nanoprobes for use in the cell compositions and methods described herein are also provided.

[0345] In some embodiments, the disclosure provides cell compositions comprising a nanoprobe configured to provide an optical signal that is detectable following transplantation of the cell composition in vivo. In some embodiments, detection of the optical signal provides a readout of the health status of the cell composition, which readout is used to inform clinical intervention(s) for sustaining or eliminating the cell composition (e.g., upon detecting uncontrolled proliferation and / or teratoma formation). In some embodiments, the nanoprobe is configured for SERS detection, wherein a SERS signal emitted from the nanoprobe is used to construct an image of the transplant that is monitored over a duration of time following transplantation. In some embodiments, images of the transplant obtained at different time points following transplantation are analyzed using an artificial intelligence algorithm,wherein a change in the images as detected by the artificial intelligence algorithm provides information regarding health status of the transplant. In some embodiments, the nanoprobe is configured to emit a SERS signal in the presence of a biomarker of health status of the SCD cell (e.g., a biomarker of inflammatory insult, nutrient deprivation, and / or hypoxia). In some embodiments, the biomarker is a target nucleic acid described herein. In some embodiments, the nanoprobe comprises a metallic nanoparticle and an optical reporter. In some embodiments, the nanoprobe further comprises a nucleic acid probe described herein.

[0346] In some embodiments, the detection principle of the nanoprobes of the disclosure is based on a plasmonic enhancement mechanism near the metallic nanoparticle.Electromagnetic enhancements are divided into two main classes: a) enhancements that occur only in the presence of a radiation field, and b) enhancements that occur even without a radiation field. When a nanostructured metallic surface is irradiated by an electromagnetic field (e.g., a laser beam), electrons within the conduction band begin to oscillate at a frequency equal to that of the incident light. These oscillating electrons, called “surface plasmons,” produce a secondary electric field which adds to the incident field. If these oscillating electrons are spatially confined, as is the case for isolated metallic nanospheres or roughened metallic surfaces (nanostructures), there is a characteristic frequency (the plasmon frequency) at which there is a resonant response of the collective oscillations to the incident field. This condition yields intense localized field enhancements that can interact with molecules on or near the metal surface. Secondary fields are typically most concentrated at points of high curvature on the roughened metal surface.

[0347] Accordingly, an effective type of plasmonic-active substrate consists of nanostructured metal particles, protrusions, or rough surfaces of metallic materials. When exposed to incident light at the plasmon frequency, the surface of metallic nanoparticles becomes polarized, resulting in large field induced polarizations at the surface. As a result, the effective electromagnetic field experienced by molecules (e.g., a Raman optical reporter) proximal to the surface of the metallic nanoparticle is much larger than the actual applied field. These local fields increase emission intensity of Raman scattering. As is understood in the art, the Raman-active analyte molecule is not required to be in contact with the metallic surface for polarization to occur, but can be located anywhere within the range of the enhanced local field.

[0348] Raman scattering generally refers to the inelastic scattering of a photon incident on a molecule. Photons that are inelastically scattered have an optical frequency (v,), which is different than the frequency of the incident light (vO). The difference in energy (AE) betweenthe incident light and the inelastically scattered light can be represented as (AE) = h|vO - v,|, wherein h is Planck's constant, and corresponds to energies that are absorbed by the molecule. The incident radiation can be of any frequency vO, but typically is monochromatic radiation in the visible or near-infrared spectral region. The absolute difference |v0 - v,| is an infrared, e.g., vibrational, frequency. The frequency v, of the "Raman scattered" radiation can be greater than or less than vO, but the amount of light with frequency v, < vO (Stokes radiation) is greater than that with frequency v, > vO (anti-Stokes radiation).

[0349] The Raman spectra typically cover vibrational energies from 300-3500 cm’1, making it possible to measure multiple (e.g., a dozen or more) optical reporters simultaneously using a single light source. Indeed, Raman spectroscopy generates a fingerprint-like vibrational spectrum having features that are much narrower than typical fluorescence. However, signals in Raman spectra are weak, precluding their use for applications such as single molecule detection. However, as is understood by the skilled artisan, the technique for enhancing the Raman scattering effect by bringing optical reporters proximal to the surface of a metallic nanostructures that generates a surface plasmon is referred to as “surface-enhanced Raman scattering” or “SERS.” The SERS effect can enhance the efficiency of light emitted from molecules adsorbed at or near the surface of the metallic nanostructures. The intensity of the normally weak Raman scattering process is increased by factors as large as 1013or 1015for compounds adsorbed onto a SERS substrate, allowing for single-molecule detection.Nanoprobes for SERS Detection and / or Photothermal Irradiation

[0350] Nanoprobes configured to emit an optical signal for SERS detection are provided for use in the cell compositions and methods described herein. In some embodiments, the nanoprobe is configured to induce thermal damage of a cell containing the nanoprobe upon exposure to photothermal irradiation.

[0351] In some embodiments, the nanoprobe comprises a metallic nanoparticle and an optical reporter. In some embodiments, the optical reporter is operably linked to the surface of the metallic nanoparticle, such that the optical reporter is within the local SPR field of the metallic nanoparticle upon irradiation. In some embodiments, the optical reporter is operably linked to the surface of the metallic nanoparticle by adsorption. In some embodiments, the optical reporter is operably linked to the surface of the metallic nanoparticle by a linker.

[0352] In some embodiments, the nanoprobe comprises the metallic nanoparticle and a plurality of optical reporter molecules, each operably linked to the surface of the metallicnanoparticle. In some embodiments, the optical reporter molecules of the plurality comprise the same electromagnetic properties (e.g., the same excitation and / or emission wavelengths). In some embodiments, the optical reporter molecules of the plurality comprise at least two different electromagnetic properties (e.g., different excitation and / or emission wavelengths). For example, in some embodiments, the nanoprobe comprises a metallic nanoparticle, a first optical reporter, and a second optical reporter, wherein the first optical reporter and the second optical reporter comprise different electromagnetic properties (e.g., different excitation and / or emission wavelengths).

[0353] In some embodiments, the nanoprobe comprises a metallic nanoparticle and an optical reporter, wherein the metallic nanoparticle is contained in a coating. In some embodiments, the coating comprises silica. In some embodiments, the coating comprises a polymeric matrix. In some embodiments, the optical reporter is operably linked to the surface of the coating. In some embodiments, the optical reporter is operably linked to the surface of the coating by adsorption. In some embodiments, the optical reporter is operably linked to the surface of the coating by a linker. In some embodiments, the optical reporter is sufficiently proximal to a surface of the metallic nanoparticle to be within an SPR field of the metallic nanoparticle upon irradiation.

[0354] In some embodiments, the nanoprobe comprises a component operably linked to the surface of the metallic nanoparticle. In some embodiments, the component comprises a synthetic polymer, a cell penetrating peptide, a membrane anchor, a bioreceptor, or a combination thereof. In some embodiments, the component is operably linked to the surface of the metallic nanoparticle by adsorption. In some embodiments, the component is operably linked to the surface of the metallic nanoparticle by a linker.

[0355] In some embodiments, the nanoprobe comprises the synthetic polymer operably linked to the surface of the metallic nanoparticle. In some embodiments, the nanoprobe comprises the cell penetrating peptide operably linked to the surface of the metallic nanoparticle. In some embodiments, the nanoprobe comprises the membrane anchor operably linked to the surface of the metallic nanoparticle. In some embodiments, the nanoprobe comprises the bioreceptor operably linked to the surface of the metallic nanoparticle. In some embodiments, the nanoprobe comprises the synthetic polymer and the cell penetrating peptide operably linked to the surface of the metallic nanoparticle. In some embodiments, the nanoprobe comprises the synthetic polymer and the membrane anchor operably linked to the surface of the metallic nanoparticle. In some embodiments, the nanoprobe comprises thesynthetic polymer and the membrane anchor operably linked to the surface of the metallic nanoparticle.Nanoprobes for Target Nucleic Acid Detection

[0356] Nanoprobes configured for detection of a target nucleic acid described herein (e.g., a DNA, mRNA, or noncoding RNA described herein) are provided for use in the cell compositions and methods of the disclosure. In some embodiments, the target nucleic acid is a biomarker of a cellular phenotype and / or differentiation state. In some embodiments, the biomarker is characterized by an altered (e.g., increased or decreased) expression level in a target cell upon a change in a cellular phenotype and / or differentiation state of the target cell. In some embodiments, the biomarker is characterized by an increased expression level in a target cell upon a change in a cellular phenotype and / or differentiation state of the target cell. In some embodiments, the biomarker is characterized by a decreased expression level in a target cell upon a change in a cellular phenotype and / or differentiation state of the target cell.

[0357] In some embodiments, the disclosure provides nanoprobes designed to generate a SERS signal in the presence of a target nucleic acid (e.g., a DNA or an RNA). In some embodiments, the nanoprobe comprises (a) a metallic nanoparticle; (b) a nucleic acid probe comprising (i) an oligonucleotide configured comprising a nucleotide sequence capable of forming a stem-loop structure (also referred to herein as the “stem loop strand”); and (ii) an optical reporter, wherein the stem loop strand comprises a 5'end or a 3'end operably linked to a surface of the metallic nanoparticle, and the opposite end operably linked to the optical reporter. In some embodiments the nucleic acid probe further comprises a second oligonucleotide comprising a first region of complementarity capable of forming a duplex with the stem loop strand and a second region of complementarity capable of binding to a target nucleic acid sequence (also referred to herein as the “placeholder strand”).

[0358] In some embodiments, the nanoprobe is present in a “closed” configuration, wherein the stem loop strand forms the stem loop structure. The “closed” configuration is also referred to as the “on” configuration herein. In some embodiments, the formation of the stem loop structure brings the optical reporter into proximity of the surface of the metallic nanoparticle. In some embodiments, the nanoprobe is present in an “open” configuration, wherein the stem loop structure is unfolded, thereby maintaining the optical reporter at a distance of at least about 5 nm to about 20 nm from the surface of the metallic nanoparticle. The “open” configuration is also referred to as the “off’ configuration herein. In someembodiments, the stem loop strand comprises an extended length sufficient to remove the optical reporter from the surface of the metallic nanoparticle in the open configuration by a distance of at least about 10 nm.

[0359] Without being bound by theory, SERS is substantially increased as the optical reporter approaches or attaches to the surface of the metallic nanoparticle. Accordingly, in some embodiments, the nanoprobe emits a SERS signal that is significantly increased in the “closed” or “on” configuration as compared to the “open” or “off’ configuration. In some embodiments, the SERS signal is detectable using Raman spectroscopy. In some embodiments, the SERS signal in the closed configuration is at least about 3, about 5, about 10, about 50, or about 100 times higher compared to the SERS signal in the open configuration.

[0360] In some embodiments, the metallic nanoprobe further comprises a second optical reporter. In some embodiments, the second optical reporter is operably linked to the surface of the metallic nanoparticle, such that the second optical reporter is within the local SPR field enhancement upon irradiation. In some embodiments, the second optical reporter is operably linked to the surface of the metallic nanoparticle by adsorption. In some embodiments, the second optical reporter is operably linked to the surface of the metallic nanoparticle by a linker. In some embodiments, the first optical reporter emits a signal at a first Raman shift (cm'1) and the second optical reporter emits a signal at a second Raman shift (cm'1), wherein the first Raman shift and the second Raman shift are different. In some embodiments, the signal at the first Raman shift is increased in the closed configuration as compared to the open configuration. In some embodiments, the signal at the second Raman shift is substantially similar in the open and the closed configuration. In some embodiments, detection of the target nucleic acid comprises determining a ratio between the signal at the first Raman shift and the signal at the second Raman shift, wherein a change in the ratio (e.g., an increase or decrease in the ratio) indicates the presence or absence of the target nucleic acid.

[0361] In some embodiments, the metallic nanoparticle comprises a coating. In some embodiments, the metallic nanoparticle comprises a silica coating. In some embodiments, the metallic nanoparticle comprises a polymeric coating. In some embodiments, the nucleic acid probe is operably linked to a surface of the metallic nanoparticle comprising a coating, wherein the nucleic acid probe is directly linked to the metallic nanoparticle or to the coating. In some embodiments, the second optical reporter is directly linked to the metallic nanoparticle or to the coating.

[0362] In some embodiments, the nanoprobe comprises a component operably linked to the surface of the metallic nanoparticle. In some embodiments, the component comprises a synthetic polymer, a cell penetrating peptide, a membrane anchor, a bioreceptor, or a combination thereof. In some embodiments, the component is operably linked to the surface of the metallic nanoparticle by adsorption. In some embodiments, the component is operably linked to the surface of the metallic nanoparticle by a linker.

[0363] In some embodiments, the nanoprobe comprises the synthetic polymer operably linked to the surface of the metallic nanoparticle. In some embodiments, the nanoprobe comprises the cell penetrating peptide operably linked to the surface of the metallic nanoparticle. In some embodiments, the nanoprobe comprises the membrane anchor operably linked to the surface of the metallic nanoparticle. In some embodiments, the nanoprobe comprises the bioreceptor operably linked to the surface of the metallic nanoparticle. In some embodiments, the nanoprobe comprises the synthetic polymer and the cell penetrating peptide operably linked to the surface of the metallic nanoparticle. In some embodiments, the nanoprobe comprises the synthetic polymer and the membrane anchor operably linked to the surface of the metallic nanoparticle. In some embodiments, the nanoprobe comprises the synthetic polymer and the membrane anchor operably linked to the surface of the metallic nanoparticle.On-to-Off Nanoprobes for Target Nucleic Acid Detection

[0364] In some embodiments, the disclosure provides a nanoprobe configured for detection of a target nucleic acid described herein, wherein the nanoprobe emits a detectable signal (e.g., an optical signal) in the absence of the target nucleic acid.

[0365] In some embodiments, the nanoprobe comprises a metallic nanoparticle and a nucleic acid probe, wherein the nucleic acid probe comprises (i) an oligonucleotide comprising a nucleotide sequence capable of forming a stem-loop structure, wherein the stem-loop structure has an open and closed configuration, and (ii) an optical reporter, wherein the oligonucleotide comprises a first end operably linked to the metallic nanoparticle and an opposing, second end operably linked to the optical reporter. In some embodiments, the nucleotide sequence comprises a region of complimentary to a target sequence in the target nucleic acid. In some embodiments, the stem-loop structure is in the closed configuration in the absence of the target nucleic acid and in the open configuration in the presence of the target nucleic acid. In some embodiments, the optical reporter is in proximity of a surface ofthe metallic nanoparticle in the closed configuration. In some embodiments, the optical reporter is within an SPR field of the metallic nanoparticle in the closed configuration and upon irradiation.

[0366] In some embodiments, the metallic nanoprobe further comprises a second optical reporter. In some embodiments, the second optical reporter is operably linked to the surface of the metallic nanoparticle, such that the second optical reporter is within the local SPR field enhancement upon irradiation. In some embodiments, the optical reporter emits a signal at a first Raman shift (cm’1) and the second optical reporter emits a signal at a second Raman shift (cm’1), wherein the first Raman shift and the second Raman shift are different. In some embodiments, the signal at the first Raman shift is increased in the closed configuration as compared to the open configuration. In some embodiments, the signal at the second Raman shift is substantially similar in the open and the closed configuration. In some embodiments, detection of the target nucleic acid comprises determining a ratio between the signal at the first Raman shift and the signal at the second Raman shift, wherein a decrease in the ratio indicates the presence of the target nucleic acid.Off-to-On Nanoprobes for Target Nucleic Acid Detection

[0367] In some embodiments, the disclosure provides a nanoprobe configured for detection of a target nucleic acid described herein, wherein the nanoprobe emits a detectable signal (e.g., an optical signal) in the presence of the target nucleic acid.

[0368] In some embodiments, the nanoprobe comprises a metallic nanoparticle and a nucleic acid probe comprising (i) a stem-loop oligonucleotide comprising a nucleotide sequence capable of forming a stem-loop structure, wherein the stem-loop structure has an open and closed configuration, (ii) an optical reporter, and (iii) a placeholder oligonucleotide comprising a first region of complementarity to a sequence in the stem-loop oligonucleotide and a second region of complementarity to a target sequence in the target nucleic acid, wherein the oligonucleotide comprises a first end operably linked to the metallic nanoparticle and an opposing, second end operably linked to the optical reporter.

[0369] In some embodiments, the first region of complementarity overlaps the second region of complementarity. In some embodiments, the first region of complementarity hybridizes to the sequence in the stem loop oligonucleotide by base-pairing, thereby maintaining the stem loop oligonucleotide in the open configuration. In some embodiments, the second region of complementarity hybridizes to target sequence in the presence of the target nucleic acid,thereby displacing the stem loop oligonucleotide to generate the closed configuration. In some embodiments, the stem-loop structure is in the open configuration in the absence of the target nucleic acid and in the closed configuration in the presence of the target nucleic acid. In some embodiments, the optical reporter is in proximity of a surface of the metallic nanoparticle in the closed configuration. In some embodiments, the optical reporter is within an SPR field of the metallic nanoparticle in the closed configuration and upon irradiation.

[0370] In regards to the placeholder strand, it is to be understood that in certain embodiments, the second region of complementarity (that binds to the target nucleic acid) overlaps with the first region of complementarity (that binds to the stem loop strand). Accordingly, in some embodiments, the portion of the stem loop strand that forms a duplex with the first region of complementarity comprises a nucleotide sequence that is substantially similar to or identical to the target sequence or a portion thereof present in the target nucleic acid. In some embodiments, the entire length of the stem loop strand that forms the duplex region is substantially similar to or identical to the target sequence or a portion thereof . In some embodiments, a 5' contiguous portion of the stem loop strand that forms the duplex region is substantially similar to or identical to the target sequence or a portion thereof. In some embodiments, a 3' contiguous portion of the stem loop strand that forms the duplex region is substantially similar to or identical to the target sequence or a portion thereof. Wherein the target nucleic acid is an RNA, the portion of the stem loops strand that is substantially similar to or identical to the target sequence or a portion thereof may have thymine bases (or analogs thereof) in place of uridine bases.

[0371] In some embodiments, the placeholder strand comprises an overhang when hybridized to the stem loop strand. As used herein, an "overhang" refers to unpaired nucleotides present in the placeholder strand when hybridized to the stem loop strand via the duplex region. Without being bound by theory, the overhang functions as an initial site for binding to the target nucleic acid that displaces the placeholder strand from the stem loop strand via a nonenzymatic strand-displacement process (see, e.g., Bath, et al (2007) Nat Nanotechnol 2:274-284; Zhang, et al (2011) Nat Chem 3: 103-113).

[0372] In some embodiments, the metallic nanoprobe further comprises a second optical reporter. In some embodiments, the second optical reporter is operably linked to the surface of the metallic nanoparticle, such that the second optical reporter is within the local SPR field enhancement upon irradiation. In some embodiments, the optical reporter emits a signal at a first Raman shift (cm’1) and the second optical reporter emits a signal at a second Raman shift (cm’1), wherein the first Raman shift and the second Raman shift are different. In someembodiments, the signal at the first Raman shift is increased in the closed configuration as compared to the open configuration. In some embodiments, the signal at the second Raman shift is substantially similar in the open and the closed configuration. In some embodiments, detection of the target nucleic acid comprises determining a ratio between the signal at the first Raman shift and the signal at the second Raman shift, wherein an increase in the ratio indicates the presence of the target nucleic acid.

[0373] In some embodiments, the off-to-on nanoprobe is one described in US Pat 10633695, hereby incorporated by reference.Target Nucleic Acids

[0374] In some embodiments, the target nucleic acid is a DNA (e.g., gene, plasmid, or cell- free DNA). In some embodiments, the DNA comprises an altered (e.g., increased or decreased) expression level in a cell composition described herein upon a change in a cellular phenotype and / or differentiation state.

[0375] In some embodiments, the target nucleic acid that is differentially expressed is an RNA (e.g., coding RNAs such as mRNA, and microRNA, siRNA, shRNA, piRNA, snRNA, snoRNA, exRNA or other non-coding RNA). In some embodiments, the RNA comprises an altered (e.g., increased or decreased) expression level in a cell composition described herein upon a change in a cellular phenotype and / or differentiation state.

[0376] In some embodiments, the target nucleic acid is a microRNA. As is understood by the skilled artisan, microRNAs (or miRNA) are 19-25 nucleotide long (commonly 19-23 nucleotides long, most typically 22 nucleotides long) noncoding RNAs that bind to a corresponding miRNA target sequence present in the 3'UTR of nucleic acid molecules and post-translationally down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. A microRNA sequence comprises a “seed” region or sequence, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which sequence has perfect Watson-Crick complementarity to the miRNA target sequence. microRNAs derive enzymatically from regions of RNA transcripts that fold back on themselves to form short hairpin structures often termed a pre-miRNA (precursor-miRNA). This precursor-mRNA is processed in the nucleus and subsequently transported to the cytoplasm where it is further processed by DICER (a RNase III enzyme), to form a mature microRNA of approximately 22 nucleotides. The mature microRNA is then incorporated into a ribonuclear particle to form the RNA-induced silencing complex, RISC, which mediatesgene silencing. Art-recognized nomenclature for mature miRNAs typically designates the arm of the pre-miRNA from which the mature miRNA derives; “5p” means the microRNA is from the 5 prime arm of the pre-miRNA hairpin and “3p” means the microRNA is from the 3 prime end of the pre-miRNA hairpin. A miR referred to by number herein can refer to either of the two mature microRNAs originating from opposite arms of the same pre-miRNA (e.g., either the 3p or 5p microRNA). All miRs referred to herein are intended to include both the 3p and 5p arms / sequences, unless particularly specified by the 3p or 5p designation.

[0377] In some embodiments, the microRNA comprises an altered (e.g., increased or decreased) expression in a cell composition described herein upon a change in cellular phenotype and / or differentiation state. In some embodiments, the cellular phenotype and / or differentiation state is induced by hypoxia, presence of reactive oxygen species, nutrient deprivation, oxidative stress, metabolic stress, and / or inflammation.

[0378] In some embodiments, a method to identify a microRNA comprising an altered (e.g., increased or decreased) expression level in a cell composition described herein upon a change in cellular phenotype and / or differentiation state comprises quantifying miRNA expression levels in the cell composition contacted with culture conditions that induce the change in cellular phenotype and / or differentiation state as compared to a control cell not contacted with the culture conditions. In some embodiments, the culture conditions comprise an inflammatory factor (e.g., a cytokine, an interferon, and / or a chemokine). In some embodiments, the culture conditions comprise hypoxia. In some embodiments, the culture conditions comprise reduced nutrients. In some embodiments, the culture conditions comprise reactive oxygen species. Methods to quantify microRNAs expressed by a cell are known in the art. Non-limiting examples include Taqman microRNA analysis, RNA sequencing, microarray analysis real-time PCR, North blots, in situ hybridization, solution hybridization, and quantitative reverse transcription PCR.

[0379] In some embodiments, the microRNA comprises an increased expression level in a cell composition described herein upon contacting with culture conditions comprising an inflammatory factor (e.g., a cytokine, an interferon, and / or a chemokine) as compared to a cell composition not contacted with the culture conditions. In some embodiments, the cell composition comprises insulin producing cells (e.g., SCD P cells).

[0380] In some embodiments, the culture conditions comprise an interferon, wherein the microRNA(s) comprising an increased expression level are selected from miR-224, miR- 302b, miR-302a, miR-203a, miR- 146b, miR-21, and a combination thereof. In someembodiments, the miroRNA(s) are selected from miR-224-5p, miR-302b-3p, miR-302a-5p, miR-203a-3p, miR-146b-5p, and a combination thereof.

[0381] In some embodiments, the culture conditions comprise a cytokine, wherein the microRNA(s) comprising an increased expression level are selected from miR-203b, miR- 302a, miR-155, miR-146a, miR-302a, miR-21, and a combination thereof. In some embodiments, the microRNA(s) comprising an increased expression level are selected from miR-203b-3p, miR-302a-5p, miR-155-5p, miR-146a-5p, miR-302a-3p, miR-21-5p, and a combination thereof.

[0382] In some embodiments, the culture conditions comprises hypoxia, wherein the microRNA(s) comprising an increased expression level are selected from miR-210, miR-124, miR-1246, miR-155, miR-205, and a combination thereof. In some embodiments, the microRNA(s) comprising an increased expression level are selected from miR-210-3p, miR- 124-5p, miR-1246-3p, miR-1246-5p, miR-155-5p, miR-205-5p, and a combination thereof.

[0383] In some embodiments, a target nucleic acid of the disclosure is one set forth in Table 8A. In some embodiments, the target sequence comprising the nucleic acid sequence of any one of SEQ ID NO: 4-19.Table 8A: Exemplary target nucleic acids of the disclosureNanoprobe ComponentsNucleic Acid Probes(i) Oligonucleotides

[0384] The nucleic acids described herein are made using methods known in the art, de novo synthesis using any of a number of procedures known in the art including for example the b- cyanoethyl phosphorami di te method (Beaucage and Caruthers Tet. Let. 22: 1859, 1981), and the nucleoside H-phosphonate method (Garegg et al., Tet. Let. 27:4051-4054, 1986; Froehler et al., Nucl. Acid. Res. 14:5399-5407, 1986; Garegg et al., Tet. Let. 27:4055-4058, 1986, Gaffney et al., Tet. Let. 29:2619-2622, 1988). These chemistries can be performed by a variety of automated nucleic acid synthesizers available in the market. These nucleic acids are referred to as synthetic nucleic acids. Alternatively, oligonucleotides may be generated from larger nucleic acids such as but not limited to plasmids. Nucleic acids can be prepared from existing nucleic acid sequences (e.g., genomic or cDNA) using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases. Nucleic acids prepared in this manner are referred to as isolated nucleic acid. An isolated nucleic acid generally refers to a nucleic acid which is separated from components which it is normally associated with in nature. As an example, an isolated nucleic acid may be one which is separated from a cell, from a nucleus, from mitochondria, or from chromatin.(a) Stem Loop Strand

[0385] In some embodiments, the stem loop strand comprises a 5' end operably linked to the metallic nanoparticle and a 3' end operably linked to an optical reporter. In some embodiments, the stem loop strand comprises a 3' end operably linked to the metallic nanoparticle and a 5' end operably linked to an optical reporter. In some embodiments, the stem loop strand is operably linked to the metallic nanoparticle, the optical reporter, or both by a linker described herein.

[0386] In some embodiments, the stem loop strand has a length such that the distance between the optical reporter and the surface of metallic nanoparticle is sufficient (e.g., greater than 10 nm) to minimize or reduce background SERS signal when the nanoprobe is in an open configuration.

[0387] In some embodiments, the stem loop strand is about 10 to about 100 nucleotides in length. In some embodiments, the stem loop strand is about 10 nucleotides in length. In some embodiments, the stem loop strand is about 15 nucleotides in length. In some embodiments, the stem loop strand is about 20 nucleotides in length. In some embodiments, the stem loop strand is about 30 nucleotides in length. In some embodiments, the stem loop strand is about31 nucleotides in length. In some embodiments, the stem loop strand is about 32 nucleotides in length. In some embodiments, the stem loop strand is about 33 nucleotides in length. In some embodiments, the stem loop strand is about 34 nucleotides in length. In some embodiments, the stem loop strand is about 35 nucleotides in length. In some embodiments, the stem loop strand is about 40 nucleotides in length. In some embodiments, the stem loop strand is about 50 nucleotides in length. In some embodiments, the stem loop strand is about 60 nucleotides in length. In some embodiments, the stem loop strand is about 70 nucleotides in length. In some embodiments, the stem loop strand is about 80 nucleotides in length. In some embodiments, the stem loop strand is about 90 nucleotides in length. In some embodiments, the stem loop strand is about 100 nucleotides in length.

[0388] In some embodiments, the stem loop strand comprises a stem loop structure. In some embodiments, the stem loop structure comprises a nucleotide sequence according to the formula: 5'-Sl-L-S2-3', wherein SI is capable of hybridizing with S2 to form a stem, and wherein L forms a single-stranded loop between SI and S2.

[0389] In some embodiments, SI and S2 are 1 to 15 nucleotides in length and have the same length. In some embodiments, SI and S2 are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length and have the same length. In some embodiments, SI and S2 are 4 nucleotides in length and have the same length. In some embodiments, SI and S2 are 5 nucleotides in length and have the same length. In some embodiments, SI and S2 are 6 nucleotides in length and have the same length.

[0390] In some embodiments, SI and S2 hybridize to form a duplex of 2 nucleotides in length. In some embodiments, SI and S2 hybridize to form a duplex of 3 nucleotides in length. In some embodiments, SI and S2 hybridize to form a duplex of 4 nucleotides in length. In some embodiments, SI and S2 hybridize to form a duplex of 5 nucleotides in length. In some embodiments, SI and S2 hybridize to form a duplex of 6 nucleotides in length. In some embodiments, SI and S2 hybridize to form a duplex of 7 nucleotides in length. In some embodiments, SI and S2 hybridize to form a duplex of 8 nucleotides in length. In some embodiments, SI and S2 hybridize to form a duplex of 9 nucleotides in length. In some embodiments, SI and S2 hybridize to form a duplex of 10 nucleotides in length. In some embodiments, SI and S2 hybridize to form a duplex of 11 nucleotides in length. In some embodiments, SI and S2 hybridize to form a duplex of 12 nucleotides in length. In some embodiments, SI and S2 hybridize to form a duplex of 13 nucleotides in length. In some embodiments, SI and S2 hybridize to form a duplex of 14 nucleotides in length.

[0391] In some embodiments, the loop (L) of the stem loop structure is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the loop (L) of the stem-loop structure is about 5-10 nucleotides in length. In some embodiments, the loop (L) of the stem-loop structure is about 10-15 nucleotides in length. In some embodiments, the loop (L) of the stem-loop structure is about 15-20 nucleotides in length. In some embodiments, the loop (L) of the stem-loop structure is about 20-25 nucleotides in length. In some embodiments, the loop (L) of the stem-loop structure is about 25-30 nucleotides in length. In some embodiments, the loop (L) of the stem-loop structure is about 18 nucleotides in length. In some embodiments, the loop (L) of the stem-loop structure is about 19 nucleotides in length. In some embodiments, the loop (L) of the stem-loop structure is about 20 nucleotides in length.

[0392] In some embodiments, a portion of the stem loop strand forms a duplex region with the placeholder strand. In some embodiments, the portion of the stem loop strand that forms the duplex region comprises all or part of the stem loop structure. In some embodiments, the duplex region spans the full-length of the stem loop strand. In some embodiments, the duplex region spans the full-length of the stem-loop structure. In some embodiments, the duplex region spans the full-length of SI and a 5' portion of L. In some embodiments, the duplex region spans a 3' portion of SI and a 5' portion of L. In some embodiments, the duplex region spans the full-length of L and neither SI or S2. In some embodiments, the duplex region spans the full-length of L and a 5' portion of S2. In some embodiments, the duplex region spans the full-length of L and the full-length of S2. In some embodiments, the duplex region spans a 3' portion of L and the full-length of S2. In some embodiments, the duplex region spans a 3' portion of L and a 5' portion of S2. In some embodiments, the duplex region spans the full-length of SI and the full-length of L.

[0393] In some embodiments, the entire portion of the stem loop strand that forms the duplex region with the placeholder strand is substantially similar to or identical to all or part of the target sequence of the target nucleic acid. In some embodiments, a contiguous portion (e.g., 5-30 nucleotides in length) of the stem loop strand present at the 3'end of the duplex region is substantially similar to or identical to all or part of the target sequence of the target nucleic acid. In some embodiments, a contiguous portion (e.g., 5-30 nucleotides in length) of the stem loop strand present at the 5'end of the duplex region is substantially similar to or identical to all or part of the target sequence of the target nucleic acid. Wherein the target nucleic acid is an RNA (e.g., microRNA) described herein, the disclosure provides embodiments with thymine nucleobases in place of uridine nucleobases for the portion of thestem loop strand that is substantially similar to or identical to all or part of the target sequence of the target nucleic acid.(b) Placeholder Strand

[0394] In some embodiments, the placeholder strand is about 10 to about 100 nucleotides in length. In some embodiments, the placeholder strand is about 10 nucleotides in length. In some embodiments, the placeholder strand is about 15 nucleotides in length. In some embodiments, the placeholder strand is about 20 nucleotides in length. In some embodiments, the placeholder strand is about 25 nucleotides in length. In some embodiments, the placeholder strand is about 26 nucleotides in length. In some embodiments, the placeholder strand is about 27 nucleotides in length. In some embodiments, the placeholder strand is about 28 nucleotides in length. In some embodiments, the placeholder strand is about 29 nucleotides in length. In some embodiments, the placeholder strand is about 30 nucleotides in length. In some embodiments, the placeholder strand is about 40 nucleotides in length. In some embodiments, the placeholder strand is about 50 nucleotides in length. In some embodiments, the placeholder strand is about 60 nucleotides in length. In some embodiments, the placeholder strand is about 70 nucleotides in length. In some embodiments, the placeholder strand is about 80 nucleotides in length. In some embodiments, the placeholder strand is about 90 nucleotides in length. In some embodiments, the placeholder strand is about 100 nucleotides in length.

[0395] In some embodiments, the placeholder strand forms an overhang when hybridized to from the duplex region with the stem loop strand. In some embodiments, the overhang is present at the 5'end of the duplex region. In some embodiments, the overhang is present at the 3'end of the duplex region. In some embodiments, the overhang is 5-15 nucleotides in length. In some embodiments, the overhang is 5 nucleotides in length. In some embodiments, the overhang is 6 nucleotides in length. In some embodiments, the overhang is 7 nucleotides in length. In some embodiments, the overhang is 8 nucleotides in length. In some embodiments, the overhang is 9 nucleotides in length. In some embodiments, the overhang is 10 nucleotides in length.(c) Linkers

[0396] In some embodiments, the stem loop strand is operably linked via a linker to the metallic nanoparticle, the optical reporter, or both. In some embodiments, the linker is anoligonucleotide linker, a peptide linker, a polymer, a hydrocarbon linker, or a combination thereof.

[0397] In some embodiments, the linker is an oligonucleotide. The oligonucleotide can be of any sequence, for example, the sequence of the oligonucleotide can be a random sequence, or a sequence specifically chosen for its molecular or biochemical properties (e.g., highly polar). In some embodiments, the linker includes one or more series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof. In some embodiments, the linker consists of a series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analog thereof.

[0398] In some embodiments, the linker is an oligonucleotide of consecutive adenine nucleotides or analogs thereof. In some embodiments, the oligonucleotide is 1-100 nt in length. In some embodiments, the oligonucleotide is 1-50 nt in length. In some embodiments, the oligonucleotide is 1-10 nt in length. In some embodiments, the oligonucleotide is 1-7 nt in length. In some embodiments, the oligonucleotide is 1 nt in length. In some embodiments, the oligonucleotide is 2 nt in length. In some embodiments, the oligonucleotide is 3 nt in length. In some embodiments, the oligonucleotide is 4 nt in length. In some embodiments, the oligonucleotide is 5 nt in length. In some embodiments, the oligonucleotide is 6 nt in length. In some embodiments, the oligonucleotide is 7 nt in length.

[0399] In some embodiments, the linker comprises one or more ethylene glycol (EG) units, more preferably 2 or more EG units (i.e., polyethylene glycol (PEG)).

[0400] In some embodiments, the linker comprises a hydrocarbon chain. In some embodiments, the hydrocarbon chain is saturated. In some embodiments, the hydrocarbon chain is unsaturated. In some embodiments, the hydrocarbon chain is straight or branched. In some embodiments, the linker is a C3-C30 hydrocarbon chain.(ii) Nucleic Acid Modifications

[0401] In some embodiments, the nucleic acid probes of the disclosure comprise oligonucleotides that are DNA or RNA nucleotides comprising a heterocyclic base (nucleic acid base), a sugar moiety attached to the heterocyclic base, and a phosphate moiety which esterifies a hydroxyl function of the sugar moiety. The principal naturally-occurring nucleotides comprise uracil, thymine, cytosine, adenine and guanine as the heterocyclic bases, and ribose or deoxyribose sugar linked by phosphodiester bonds.

[0402] In some embodiments, the oligonucleotides are composed of nucleotide analogs that have been chemically modified to improve stability, half-life, or specificity or affinity for a target receptor, relative to a DNA or RNA counterpart. The chemical modifications include chemical modification of nucleobases, sugar moieties, nucleotide linkages, or combinations thereof. As used herein “modified nucleotide" or "chemically modified nucleotide" defines a nucleotide that has a chemical modification of one or more of the heterocyclic base, sugar moiety or phosphate moiety constituents. In some embodiments, the charge of the modified nucleotide is reduced compared to DNA or RNA oligonucleotides of the same nucleobase sequence. For example, the oligonucleotide can have low negative charge, no charge, or positive charge. Typically, nucleoside analogs support bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, where the analog backbone presents the bases in a manner to permit such hydrogen bonding in a sequence-specific fashion between the oligonucleotide analog molecule and bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). In some embodiments, the analogs have a substantially uncharged, phosphorus containing backbone.(a) Heterocyclic Bases

[0403] The principal naturally-occurring nucleotides include uracil, thymine, cytosine, adenine and guanine as the heterocyclic bases. The oligonucleotides can include chemical modifications to their nucleobase constituents. Chemical modifications of heterocyclic bases or heterocyclic base analogs may be effective to increase the binding affinity or stability in binding a target sequence. Chemically-modified heterocyclic bases include, but are not limited to, inosine, 5-(l-propynyl) uracil (pU), 5-(l-propynyl) cytosine (pC), 5- methylcytosine, 8-oxo-adenine, pseudocytosine, pseudoisocytosine, 5 and 2-ammo-5-(2'- deoxy-beta-D-ribofuranosyl)pyridine (2-aminopyridine), and various pyrrolo- and pyrazolopyrimidine derivatives.(b) Sugar Modifications

[0404] Oligonucleotides can also contain nucleotides with modified sugar moieties or sugar moiety analogs. Sugar moiety modifications include, but are not limited to, 2'-O-aminoetoxy, 2'-O-amonioethyl (2'-OAE), 2'-O- methoxy, 2'-O-methyl, 2-guanidoethyl (2'-OGE), 2'-O,4'- C-methylene (LNA), 2'-O-(methoxy ethyl) (2'-0ME) and 2'-O-methylacetamido) (2'- OMA). 2'-O-aminoethyl sugar moiety substitutions are especially preferred because they areprotonated at neutral pH and thus suppress the charge repulsion between the TFO and the target duplex. This modification stabilizes the C3'-endo conformation of the ribose or dexyribose and also forms a bridge with the i-1 phosphate in the purine strand of the duplex.

[0405] In some embodiments, the oligonucleotide is a morpholino oligonucleotide. Morpholino oligonucleotides are typically composed of two more morpholino monomers containing purine or pyrimidine base-pairing moieties effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, which are linked together by phosphorus- containing linkages, one to three atoms long, joining the morpholino nitrogen of one monomer to the 5' exocyclic carbon of an adjacent monomer. The purine or pyrimidine basepairing moiety is typically adenine, cytosine, guanine, uracil or thymine. The synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337.

[0406] Important properties of the morpholino-based subunits typically include: the ability to be linked in a oligomeric form by stable, uncharged backbone linkages; the ability to support a nucleotide base (e.g. adenine, cytosine, guanine, thymidine, uracil or inosine) such that the polymer formed can hybridize with a complementary -base target nucleic acid, including target RNA, with high Tm, even with oligomers as short as 10-14 bases; the ability of the oligomer to be actively transported into mammalian cells; and the ability of an oligomerRNA heteroduplex to resist RNAse degradation.

[0407] In some embodiments, oligonucleotides employ morpholino-based subunits bearing base-pairing moieties, joined by uncharged linkages, as described above.(c) Internucleotide Linkages

[0408] Oligonucleotides connected by an internucleotide bond that refers to a chemical linkage between two nucleoside moieties. Modifications to the phosphate backbone of DNA or RNA oligonucleotides may increase the binding affinity or stability oligonucleotides, or reduce the susceptibility of oligonucleotides to nuclease digestion. Cationic modifications, including, but not limited to, diethyl-ethylenediamide (DEED) or dimethylaminopropylamine (DMAP) may be especially useful due to decrease electrostatic repulsion between the oligonucleotide and a target. Modifications of the phosphate backbone may also include the substitution of a sulfur atom for one of the non-bridging oxygens in the phosphodiester linkage. This substitution creates a phosphorothioate intemucleoside linkagein place of the phosphodiester linkage. Oligonucleotides containing phosphorothioate internucleoside linkages have been shown to be more stable in vivo.

[0409] Examples of modified nucleotides with reduced charge include modified internucleotide linkages such as phosphate analogs having achiral and uncharged intersubunit linkages (e.g., Sterchak, E. P. et al., Organic Chem., 52:4202, (1987)), and uncharged morpholino-based polymers having achiral intersubunit linkages (see, e.g., U.S. Pat. No. 5,034,506), as discussed above. Some intemucleotide linkage analogs include morpholidate, acetal, and polyamide-linked heterocycles.

[0410] In another embodiment, the oligonucleotides are composed of locked nucleic acids. Locked nucleic acids (LNA) are modified RNA nucleotides (see, for example, Braasch, et al., Chem. Biol., 8(1): 1-7 (2001)). LNAs form hybrids with DNA which are more stable than DNA / DNA hybrids, a property similar to that of peptide nucleic acid (PNA) / DNA hybrids.

[0411] Therefore, LNA can be used just as PNA molecules would be. LNA binding efficiency can be increased in some embodiments by adding positive charges to it. Commercial nucleic acid synthesizers and standard phosphoramidite chemistry are used to make LNAs.

[0412] In some embodiments, the oligonucleotides are composed of peptide nucleic acids. Peptide nucleic acids (PNAs) are synthetic DNA mimics in which the phosphate backbone of the oligonucleotide is replaced in its entirety by repeating N-(2-aminoethyl)-glycine units and phosphodiester bonds are typically replaced by peptide bonds. The various heterocyclic bases are linked to the backbone by methylene carbonyl bonds. PNAs maintain spacing of heterocyclic bases that is similar to conventional DNA oligonucleotides, but are achiral and neutrally charged molecules. Peptide nucleic acids are comprised of peptide nucleic acid monomers.

[0413] Other backbone modifications include peptide and amino acid variations and modifications. Thus, the backbone constituents of oligonucleotides such as PNA may be peptide linkages, or alternatively, they may be non-peptide peptide linkages. Examples include acetyl caps, amino spacers such as 8-amino-3,6-dioxaoctanoic acid (referred to herein as O- linkers), amino acids such as lysine are particularly useful if positive charges are desired in the PNA, and the like. Methods for the chemical assembly of PNAs are well known. See, for example, U.S. Patent Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 and 5,786,571.

[0414] Oligonucleotides optionally include one or more terminal residues or modifications at either or both end to increase stability, and / or affinity of the oligonucleotide for its target.Commonly used positively charged moieties include the amino acids lysine and arginine, although other positively charged moieties may also be useful. Oligonucleotides may further be modified to be end capped to prevent degradation using a propylamine group. Procedures for 3' or 5' capping oligonucleotides are well known in the art.(iii) Optical Reporter

[0415] In some embodiments, the optical reporter is a SERS reporter molecule.

[0416] In some embodiments, the SERS reporter molecule is excited by a wavelength in the near-infrared (NIR) region of the light spectrum, which ranges from about 700 nm to about 1700 nm. In some embodiments, the SERS reporter molecule is excited by a wavelength in the visible region of the light spectrum, ranging from about 532 nm to about 700 nm. In some embodiments, the SERS reporter molecule emits a Raman spectroscopic signal at one or more wavenumbers within a range from about 100 cm’1and about 4000 cm’1. In some embodiments, the SERS reporter molecule emits a Raman spectroscopic signal at a wavenumbers of about 100 cm’1, about 200 cm’1, about 300 cm’1, about 400 cm’1, about 500 cm’1, about 600 cm’1, about 700 cm’1, about 800 cm’1, about 900 cm’1, about 1000 cm’1, 1100 cm’1, about 1200 cm’1, about 1300 cm’1, about 1400 cm’1, about 1500 cm’1, about 1600 cm’1, about 1700 cm’1, about 1800 cm’1, about 1900 cm’1, about 2000 cm’1, 2100 cm’1, about 2200 cm’1, about 2300 cm’1, about 2400 cm’1, about 2500 cm’1, about 2600 cm’1, about 2700 cm’1, about 2800 cm’1, about 2900 cm’1, about 3000 cm’1, 3100 cm’1, about 3200 cm’1, about 3300 cm’1, about 3400 cm’1, about 3500 cm’1, about 3600 cm’1, about 3700 cm’1, about 3800 cm’1, about 3900 cm’1, or about 4000 cm’1.

[0417] In some embodiments, the SERS reporter molecule is selected from 4- mercaptopyridine (4-MP), trans-4,4'bis(pyridyl)ethylene (BPE), quinolinethiol, 1 ,4- phenyldiisocyanide, mercaptobenzamidazole, 4-cyanopyridine, 1 ',3, 3, 3', 3'- hexam ethylindotricarbocyanine iodide, 3,3'-diethyltiatricarbocyanine, malachite green isothiocyanate, bis-(pyridyl)acetylenes, Bodipy, 4,4'-dipyridyl (DIPY), D8-4,4'-dipyridyl (d8DIPY), trans- 1 ,2-bis(4-pyridyl)-ethylene (BPE), quinoline thiol, 2-quinolinethiol (QSH), 1 ,2-dil(4- pyridyl)acetylene (BP A), 4-azobis(pyridine) (4-AZP), GM19, 1 -(4-pyridyl)-l- cyano-2-(2- fluoro-4- pyridyl)-ethylene (CNFBPE), 1 -cyano- 1 -(4-quinolinyl)-2-(4-pyridyl)- ethylene (CQPE), dye 10, 4- (4-hydroxyphenylazo)pyridine, CyNAMLA-381, 3,3'- Diethylthiadicarbocyanine iodide (DTDC), 3,3 '-di ethylthiatricarbocyanine iodide (DTTC), l,l',3,3,3',3'-Hexamethylindotricarbocyanine iodide (HITC), CY3 dye, CY3.5 dye, CY5.5dye, CY7 dye, CY7.5 dye, a positively-charged hydrophobic near infrared (NIR) dye, IR-780, IR-792, IR-797, IR-813, methylene blue hydrate (MB), 4-mercaptobenzoic acid (4-MBA), 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), 4-aminothiophenol (4ATP), fluorescein, fluorescein isothiocyanate (FITC), thionine dyes, rhodamine-based dye, crystal violet, a fluorescence label, and an absorbance label.

[0418] In some embodiments, the SERS reporter molecule comprises an ATTO fluorescent label (e.g., an ATTO fluorescent label from ATTO-TEC). In some embodiments, the ATTO fluorescent label comprises ATTO 700.

[0419] In some embodiments, the SERS reporter molecule comprises a Black Hole Quencher™ dye.Nanoparticles

[0420] Further described herein are nanoparticles suitable for use in nanoprobes of the disclosure.

[0421] In some embodiments, the nanoparticle is a SERS nanoparticle having a surface that induces, generates, or otherwise supports SERS or surface-enhanced resonance Raman light scattering (SERRS). In some embodiments, the SERS nanoparticle generates a surface plasmon when irradiated with incident light, wherein the surface plasmon induces, generates, or otherwise supports SERS or SERRS for optical reporters proximal to the nanoparticle surface. In some embodiments, the surface of the SERS nanoparticle that supports SERS is directly adsorbed to a SERS reporter molecule or within close proximity (e.g., within about 50 angstroms).

[0422] A number of surfaces are capable of producing a SERS signal, including roughened surfaces, textured surfaces, and other surfaces, including smooth surfaces. In some embodiments, the surface of the SERS nanoparticle comprises a metal. In some embodiments, the SERS nanoparticle comprises a surface capable of sustaining plasmons or plasmon resonance. Materials such as Group 11 metals (e.g., copper (Cu), silver (Ag), and gold (Au)), aluminum (Al), and high conductivity metal oxides (e.g., indium tin oxide, zinc oxide, tungsten oxide) are capable of supporting surface plasmon resonance. In some embodiments, the surface of the SERS nanoparticle comprises one or more metal selected from Cu, Ag, Au, and Al. In some embodiments, the surface of the SERS nanoparticle comprises a metal oxide.

[0423] In some embodiments, the surface of the SERS nanoparticle that induces, causes, or otherwise supports SERS or SERRS is an internal surface of the SERS nanoparticle (e.g., a surface of a core of the SERS nanoparticle). In some embodiments, the SERS nanoparticle comprises an outer shell and a core comprising a material that supports SERS. In some embodiments, the core of the SERS nanoparticle comprises a metal. In some embodiments, the core of the SERS nanoparticle comprises a single metal element (e.g., Cu, Ag, Au, or Al). In other embodiments, the core of the SERS nanoparticle includes a combination of at least two elements, such as an alloy, for example, a binary alloy (e.g., a metal oxide). In some embodiments, the core of the SERS nanoparticle is solid, semi-porous, porous, or hollow. In some embodiments, the core of the SERS nanoparticle is magnetic. In some embodiments, the shell of the SERS nanoparticle comprises silicon dioxide.

[0424] In some embodiments, the SERS nanoparticle comprises at least one dimension in the range of about 1 nm to about 1000 nm, about 20 nm to about 750 nm, or about 50 nm to about 500 nm. In some embodiments, the SERS nanoparticle comprises at least one dimension of about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm. Methods for measuring the size of nanoparticles are known in the art, and include, but are not limited to, electron microscopy (EM), dynamic light scattering (DLS), disc centrifugation, nanoparticle tracking analysis, tunable resistive pulse sensing, and atomic force microscopy (AFM).

[0425] In some embodiments, the SERS nanoparticle is spherical. In some embodiments, the SERS nanoparticle is non-spherical. In some embodiments, the SERS nanoparticle has a shape selected from spherical, spheroid, rod-shaped, disk-shaped, pyramid-shaped, cubeshaped, cylinder-shaped, nanohelical-shaped, nanospring-shaped, nanoring-shaped, arrowshaped, teardrop-shaped, tetrapod-shaped, prism-shaped, and star-shaped. In some embodiments, the SERS nanoparticle has an amorphous shape. Methods for measuring the shape of nanoparticles are known in the art, and include, but are not limited to, EM and AFM.

[0426] In some embodiments, the SERS nanoparticle is selected from silver nanoparticles, gold nanoparticles, silver nanostars, gold nanostars, silver-coated gold nanostars, bimetallic nanoparticles, multi-metallic nanoparticles, dielectric nanoparticle cores covered with metal nanoshells, or multi-nanoparticle structures.

[0427] In some embodiments, the SERS nanoparticle is one described in US Pat No 10633695, US Pat 9987358, US Pat 10633695, each of which are hereby incorporated by reference.

[0428] In some embodiments, the SERS nanoparticle is a gold nanostar (also referred to as a “Au nanostar” or “AuNS”). As used herein, the term “nanostar” or “NS” refers a nanoparticle which has a single core section with two or more protrusions emitting from the core section of the nanoparticle. In some embodiments, the protrusion has a shape that is conical or pyramidal. Methods for making AuNS are known in the art, and further described in US Pat. No. 9,561,292 and US Pat. No. 9,789,154 (each of which are incorporated by reference herein) and in the Examples section below.

[0429] In some embodiments, the SERS nanoparticle is a silver-coated gold nanostar. In some embodiments, the silver-coated gold nanostar is produced by reducing aqueous silver (Ag+) to solid silver)(AgO) onto gold nanostar seeds under conditions such that the silver- coated gold nanostars are produced. Methods for making silver-coated gold nanostars are known in the art, and further described in US Pat. No. 10,633,695 (incorporated by reference herein).Other Components

[0430] Also described herein are components operably linked to the metallic nanoparticles described herein to generate nanoprobes of the disclosure.

[0431] In some embodiments, the component comprises a synthetic polymer. In some embodiments, the synthetic polymer is biocompatible (e.g., non-immunogenic, biodegradable, and / or non-toxic in vivo). In some embodiments, the component is operably linked to a surface of the metallic nanoparticle by adsorption. In some embodiments, the component is operably linked to a surface of the metallic nanoparticle by a thiol linkage. In some embodiments, the synthetic polymer comprises polyethylene glycol (PEG). In some embodiments, the PEG comprises a thiol group to operably link the polymer to the metallic surface. In some embodiments, the PEG is less than 5 kDa. In some embodiments, the PEG is less than 10 kDa. In some embodiments, the PEG is less than 15 kDa. In some embodiments, the PEG is about 5kDa to about lOkDa, about 5kDa to about 15kDa, about lOkDa to about 15kDa, or about 15kDa to about 20kDa. In some embodiments, the synthetic polymer comprises poly(acrylamide) (PAM). In some embodiments, the synthetic polymer comprises poly(N-isopropylacrylamide) (PNIPAM). In some embodiments, the PAM or PNIPAM is less than 5 kDa. In some embodiments, the PAM or PNIPAM is less than 10 kDa. In some embodiments, the PAM or PNIPAM is less than 15 kDa. In some embodiments, the PAM orPNIPAM is about 5kDa to about lOkDa, about 5kDa to about 15kDa, about lOkDa to about 15kDa, or about 15kDa to about 20kDa.

[0432] In some embodiments, the component comprises a cell penetrating peptide. Cell penetrating peptides are short polypeptides that when operably linked to a cargo, facilitate transport of the cargo across a cellular membrane that is more efficient than the free cargo (not operably linked to the cell penetrating peptide). In some embodiments, the cell penetrating peptide is a polypeptide sequence of less than about 50, 45, 40, 35, 30, 25, 20, 15, or 10 amino acids in length. In some embodiments, a plurality of amino acid residues of the cell penetrating peptide are cationic. In some embodiments, the cell penetrating peptide comprises an human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription (TAT) peptide. In some embodiments, the HIV-1 TAT peptide comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 47). In some embodiments, the cell penetrating peptide comprises an HIV-1 TAT peptide comprises and N-terminal or a C-terminal cysteine. In some embodiments, the cysteine is used to conjugate the HIV-1 TAT peptide to a metallic nanoparticle described herein.

[0433] In some embodiments, the component comprises a membrane anchor. Membrane anchors refer to compounds that when operably linked to a cargo, facilitate an increased level of attachment and / or insertion of the cargo to a cellular membrane as compared to the free cargo (not operably linked to the membrane anchor). In some embodiments, the membrane anchor comprises a lipid, a cholesterol, a cationic polymer, a bioreceptor, or a combination thereof. In some embodiments, the membrane anchor comprises a cationic polymer. In some embodiments, the cationic polymer comprises poly-lysine.

[0434] In some embodiments, the component comprises a bioreceptor. In some embodiments, the bioreceptor comprises an antibody or antigen binding domain thereof, wherein the antibody targets a cell surface receptor.

[0435] In some embodiments, the component comprises a synthetic peptide. In some embodiments, the synthetic peptide is a targeting peptide (e.g., a peptide ligand for a cell surface receptor). In some embodiments, the synthetic peptide is a membrane destabilizing peptide. In some embodiments, the synthetic peptide is a endosomolytic peptide. In some embodiments, the synthetic peptide is a cell penetrating peptide.Making Nanoprobes

[0436] Known methods can be employed to immobilize the nucleic acid probes to metal nanoparticles to prepare the nanoprobes of the present disclosure. Exemplary methods are described in the Examples section herein.

[0437] The immobilization of biomolecules (such as, e.g., DNA, RNA, LNA, proteins, antibodies, etc.) to a solid support can use a wide variety of methods published in the literature. Binding can be performed through covalent bonds usually takes advantage of reactive groups such as amine ( — NH2) or sulfide ( — SH) that naturally are present or can be incorporated into the biomolecule structure. Amines can react with carboxylic acid or ester moieties in high yield to form stable amide bonds. Thiols can participate in maleimide coupling, yielding stable dialkylsulfides.

[0438] In some embodiments, the nanoparticle comprises gold nanoparticles (e.g., AuNS) or silver-coated gold nanoparticles (e.g., silver-coated AuNS). The majority of immobilization schemes involving gold or silver surfaces utilize a prior derivatization of the surface with alkylthiols, forming stable linkages. Alkylthiols readily form self-assembled monolayers (SAM) onto silver surfaces in micromolar concentrations. The end of the alkylthiol chain can be used to bind biomolecules, or can be easily modified to do so. The length of the alkylthiol chain has been found to be an important parameter, keeping the biomolecules away from the surface. Furthermore, to avoid direct, non-specific DNA adsorption onto the surface, alkylthiols can be used to block further access to the surface, allowing only covalent immobilization through the linker (Steel, A. B. J. Anal. Chem. (1998) 70, 4670-7; Herne, T. M. J. Am. Chem. Soc. (1997) 119, 8916-20).

[0439] Silver surfaces have been found to exhibit controlled self-assembly kinetics when exposed to dilute ethanolic solutions of alkylthiols. The tilt angle formed between the surface and the hydrocarbon tail ranges from 0 to 15 degrees. There is also a larger thiol packing density on silver, when compared to gold (Burges, J. D. Langmuir 1997, 13, 3781-6). After SAM formation on gold / silver nanoparticles, alkylthiols can be covalently coupled to biomolecules. The majority of synthetic techniques for the covalent immobilization of biomolecules utilize free amine groups of a polypeptide (enzymes, antibodies, antigens, etc) or of amino-labeled DNA strands, to react with a carboxylic acid moiety forming amide bonds. As a general rule, a more active intermediate (labile ester) is first formed with the carboxylic acid moiety and in a later stage reacted with the free amine, increasing the coupling yield. Coupling procedures for use in the present disclosure are further described below.

[0440] Additional immobilization schemes involve functionalizing the gold or silver surface with a polyethylene glycol (PEG) comprising a terminal reactive group (e.g., amine, azide, carboxylic acid, N-hydroxysuccinimide, etc.). Methods for preparing PEG-functionalized gold nanostructures (e.g., PEG-functionalized gold nanostars) are known in the art. For example, a suitable method comprises combining the gold nanostructure with a PEG having a thiol at one terminus and a reactive group at the other terminus, wherein the thiolated terminus appends to the gold surface and displays the reactive group for subsequent conjugation, e.g., to an oligonucleotide described herein. Any PEG molecular weight known in the art for preparation of PEG-functionalized nanostructures is suitable for use in the present disclosure, e.g., PEG2k, PEG6k, PEGlOk.(i) Binding Procedure Using N-Hydroxysuccinimide (NHS) and its Derivatives.

[0441] The coupling approach involves the esterification under mild conditions of a carboxylic acid with a labile group, an N-hydroxysuccinimide (NHS) derivative, and further reaction with free amine groups in a polypeptide (enzymes, antibodies, antigens, etc) or amine-labeled DNA, producing a stable amide (Boncheva, M. Langmuir (1999), 15, 4317- 20). NHS reacts almost exclusively with primary amine groups. Covalent immobilization can be achieved in as little as 30 minutes. Since H2O competes with amines in reactions involving these very labile esters, it is important to consider the hydrolysis kinetics of the available esters used in this type of coupling. The derivative of NHS O — (N-succinimidyl)- N,N,N,N-tetramethyluronium tetrafluoroborate, increase the coupling yield by utilizing a leaving group that is converted to urea during the carboxylic acid activation, hence favorably increasing the negative enthalpy of the reaction.(ii) Binding Procedure Using Maleimide.

[0442] Maleimide can be used to immobilize biomolecules through available — SH moieties. Coupling schemes with maleimide have been proven useful for the site-specific immobilization of antibodies, Fab fragments, peptides, and SH-modified DNA strands. Sample preparation for the maleimide coupling of a protein involves the simple reduction of disulfide bonds between two cysteine residues with a mild reducing agent, such as dithiothreitol, 2-mercaptoethanol or tris(2-carboxyethyl)phosphine hydrochloride. However, disulfide reduction will usually lead to the protein losing its natural conformation, and might impair enzymatic activity or antibody recognition. The modification of primary amine groupswith 2-iminothiolane hydrochloride (Traut's reagent) to introduce sulfydryl groups is an alternative for biomolecules lacking them. Free sulfhydryls are immobilized to the maleimide surface by an addition reaction to unsaturated carbon-carbon bonds (Jordan, C. E., et al., 1997).(iii) Binding Procedure Using Carbodiimide.

[0443] Surfaces modified with mercaptoalkyldiols can be activated with 1,1'- carbonyldiimidazole (CDI) to form a carbonylimidazole intermediate. A biomolecule with an available amine group displaces the imidazole to form a carbamate linkage to the alkylthiol tethered to the surface (Potyrailo, R. A., et al., 1998).Contacting

[0444] In some embodiments, the methods of the disclosure comprise contacting a population comprising cells with one or more nanoprobes described herein. Suitable methods for performing the contacting include any known method for introducing nanostructures (e.g., nanoprobes described herein) to a population comprising cells, e.g., in a manner that enables nanostructure uptake in a plurality of cells in the population and / or labeling of the cell surface of a plurality of cells in the population with the nanostructures.

[0445] In some embodiments, the contacting is performed in a manner that promotes internalization of the nanoprobes by cells in the population. For example, in some embodiments, the contacting is performed using transfection. As used herein, the term “transfection” refers to the process of delivering, passively or actively, nanoparticles (e.g., nanoprobes as described herein) into eukaryotic cells. Delivery can be achieved by using any number of known means, including but not limited to, passive uptake, chemi cal -reagent based uptake, mechanical bombardment, direct injection, and electroporation. In some embodiments, the delivery comprises passive uptake, wherein internalization of the nanoprobes occurs through endocytic or phagocytic pathways.

[0446] In some embodiments, the contacting is performed by transfecting the population comprising cells with one or a combination of nanoprobes described herein using electroporation. Methods for performing electroporation are known in the art, and include introducing an electric field to the population comprising cells present in a cuvette, microchannel, micro-capillary, nano-channel, or micro-well array (see, e.g., Kim, et al (2017) J. Mater. Chem. 5:2726). In some embodiments, the contacting comprises incubating thepopulation comprising cells with one or a combination of nanoprobes described herein and performing electroporation for a sufficient duration to enable uptake of the nanoprobes. In some embodiments, the electroporation is performed using any method known in the art or described in the Examples section herein.

[0447] In some embodiments, the contacting is performed in a manner that conjugates the nanoprobes onto the surface of the cells in the population, e.g., via covalent or non-covalent conjugation. In some embodiments, the nanoprobes are covalently-conjugated to the surface of the cells. In some embodiments, the nanoprobes are non-covalently-conjugated to the surface of the cells, e.g., via absorption onto the cell surface.

[0448] In some embodiments, the disclosure provides a method for inducing nanoprobe internalization by a plurality of cells (e.g., SCD cells and / or insulin producing cells) of a cell composition described herein, wherein the plurality are formed into cell clusters. In some embodiments, the method comprises dissociating the cell clusters to form a cell suspension, transfecting the cell suspension with a nanoprobe described herein (e.g., via electroporation), and culturing the cell suspension for a duration sufficient to allow cell clusters to reform. Methods for dissociating cell clusters known in the art are suitable for use herein.Exemplary Cell Compositions

[0449] In some embodiments, the disclosure provides a cell composition comprising an insulin producing cell described herein, wherein the insulin producing cell comprises a nanoprobe described herein. In some embodiments, the nanoprobe is operably linked to a surface of the insulin producing cell. In some embodiments, the nanoprobe is contained within the insulin producing cell.

[0450] In some embodiments, the disclosure provides a cell composition comprising an insulin producing cell, wherein the insulin producing cell comprises a nanoprobe, wherein the nanoprobe comprises a metallic nanoparticle and an optical reporter, and wherein the optical reporter is operably linked to a surface of the metallic nanoparticle. In some embodiments, the metallic nanoparticle comprises a gold nanoparticle. In some embodiments, the metallic nanoparticle comprises a gold nanostar. In some embodiments, the optical reporter is a SERS reporter molecule. In some embodiments, the nanoprobe further comprises a coating encapsulating the metallic nanoparticle. In some embodiments, coating is silica. In some embodiments, the coating is a polymer. In some embodiments, the polymer is PEG. In some embodiments, the nanoprobe further comprises a component operably linked to a surface ofthe metallic nanoparticle. In some embodiments, the component comprises PEG. In some embodiments, the component comprises poly -lysine. In some embodiments, the component comprises PEG and poly-lysine. In some embodiments, the cell composition comprises at least about 10 to about 100, about 10 to about 500, about 100 to about 500, about 100 to about IxlO3, about 500 to about 5xl03, about lxl03to about 5xl03, about 2xl03to about 5xl03, or about 3xl03to about 5xl03nanoprobes per insulin producing cell. In some embodiments, the cell composition comprises at least about IxlO3to about 5xl03, about 2xl03to about 5xl03, or about 3xl03to about 5xl03nanoprobes per insulin producing cell.

[0451] In some embodiments, the disclosure provides a cell composition comprising an insulin producing cell, wherein the insulin producing cell comprises a nanoprobe configured to detect a target nucleic acid described herein.

[0452] In some embodiments, the disclosure provides a cell composition comprising an insulin producing cell, wherein the insulin producing cell comprises a nanoprobe, wherein the nanoprobe comprises (i) a metallic nanoparticle, (ii) a nucleic acid probe comprising (a) a stem-loop oligonucleotide comprising a nucleotide sequence capable of forming a stem-loop structure, wherein the stem-loop structure comprises an open and closed configuration, (b) a first optical reporter, and (c) a placeholder oligonucleotide comprising a first region of complementarity to a sequence in the stem-loop oligonucleotide and a second region of complementarity to a target sequence in the target nucleic acid, wherein the oligonucleotide comprises a first end operably linked to the metallic nanoparticle and an opposing, second end operably linked to the optical reporter; and (iii) a second optical reporter operably linked to a surface of the metallic nanoparticle. In some embodiments, the first optical reporter is a SERS reporter molecule. In some embodiments, the first optical label emits a signal at a first Raman shift (cm'1), wherein the signal is increased in the closed configuration relative to the open configuration. In some embodiments, the second optical reporter is a SERS reporter molecule. In some embodiments, the second optical label emits a signal at a second, different Raman shift (cm'1) that is substantially similar in the open and closed configuration. In some embodiments, the metallic nanoparticle is a gold nanoparticle. In some embodiments, the metallic nanoparticle is a gold nanostar. In some embodiments, the nanoprobe further comprises a coating. In some embodiments, coating is silica. In some embodiments, the coating is a polymer. In some embodiments, the metallic nanoparticle is contained within the coating. In some embodiments, the metallic nanoparticle and the nucleic acid probe is contained within the coating. In some embodiments, the metallic nanoparticle, the nucleic acid probe, and the second optical reporter are contained within the coating. In someembodiments, the second optical reporter is operably linked to a surface of the coating. In some embodiments, the nucleic acid probe is operably linked to a surface of the coating. In some embodiments, the nanoprobe further comprises a component operably linked to a surface of the metallic nanoparticle. In some embodiments, the component comprises PEG. In some embodiments, the component comprises poly-lysine. In some embodiments, the cell composition comprises at least about 10 to about 100, about 10 to about 500, about 100 to about 500, about 100 to about IxlO3, about 500 to about 5xl03, about lxl03to about 5xl03, about 2xl03to about 5xl03, or about 3xl03to about 5xl03nanoprobes per insulin producing cell. In some embodiments, the cell composition comprises at least about 10 to about 100, about 10 to about 500, about 100 to about 500, about 100 to about IxlO3, about 500 to about 5xl03nanoprobes per insulin producing cell.

[0453] In some embodiments, the insulin producing cell comprises a nanoprobe comprising any one of the nucleic acid sequences provided in Table 8B. In Table 8B, the underlined bases indicate locked nucleic acid (LNA) bases.Table 8B: Exemplary nanoprobes of the disclosure

[0454] In some embodiments, the insulin-producing cell comprises a nanoprobe comprising the nucleic acid sequence of any one of SEQ ID NOS: 20-46.

[0455] In some embodiments, the insulin producing cell comprises a nanoprobe for detecting miR-21, wherein the nanoprobe comprises (i) a metallic nanoparticle, (ii) a nucleic acid probe comprising (a) a stem-loop oligonucleotide comprising a sequence having at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity to SEQ ID NOS: 20 or 22, (b) a first optical reporter, and (c) a placeholder oligonucleotide comprising a sequence having at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity SEQ ID NOS: 21 or 23, wherein the oligonucleotide comprises a first end operably linked to the metallic nanoparticle and an opposing, second end operably linked to the first optical reporter; and optionally (iii) a second optical reporter operably linked to a surface of the metallic nanoparticle.

[0456] In some embodiments, the insulin producing cell comprises a nanoprobe for detecting miR-146a, wherein the nanoprobe comprises (i) a metallic nanoparticle, (ii) a nucleic acid probe comprising (a) a stem-loop oligonucleotide comprising a sequence having at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity to SEQ ID NOS: 24 or 26, (b) a first optical reporter, and (c) a placeholder oligonucleotide comprising a sequence having at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity SEQ ID NOS: 25 or 27, wherein the oligonucleotide comprises a first endoperably linked to the metallic nanoparticle and an opposing, second end operably linked to the first optical reporter; and optionally (iii) a second optical reporter operably linked to a surface of the metallic nanoparticle.

[0457] In some embodiments, the insulin producing cell comprises a nanoprobe for detecting miR-224, wherein the nanoprobe comprises (i) a metallic nanoparticle, (ii) a nucleic acid probe comprising (a) a stem-loop oligonucleotide comprising a sequence having at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity to SEQ ID NO: 28, (b) a first optical reporter, and (c) a placeholder oligonucleotide comprising a sequence having at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity SEQ ID NO: 29, wherein the oligonucleotide comprises a first end operably linked to the metallic nanoparticle and an opposing, second end operably linked to the first optical reporter; and optionally (iii) a second optical reporter operably linked to a surface of the metallic nanoparticle.

[0458] In some embodiments, the insulin producing cell comprises a nanoprobe for detecting miR-302a, wherein the nanoprobe comprises (i) a metallic nanoparticle, (ii) a nucleic acid probe comprising (a) a stem-loop oligonucleotide comprising a sequence having at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity to SEQ ID NO: 30, (b) a first optical reporter, and (c) a placeholder oligonucleotide comprising a sequence having at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity SEQ ID NO: 31, wherein the oligonucleotide comprises a first end operably linked to the metallic nanoparticle and an opposing, second end operably linked to the first optical reporter; and optionally (iii) a second optical reporter operably linked to a surface of the metallic nanoparticle.

[0459] In some embodiments, the insulin producing cell comprises a nanoprobe for detecting miR-210, wherein the nanoprobe comprises (i) a metallic nanoparticle, (ii) a nucleic acid probe comprising (a) a stem-loop oligonucleotide comprising a sequence having at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity to SEQ ID NOS: 32-35, (b) a first optical reporter, and (c) a placeholder oligonucleotide comprising a sequence having at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity SEQ ID NO: 36, wherein the oligonucleotide comprises a first end operably linked to the metallic nanoparticle and an opposing, second end operably linked to the first optical reporter; and optionally (iii) a second optical reporter operably linked to a surface of the metallic nanoparticle.

[0460] In some embodiments, the insulin producing cell comprises a nanoprobe for detecting miR-155, wherein the nanoprobe comprises (i) a metallic nanoparticle, (ii) a nucleic acid probe comprising (a) a stem-loop oligonucleotide comprising a sequence having at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity to SEQ ID NOS: 37-40, (b) a first optical reporter, and (c) a placeholder oligonucleotide comprising a sequence having at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity SEQ ID NO: 41, wherein the oligonucleotide comprises a first end operably linked to the metallic nanoparticle and an opposing, second end operably linked to the first optical reporter; and optionally (iii) a second optical reporter operably linked to a surface of the metallic nanoparticle.

[0461] In some embodiments, the insulin producing cell comprises a nanoprobe for detecting miR-124, wherein the nanoprobe comprises (i) a metallic nanoparticle, (ii) a nucleic acid probe comprising (a) a stem-loop oligonucleotide comprising a sequence having at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity to SEQ ID NOS: 42-45, (b) a first optical reporter, and (c) a placeholder oligonucleotide comprising a sequence having at least about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity SEQ ID NO: 46, wherein the oligonucleotide comprises a first end operably linked to the metallic nanoparticle and an opposing, second end operably linked to the first optical reporter; and optionally (iii) a second optical reporter operably linked to a surface of the metallic nanoparticle.

[0462] In some embodiments, the target nucleic acid is a miRNA biomarker for inflammation. In some embodiments, the target nucleic acid is a miRNA biomarker for cytokine-dependent inflammation. In some embodiments, the miRNA biomarker for cytokine-dependent inflammation is selected from the group consisting of miR-203b, miR- 302a, miR-155, miR-146a, miR-302a, miR-21, and a combination thereof. In some embodiments, the miRNA biomarker for cytokine-dependent inflammation is selected from the group consisting of miR-203b-3p, miR-302a-5p, miR-155-5p, miR-146a-5p, miR-302a- 3p, miR-21 -5p, and a combination thereof. In some embodiments, the miRNA biomarker for cytokine-dependent inflammation comprises miR-146a-5p. In some embodiments, the target nucleic acid is a miRNA biomarker for interferon-dependent inflammation. In some embodiments, the miRNA biomarker for interferon-dependent inflammation is selected from the group consisting of miR-224, miR-302b, miR-302a, miR-203a, miR-146b, miR-21, and a combination thereof. In some embodiments, the miRNA biomarker for interferon-dependent inflammation is selected from the group consisting of miR-224-5p, miR-302b-3p, miR-302a-5p, miR-203a-3p, miR-146b-5p, and a combination thereof. In some embodiments, the miRNA biomarker for interferon-dependent inflammation comprises miR-224-5p. In some embodiments, the target nucleic acid is a miRNA biomarker for hypoxia. In some embodiments, the miRNA biomarker for hypoxia is selected from the group consisting of miR-210, miR-124, miR-1246, miR-155, miR-205, and a combination thereof. In some embodiments, the miRNA biomarker for hypoxia is selected from the group consisting of miR-210-3p, miR-124-5p, miR-1246-3p, miR-1246-5p, miR-155-5p, miR-205-5p, and a combination thereof. In some embodiments, the miRNA biomarker comprises miR-210-3p.

[0463] In some embodiments, the cell composition further comprises a support factor described herein. In some embodiments, the cell composition a support factor selected from GABA, leptin, serotonin, PTH, HGF, osteopontin, PTHrP, VEGF, angiopoietin-1, angiopoietin-2, PDGF-AA, PDGF-BB, and a combination thereof. In some embodiments, the cell composition comprises GABA, leptin, serotonin, PTH, HGF, osteopontin, PTHrP, VEGF, angiopoietin-1, angiopoietin-2, PDGF-AA, and PDGF-BB. In some embodiments, the cell composition comprises a support factor selected from GABA, leptin, serotonin, PTH, HGF, osteopontin, PTHrP, VEGF, angiopoietin-1, angiopoietin-2, PDGF-AA, PDGF-BB, IL- 6, and a combination thereof. In some embodiments, the cell composition comprises GABA, leptin, serotonin, PTH, HGF, osteopontin, PTHrP, VEGF, angiopoietin-1, angiopoietin-2, PDGF-AA, PDGF-BB, and. In some embodiments, the cell composition comprises (i) a concentration of VEGF, wherein the concentration is about 0.1 pg / mL to about 10 pg / mL; (ii) a concentration of angiopoietin-1, wherein the concentration is about 10 pg / mL to about 1,000 pg / mL; (iii) a concentration of angiopoietin-2, wherein the concentration is about 10 pg / mL to about 1,000 pg / mL; (iv) a concentration of PDGF-AA, wherein the concentration is about 0.1 pg / mL to about 10 pg / mL; (v) a concentration of PDGF-BB, wherein the concentration is about 10 pg / mL to about 1,000 pg / mL; (vi) a concentration of GABA, wherein the concentration is about 0.1 pM to about 50 pm; (vii) a concentration of leptin, wherein the concentration is about 1 pM to about 500 pm; (viii) a concentration of serotonin, wherein the concentration is about 1 pM to about 500 pm; (ix) a concentration of PTH, wherein the concentration is about 0.1 pg / mL to about 10 pg / mL; (x) a concentration of HGF, wherein the concentraiton is about 10 ng / mL to about 1,000 ng / mL; (xi) a concentration of osteopontin, wherein the concentration is about 10 ng / mL to about 1,000 ng / mL; (xii) a concentration of PTHrP, wherein the concentration is about 1 ng / mL to about 500 ng / mL; or (xiii) any combination of (i)-(xii). In some embodiments, the cell composition comprises (i)-(xii).

[0464] In some embodiments, the insulin producing cells are differentiated from pluripotent stem cells according to a method described herein. In some embodiments, the insulin producing cells comprise SCD P cells. In some embodiments, the cell composition further comprises SCD a cells and / or SCD 5 cells. In some embodiments, a plurality of cells of the composition are formed into cell clusters. In some embodiments, the cell clusters comprise about 100 to about 10,000 cells per cluster. In some embodiments, the cell clusters comprise about 100, about 200, about 300, about 400, about 500, about 800, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 cells per cluster. In some embodiments, the cell clusters comprise about 300 to about 1000 cells per cluster. In some embodiments, the cell clusters comprise a longest diameter of about 50 pm to about 500 pm. In some embodiments, the cell clusters comprise a longest diameter of about 100 pm to about 200 pm.

[0465] In some embodiments, the insulin producing cells of the cell composition further comprise a genetic modification described herein. In some embodiments, the insulin producing cells comprise a genomic disruption of an MHCI molecule, an MHCII molecule, a regulator of MHCI transcription, or a regulator of MHCII transcription. In some embodiments the insulin producing cells comprise a genomic disruption of Tet2. In some embodiments, the insulin producing cells comprise a gene insertion, wherein the gene encodes a tolerogenic factor described herein or a survival factor described herein. In some embodiments, the tolerogenic factor comprises a checkpoint molecule described herein.Pharmaceutical Compositions

[0466] The present disclosure provides pharmaceutical compositions comprising the nanoprobes and / or the cell compositions described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions are formulated for administration to a subject to treat, prevent, or mitigate one or more symptoms of a disease or disorder (e.g., diabetes).

[0467] In some embodiments, the pharmaceutical composition comprises a population of cells and a pharmaceutically acceptable carrier, wherein the population of cells comprises SCD cells. In some embodiments, the SCD cells are derived from pluripotent stem cells. In some embodiments, the pluripotent stem cells are ESCs. In some embodiments, the pluripotent stem cells are an ESC cell line (e.g., a cGMP ESC cell line). In some embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs). In someembodiments, the iPSCs are reprogrammed somatic cells obtained from the subject. In some embodiments, the iPSCs are derived from reprogrammed somatic cells obtained from a health human donor.

[0468] In some embodiments, the pharmaceutical composition comprises a population of cells and a pharmaceutically acceptable carrier, wherein the population of cells comprises insulin producing cells. In some embodiments, the insulin producing cells are human donor- derived pancreatic P cells. In some embodiments, the insulin producing cells are SCD p cells.

[0469] In some embodiments, the pharmaceutical composition comprises a population of cells and a pharmaceutically acceptable carrier, wherein the population of cells comprises SCD islet cells. In some embodiments, the SCD islet cells comprise SCD p cells, SCD a cells, and / or SCD 5 cells. In some embodiments, the SCD islet cells are present in one or more clusters. In some embodiments, the SCD islet cells are present in a cell suspension.

[0470] In some embodiments, a plurality of cells of the population comprise a nanoprobe described herein. In some embodiments, the nanoprobe is configured for optical detection of the population of cells following administration to the subject. In some embodiments, the nanoprobe is configured for detection of a target nucleic acid described herein following administration to the subject.

[0471] In some embodiments, the population of cells is sorted or enriched, e.g., to increase the proportion of the population that are SCD cells and / or to remove not fully differentiated progenitor cells or pluripotent stem cells. In some embodiments, the population of cells is sorted or enriched using FACS. In some embodiments, the population of cells is sorted or enriched using a microfluidic cell sorter.

[0472] In some embodiments, the pharmaceutical composition is formulated as a liquid dosage form comprising the population of cells, and a pharmaceutically acceptable carrier. In some embodiments, the liquid dosage form is a sterile solution or suspension.

[0473] In some embodiments, the pharmaceutical composition is formulated as a solid dosage form comprising the population of cells, and a pharmaceutically acceptable carrier. In some embodiments, the solid dosage form is for oral administration. In some embodiments, the solid dosage form is a drench, lozenge, dragees, capsule, pill, tablet, bolus, powder, granule, or paste.

[0474] As used herein, the term “pharmaceutically acceptable carrier” refers to reagents, cells, compounds, materials, compositions, and / or dosage forms that are compatible with the nanoprobes and / or cell compositions described herein for administration to a subject. Exemplary pharmaceutically acceptable carriers include, but are not limited to, water, a saltsolution, alcohols, oils, gelatins, carbohydrates (e.g., lactose, amylose, starch), glycolipids, cellulose derivatives, and polymers (e.g., PEG, polyvinyl pyrrolidine). In some embodiments, the pharmaceutically acceptable carrier is sterilized. In some embodiments, pharmaceutically acceptable carrier further comprises a lubricant, preservative, stabilizer, wetting agent, emulsifier, salts (e.g., to modulate osmotic pressure), buffers, and coloring.

[0475] As used herein, the term “pharmaceutically acceptable” refers to compounds, materials, compositions, and / or dosage forms that are suitable for administering to a subject without substantial risk of toxicity, allergic response, and or other complications, as ascertained by an individual skilled in the art. The term implies that each carrier present in the pharmaceutical composition is compatible with other components of the composition, and when combined to form the composition can be administered to a subject without substantial risk.

[0476] In some embodiments, the pharmaceutical composition comprises an effective amount of the population of cells. As used herein, the term “effective amount” used in respect to a population of cells described herein refers to an amount of cells in the population that is sufficient to produce an effect following administration to a subject that is characterized by one or more therapeutic and / or prophylactic benefits. For example, in some embodiments, the pharmaceutical composition comprises an effective amount of a population of cells comprising SCD P cells, wherein the amount is sufficient to produce a measurable change in at least one symptom of diabetes (e.g., glycosylated hemoglobin level, fasting blood glucose level, hypoinsulinemia) following administration to a subject having diabetes. Determination of an effective amount is within the capability of the skilled clinician and is adapted based upon the subject’s medical history, age, condition, sex, severity of the medical condition, and administration of other pharmaceutically active agents.

[0477] In some embodiments, the data obtained from cell culture assays and animal studies, such as any described herein, is used in formulating a range of dosages for use in humans. The dosage of a pharmaceutical composition described herein can be estimated initially from preclinical data comparing the relative potency of the new formulation with the standard formulation in animal studies. A minimal cell dose to achieve diabetes remission in animal studies can be compared between old and new formulations. In these dose-finding studies, C- peptide (a non-metabolized byproduct of insulin production) concentration at baseline and after administration of glucose can also be measured and compared between the old and new formulation. Such information can be used to more accurately determine useful doses inhumans. Levels of C-peptide in plasma may be measured, for example, using enzyme-linked immunosorbent assay.

[0478] The dosages administered will vary from individual to individual; a therapeutically effective dose in humans can be estimated, for example but not limited to, by the level of enhancement of function (e.g., C-peptide concentration or minimal cell dose required for diabetes remission). Monitoring levels of co-transplanted cell introduction, the level of expression of certain genes affected by such transfer, and / or the presence or level s of the encoded product will also enable one skilled in the art to select and adjust the dosages administered. Generally, a composition including co-transplanted cells will be administered in a single dose in the range of 105-10s cells per kg body weight, preferably in the range of 106-107 cells per kg body weight. This dosage may be repeated as considered appropriate by the treating physician

[0479] In some embodiments, the pharmaceutical composition is formulated for administration via a parental route. Examples of parenteral routes of administration include, for example, intramuscular, intravenous, intradermal, subcutaneous, transderma! (topical), transmucosal, intra-peritoneal and intraomental administration. In some embodiments, the pharmaceutical composition is formulated for administration to an extrahepatic site of the subject. In some embodiments, the extrahepatic site is subcutaneous. In some embodiments, the extrahepatic site is intramuscular. Methods to formulate a pharmaceutical composition based upon the route of administration are known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 19thEd (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E. Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton Pennsylvania, 1975; Liberman, H.A. and Lachman, L. Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y. 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0480] Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, tissue preservation solution, heparin containing isotonic fluid (Plasma-LyteA, normal saline), CMRL 1066, +50 mL 25% human serum albumin containing heparin, fixed oils, polyethylene ...

Claims

CLAIMS1. A nanoprobe for detecting a target nucleic acid, comprising:(a) a plasmonic-active nanoparticle;(b) a nucleic acid probe comprising a sequence that forms a stem loop, a first end attached to the nanoparticle, and a second end attached to an optical label; and(c) a placeholder sequence comprising a first region complementary to a sequence in the target nucleic acid and a second region complementary to the sequence that forms a stem loop or a portion thereof, wherein the second region is shorter than and overlapping the first region, and wherein the target nucleic acid is a microRNA (miRNA) biomarker for inflammation and / or hypoxia.

2. The nanoprobe of claim 1, wherein the target nucleic acid is a miRNA biomarker for inflammation.

3. The nanoprobe of claim 2, wherein the target nucleic acid is a miRNA biomarker for cytokine-dependent inflammation.

4. The nanoprobe of claim 3, wherein the miRNA biomarker is selected from the group consisting of miR-203b, miR-302a, miR-155, miR-146a, miR-302a, miR-21, and a combination thereof.

5. The nanoprobe of claim 3 or 4, wherein the miRNA biomarker is selected from the group consisting of miR-203b-3p, miR-302a-5p, miR-155-5p, miR-146a-5p, miR-302a-3p, miR-21 -5p, and a combination thereof.

6. The nanoprobe of claim 5, wherein the miRNA biomarker is miR-146a-5p.

7. The nanoprobe of claim 2, wherein the target nucleic acid is a miRNA biomarker for interferon-dependent inflammation.

8. The nanoprobe of claim 7, wherein the miRNA biomarker is selected from the group consisting of miR-224, miR-302b, miR-302a, miR-203a, miR-146b, miR-21, and a combination thereof.

9. The nanoprobe of claim 7 or 8, wherein the miRNA biomarker is selected from the group consisting of miR-224-5p, miR-302b-3p, miR-302a-5p, miR-203a-3p, miR-146b-5p, and a combination thereof.

10. The nanoprobe of claim 9, wherein the miRNA biomarker is miR-224-5p.

11. The nanoprobe of claim 1, wherein the target nucleic acid is a miRNA biomarker for hypoxia.

12. The nanoprobe of claim 11, wherein the miRNA biomarker is a 5 'arm (5p) or a 3 'arm (3p) of a miRNA selected from the group consisting of miR-210, miR-124, miR-1246, miR- 155, miR-205, and a combination thereof.

13. The nanoprobe of claim 12, wherein the miRNA biomarker is selected from the group consisting of miR-210-3p, miR-124-5p, miR-1246-3p, miR-1246-5p, miR-155-5p, miR-205- 5p, and a combination thereof.

14. The nanoprobe of claim 13, wherein the miRNA biomarker is miR-210-3p.

15. The nanoprobe of claim 1, wherein:(a) the miRNA biomarker is miR-146a, wherein the nucleic acid probe comprises SEQ ID NO: 24 or 26 or sequence having at least 80% identity to SEQ ID NO: 24 or 26, and wherein the placeholder sequence comprises SEQ ID NO: 25 or 27 or a sequence having at least 80% identity to SEQ ID NO: 25 or 27;(b) the miRNA biomarker is miR-224, wherein the nucleic acid probe comprises SEQ ID NO: 28 or a sequence having at least 80% identity to SEQ ID NO: 28, and wherein the placeholder sequence comprises SEQ ID NO: 29 or a sequence having at least 80% identity to SEQ ID NO: 29;(c) the miRNA biomarker is miR-210, wherein the nucleic acid probe comprises any one of SEQ ID NOS: 32-35 a sequence having at least 80% identity to SEQ ID NOS: 32- 35, and wherein the placeholder sequence comprises SEQ ID NO: 36 or a sequence having at least 80% identity to SEQ ID NO: 36;(d) the miRNA biomarker is miR-21, wherein the nucleic acid probe comprises SEQ ID NOS: 20 or 22, or a sequence having at least 80% identity to SEQ ID NO: 20 or 22, and wherein the placeholder sequence comprises SEQ ID NO: 21 or 23 or a sequence having at least 80% identity to SEQ ID NO: 21 or 23;(e) the miRNA biomarker is miR-302a, wherein the nucleic acid probe comprises SEQ ID NOS: 30, or a sequence having at least 80% identity to SEQ ID NO: 30, and wherein the placeholder sequence comprises SEQ ID NO: 31 or a sequence having at least 80% identity to SEQ ID NO: 31;(f) the miRNA biomarker is miR-155, wherein the nucleic acid probe comprises any one of SEQ ID NOS: 37-40, or a sequence having at least 80% identity to SEQ ID NO: 37- 40, and wherein the placeholder sequence comprises SEQ ID NO: 41 or a sequence having at least 80% identity to SEQ ID NO: 41; or(g) the miRNA biomarker is miR-124, wherein the nucleic acid probe comprises any one of SEQ ID NOS: 42-45, or a sequence having at least 80% identity to SEQ ID NO: 42- 45, and wherein the placeholder sequence comprises SEQ ID NO: 46 or a sequence having at least 80% identity to SEQ ID NO: 46.

16. The nanoprobe of any one of claims 1-15, further comprising a second optical label operably linked to the nanoparticle.

17. The nanoprobe of claim 16, wherein the optical label emits a signal at a first Raman shift (cm'1), and wherein the second optical label emits a signal at a second, different Raman shift (cm'1).

18. The nanoprobe of claim 17, wherein the signal of the optical label is increased in the presence of the target nucleic acid, and wherein the signal of the second optical label is substantially similar in the presence or absence of the target nucleic acid.

19. A nanoprobe, comprising:(a) a plasmonic-active nanoparticle;(b) a nucleic acid probe comprising (a) a sequence that forms a stem loop, wherein the stem loop has an open and a closed configuration, and wherein the stem loop forms the open configuration in the presence of a second, complementary sequence, (b) a first end attached to the nanoparticle, and (c) a second end attached to an optical label, wherein the optical label emits a signal at a first Raman shift (cm'1), and wherein the signal is increased in the closed configuration relative to the open configuration; and(c) a second optical label operably linked to the nanoparticle, wherein the second optical label emits a signal at a second, different Raman shift (cm'1) that is substantially similar in the open and closed configuration.

20. The nanoprobe of claim 19, wherein the second, complementary sequence comprises a target nucleic acid.

21. The nanoprobe of claim 19, wherein the second, complementary sequence comprises a placeholder sequence, wherein the placeholder sequence comprises a first region of complementary to a sequence in a target nucleic acid and a second region of complementary to the sequence that forms a stem loop or a portion thereof, and wherein the second region is shorter than and overlapping the first region.

22. The nanoprobe of claim 20 or 21, wherein the target nucleic acid comprises a DNA or an RNA.

23. The nanoprobe of claim 22, wherein the RNA comprises a pre-mRNA, and mRNA, or a non-coding RNA.

24. The nanoprobe of claim 23, wherein the non-coding RNA is selected from a miRNA, a small interfering RNA (siRNA), a piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), a small nucleolar RNA, a ribosomal RNA (rRNA), a transfer RNA (tRNA), a small cajal body-specific RNA (scaRNA), and a circular RNA (circRNA).

25. The nanoprobe of any one of claims 20-24, wherein the target nucleic acid is a biomarker of proliferation, cell death, inflammation, hypoxia, nutrient deprivation, differentiation, teratoma formation, metabolic stress, endoplasmic reticulum stress, oxidative stress, and / or activation of a cytokine signaling pathway.

26. The nanoprobe of any one of claims 1-25, wherein the plasmonic-active nanoparticle is a nanosphere, a nanoshell, a nanorod, or a nanostar.

27. The nanoprobe of any one of claims 1-26, wherein the plasmonic-active nanoparticle comprises a single metal or more than one metal, and wherein the metal is selected from the group consisting of gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iron (Fe), copper (Cu), aluminum (Al), and zinc (Zn).

28. The nanoprobe of any one of claims 1-27, wherein the plasmonic-active nanoparticle is a silver nanosphere, gold nanosphere, silver nanoshell, gold nanoshell, silver nanorod, gold nanorod, silver nanostar, or gold nanostar.

29. The nanoprobe of any one of claims 1-28, wherein the plasmonic-active nanoparticle is a gold nanostar.

30. The nanoprobe of any one of claims 1-29, wherein the plasmonic-active nanoparticle is embedded in a silica shell.

31. The nanoprobe of any one of claims 1-30, wherein the plasmonic-active nanoparticle further comprises a component attached to a surface of the nanoparticle.

32. The nanoprobe of claim 31, wherein the component comprises a synthetic polymer, a cell penetrating peptide, a membrane anchor, a bioreceptor, a synthetic peptide, or a combination thereof.

33. The nanoprobe of claim 32, wherein the component comprises the synthetic polymer, and wherein the synthetic polymer is selected from a PEG, a N-isopropylacrylamide (NIP AM), or a combination thereof.

34. The nanoprobe of claim 32, wherein the component comprises the cell penetrating peptide, and wherein the cell penetrating peptide comprises a polycationic peptide, a human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription (TAT) peptide, a HIV- 1 cysteine-terminated TAT (cTAT) peptide, or a combination thereof.

35. The nanoprobe of claim 32, wherein the component comprises the membrane anchor, and wherein the membrane anchor is selected from a cationic polymer, a lipid, cholesterol or a derivative thereof, porphyrin, tocopherol, or a combination thereof.

36. The nanoprobe of claim 35, wherein the cationic polymer comprises poly-lysine.

37. The nanoprobe of claim 32, wherein the component comprises the bioreceptor, and wherein the bioreceptor comprises an antibody or antigen-binding domain thereof.

38. The nanoprobe of any one of claims 1-37, wherein the sequence that forms a stem loop, the placeholder sequence, or both comprise at least one modified nucleoside.

39. The nanoprobe of claim 38, wherein the at least one modified nucleoside comprises a 2'-modification or a locked nucleic acid.

40. The nanoprobe of claim 39, wherein the 2'-modification is selected from 2'-aminoethyl, 2 '-fluoro, 2'-O-methyl, and 2'-O-methoxyethyl.

41. The nanoprobe of any one of claims 1-40, wherein the sequence that forms a stem loop, the placeholder sequence, or both comprise at least one modified intemucleoside linkage.

42. The nanoprobe any one of claims 1-41, wherein the optical label comprises a Raman label, a positively-charged hydrophobic near infrared (NIR) dye, a fluorescence label, or an absorbance label.

43. The nanoprobe of any one of claims 16-42, wherein the second optical label is linked to a surface of the plasmonic active nanoparticle by a linker.

44. The nanoprobe of claim 43, wherein the second optical label comprises a Raman label, a positively-charged hydrophobic NIR dye, a fluorescence label, or an absorbance label.

45. A cell comprising the nanoprobe of any one of claims 1-44.

46. The cell of claim 45, further comprising at least one second nanoprobe for detecting a second target nucleic acid.

47. The cell of claim 45 or 46, wherein the cell is a stem cell-derived cell.

48. The cell of any one of claims 45-47, wherein the cell is an insulin producing cell.

49. A method of monitoring an engineered cell in a subject for inflammation and / or hypoxia, comprising(a) administering the engineered cell to a transplant site of the subject, wherein the engineered cell comprises a nanoprobe of any one of claims 1-18, and(b) detecting an optical signal from the optical label upon exposing the transplant site to electromagnetic radiation, wherein an increase in the optical signal indicates the presence of the mRNA biomarker of inflammation and / or hypoxia, thereby providing for monitoring of the engineered cell.

50. A method of monitoring an engineered cell in a subject for inflammation and / or hypoxia, comprising(a) administering the engineered cell to a transplant site of the subject, wherein the engineered cell comprises a nanoprobe of any one of claims 19-25, and(b) detecting the signal at the first Raman shift and the signal at the second, different Raman shift upon exposing the treatment site to electromagnetic radiation, wherein an increase in a ratio of the signal at the first Raman shift to the signal at the second, different Raman shift indicates the presence of the target nucleic acid, thereby providing for monitoring of the engineered cell.

51. The method of claim 49 or 50, wherein the engineered cell comprises a stem cell- derived cell.

52. The method of any one of claims 49-51, wherein the engineered cell comprises an insulin producing cell.

53. A method of characterizing an engineered cell at a transplant site in a subject, comprising administering the engineered cell to the subject, wherein the engineered cell comprises a nanoprobe comprising(a) a plasmonic-active nanoparticle,(b) a nucleic acid probe comprising a sequence that forms a stem loop, wherein the stem loop has an open and a closed configuration, (b) a first end attached to the nanoparticle, and (c) a second end attached to an optical label, wherein the optical label emits a signal at a first Raman shift (cm'1) that is increased in the closed configuration relative to the open configuration,(c) a placeholder sequence configured to maintain the stem loop in the open configuration in the absence of a target nucleic acid, and(d) a second optical label operably linked to the nanoparticle, wherein the second optical label emits a signal at a second, different Raman shift (cm'1) that is substantially similar in the open and closed configuration, and(e) detecting the signal and the second signal from the transplant site, wherein an increase in the signal as compared to the second signal indicates a presence of the target nucleic acid, thereby characterizing the engineered cell.

54. The method of claim 53, wherein the target nucleic acid is a biomarker of proliferation, cell death, inflammation, hypoxia, nutrient deprivation, differentiation, de-differentiation,teratoma formation, endoplasmic reticulum (ER) stress, metabolic stress, endoplasmic reticulum stress, oxidative stress, and / or activation of a cytokine signaling pathway.

55. The method of claim 53, wherein the target nucleic acid is a biomarker of inflammation or hypoxia.

56. The method of claim 53, wherein the target nucleic acid is a biomarker of teratoma formation and / or proliferation.

57. The method of claim 53, wherein the target nucleic acid is a biomarker of dedifferentiation.

58. The method of claim 53, wherein the target nucleic acid is a biomarker of metabolic stress.

59. The method of claim 53, wherein the target nucleic acid is a biomarker of ER stress.

60. The method of any one of any one of claims 53-55, further comprising administering at least one immunosuppressant to the subject.

61. The method of any one of claims 53, 54 or 56, further comprising illuminating a three- dimensional volume comprising the transplant site, or portion thereof, with a photothermal wavelength.

62. The method of any one of claims 53, 54 and 57-59, further comprising administering a repeat dose of the engineered cell.

63. The method of any one of claims 53, 54, and 59, further comprising administering a therapeutic modulator of ER stress.

64. The method of any one of claims 49-63, wherein the detecting is by surface enhanced Raman spectroscopy (SERS) or surface enhanced resonance Raman spectroscopy (SERRS).

65. The method of any one of claims 49-64, wherein the electromagnetic radiation comprises a wavelength in the NIR region.

66. The method of any one of claims 49-65, wherein the engineered cell comprises at least one additional nanoprobe, and wherein the method comprises detecting one or more signals from the at least one additional nanoprobe.

67. The method of any one of claims 49-66, wherein the transplant site is subcutaneous or intramuscular.

68. The method of any one of claims 49-67, wherein the engineered cell is a stem-cell derived cell.

69. The method of any one of claims 49-68, wherein engineered cell is an insulin producing cell.

70. The method of claim 69, wherein the subject has type I diabetes.

71. A composition, comprising:(a) an insulin producing cell comprising a nanoprobe, and(b) one or more factors selected from a proangiogenic factor, a parathyroid gland (PTG) factor, an anti-inflammatory factor, and a combination thereof.

72. The composition of claim 71, wherein the one or more factors is selected from vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, platelet derived growth factor (PDGF) AA, PDGF-BB, gamma-aminobutyric acid (GABA), leptin, serotonin, parathyroid hormone (PTH), hormone growth factor (HGF), osteopontin, parathyroid hormone-related protein (PTHrP), and a combination thereof.

73. The composition of claim 71 or 72, wherein the one or more factors comprises or consists of VEGF, angiopoietin-1, and HGF.

74. The composition of claim 71 or 72, wherein the one or more factors comprises or consists of osteopontin, PTH, and PTHrP.

75. The composition of any one of claims 71-74, wherein one or more factors are operably linked to a surface of the insulin producing cell.

76. The composition of any one of claims 71-74, wherein composition further comprises a polymer matrix, wherein the insulin producing cell and the one or more factors are encapsulated in the polymer matrix.

77. The composition of any one of claims 71-74, wherein the composition comprises the one or more factors and insulin producing cell in a liquid suspension.

78. The composition of any one of claims 71-77, wherein the insulin producing cell is differentiated from a stem cell.

79. The composition of claim 78, wherein the stem cell is a pluripotent stem cell.

80. The composition of claim 79, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.

81. The composition of any one of claims 78-80, wherein the insulin producing cell is generated from a PDX1+ / NKX6.1+ progenitor cell, and wherein the PDX1+ / NKX6.1+ progenitor cell is generated from the stem cell.

82. A composition, comprising:(a) a population comprising insulin producing cells, wherein a plurality of insulin producing cells of the population comprise a nanoprobe, and wherein the population comprises at least one function of human islets selected from the group consisting of:(b) glucose-responsive secretion of C-peptide;(c) glucose-responsive secretion of insulin;(d) insulin granule biogenesis, trafficking, and / or exocytosis; and(e) a combination of (a)-(d).

83. The composition of claim 82, wherein the at least one function is improved as compared to a control population lacking the nanoprobe.

84. The composition of claim 82 or 83, wherein the at least one function is retained under hypoxic and / or nutrient poor conditions.

85. The composition of any one of claims 82-84, wherein the population comprising insulin producing cells secrete an increased level of insulin in response to an increased level of glucose.

86. The composition of any one of claims 82-85, wherein the population comprising insulin producing cells is characterized by a glucose stimulated insulin secretion (GSIS) response.

87. The composition of claim 86, wherein the GSIS response is in vitro and / or in vivo.

88. The composition of any one of claims 82-87, wherein the insulin producing cell secretes an increased level of C-peptide in response to an increased level of glucose.

89. The composition of any one of claims 82-88, wherein a plurality of insulin producing cells of the population are present in cell clusters.

90. The composition of claim 89, wherein the cell clusters comprise a longest diameter of about 57 pm to 350 pm or about 100 pm to about 200 pm.

91. The composition of claim 89, wherein the cell clusters comprise about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 cells.

92. The composition of any one of claims 82-91, wherein the population comprising insulin producing cells is present in a liquid suspension.

93. The composition of any one of claims 82-92, wherein the population comprising insulin producing cells is encapsulated in a degradable material.

94. The composition of any one of claims 82-93, wherein the population comprising insulin producing cells is differentiated from a population comprising stem cells.

95. The composition of claim 94, wherein the stem cells are pluripotent stem cells.

96. The composition of claim 95, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

97. The composition of any one of claims 94-96, wherein the population comprising insulin producing cells is generated from PDX1+ / NKX6.1+ progenitor cells, and wherein the PDX1+ / NKX6.1+ progenitor cells are generated from the population comprising stem cells.

98. The composition of any one of claims 82-97, wherein the nanoprobe comprises a plasmonic active nanoparticle and a component attached to the surface thereof.

99. The composition of claim 98, wherein the plasmonic-active nanoparticle is a nanosphere, a nanoshell, a nanorod, or a nanostar.

100. The composition of claim 98 or 99, wherein the plasmonic-active nanoparticle comprises a single metal or more than one metal, and wherein the metal is selected from thegroup consisting of gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iron (Fe), copper (Cu), aluminum (Al), and zinc (Zn).

101. The composition of any one of claims 98-100, wherein the plasmonic-active nanoparticle is a silver nanosphere, gold nanosphere, silver nanoshell, gold nanoshell, silver nanorod, gold nanorod, silver nanostar, or gold nanostar.

102. The composition of any one of claims 98-100, wherein the plasmonic-active nanoparticle is a gold nanostar.

103. The composition of any one of claims 98-102, wherein the component comprises a synthetic polymer, a cell penetrating peptide, a membrane anchor, a bioreceptor, a nucleic acid, an optical reporter, or a combination thereof.

104. The composition of claim 103, wherein the component comprises the synthetic polymer, and wherein the synthetic polymer is selected from a PEG, a polyacrylamide (PAM), a poly(N-isopropylacrylamide) (pNIPAM), or a combination thereof.

105. The composition of claim 103, wherein the component comprises the cell penetrating peptide, and wherein the cell penetrating peptide comprises a polycationic peptide, a human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription (TAT) peptide, a HIV- 1 cysteine-terminated TAT (cTAT) peptide, or a combination thereof.

106. The composition of claim 103, wherein the component comprises the membrane anchor, and wherein the membrane anchor is selected from a cationic polymer, a lipid, cholesterol or a derivative thereof, porphyrin, tocopherol, or a combination thereof.

107. The composition of claim 106, wherein the cationic polymer comprises poly-lysine.

108. The composition of claim 103, wherein the component comprises the bioreceptor, and wherein the bioreceptor comprises and antibody or antigen-binding domain thereof.

109. The composition of claim 103, wherein the component comprises the optical reporter.

110. The composition of claim 103, wherein the component comprises the nucleic acid, optionally wherein the nucleic acid comprises a first end attached to the surface of the plasmonic-acid nanoparticle and a second end attached to an optical reporter.

111. The composition of any one of claims 98-110, wherein the plasmonic-active nanoparticle is embedded in a silica shell.

112. A method for improving performance of a population comprising insulin producing cells, comprising(a) contacting a population comprising dissociated insulin producing cells with a nanoprobe to induce uptake, and(b) reaggregating to form a population comprising nanoprobe-loaded insulin producing cells, wherein the population comprising nanoprobe-loaded insulin producing cells is characterized by at least one function of human islets selected from the group consisting of:(i) glucose-responsive secretion of C-peptide;(ii) glucose-responsive secretion of insulin;(iii) insulin granule biogenesis, trafficking, and / or exocytosis; and(iv) a combination of (i)-(iii); and wherein the at least one function is improved relative to a control population comprising insulin producing cells not contacted with the nanoprobe.

113. The method of claim 112, wherein the at least one function is retained under hypoxic and / or nutrient poor conditions.

114. The method of claim 112 or 113, wherein the insulin producing cell secretes an increased level of insulin in response to an increased level of glucose.

115. The method of any one of claims 112-114, wherein the population comprising insulin producing cells is characterized by a glucose stimulated insulin secretion (GSIS) response.

116. The method of claim 115, wherein the GSIS response is in vitro and / or in vivo.

117. The method of any one of claims 112-116, wherein the insulin producing cell secretes an increased level of C-peptide in response to an increased level of glucose.

118. The method of any one of claims 112-117, wherein the population comprising nanoprobe-loaded insulin producing cells are present in cell clusters following the reaggregating.

119. The method of claim 118, wherein the cell clusters comprise a longest diameter of about 50 pm to 350 pm.

120. The method of claim 118, wherein the cell clusters comprise a longest diameter of about 100 pm to about 200 pm.

121. The method of any one of claims 118-120, wherein the cell clusters comprise about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 cells.

122. The method of any one of claims 112-121, wherein the population comprising insulin producing cells are present in a liquid suspension.

123. The method of any one of claims 112-121, wherein the population comprising insulin producing cells are encapsulated in a degradable material.

124. The method of any one of claims 112-123, wherein the population comprises stem cell- derived islets.

125. The method of any one of claims 112-124, wherein the population comprises primary donor islets.

126. A method of controlling stem cell-derived cells at a transplant site in a subject, comprising(a) administering to the subject a population comprising stem cell-derived cells, wherein a plurality of stem cell-derived cells comprise a nanoprobe configured to emit an optical signal upon electromagnetic excitation, and(b) measuring the optical signal across a three-dimensional volume comprising the transplant site at a first time point and at least one additional time point using spectroscopy.

127. The method of claim 126, wherein the spectroscopy comprises SERS or SERRS.

128. The method of claim 126 or 127, wherein the spectroscopy comprises illuminating the three-dimensional volume with electromagnetic excitation, wherein the electromagnetic excitation comprises a wavelength in the NIR region.

129. The method of claim 128, wherein the wavelength is about 650 nm to about 900 nm.

130. The method of any one of claims 126-129, wherein the spectroscopy is performed with a laser configured to produce the wavelength, optionally a continuous wave laser.

131. The method of claim 130, wherein the laser produces the wavelength at a laser power of about 5 mW to about 20 mW.

132. The method of claim 130 or 131, wherein the laser produces the wavelength with a power density of about 5 mW / cm2to about 100 mW / cm2.

133. The method of claim 130 or 131, wherein the spectroscopy is performed with an acquisition time of about 1 second to about 20 seconds.

134. The method of any one of claims 126-133, wherein the wavelength penetrates at a tissue depth of about 1 mm to about 20 mm.

135. The method of any one of claims 126-134, wherein the first time point is immediately following the administering or within about 1-2 weeks following the administering.

136. The method of any one of claims 126-135, wherein the at least one additional time point is about is about 1 day to about 30 days following the first time point.

137. The method of any one of claims 126-135, wherein the at least one additional time point is a cycle of once per every about 1 to about 180 days following the first time point.

138. The method of any one of claims 126-135, wherein the at least one additional time point is a cycle of once per every about 5 minutes to about 24 hours following the first time point.

139. The method of any one of claims 126-138, further comprising measuring a predetermined change in the optical signal at the first time point as compared to the at least one additional time point.

140. The method of claim 139, wherein the predetermined change is determined by a machine learning algorithm trained on a dataset of transplant images.

141. The method of claim 139 or 140, wherein the predetermined change is a marker of teratoma formation or proliferation.

142. The method of any one of claims 139-141, further comprising illuminating the three- dimensional volume, or a portion thereof, with a photothermal wavelength upon measuring the predetermined change.

143. The method of claim 142, wherein the photothermal wavelength induces thermal damage to the population comprising stem cell-derived cells.

144. The method of claim 142 or 143, wherein the photothermal wavelength for inducing thermal damage is about 650 nm to about 900 nm.

145. The method of any one of claims 142-144, wherein the illuminating is performed with a laser configured to produce the photothermal wavelength for inducing thermal damage, optionally wherein the laser is a continuous wave laser.

146. The method of claim 145, wherein the laser produces the photothermal wavelength for inducing thermal damage at a laser power of about 20 mW to about 500 mW.

147. The method of claim 145 or 146, wherein the laser produces the photothermal wavelength for inducing thermal damage at a power density of about 0.1 W / cm2to about 2 W / cm2.

148. The method of any one of claims 142-147, wherein the illuminating is performed with an irradiation time of about 30 seconds to about 30 minutes.

149. The method of any one of claims 142-148, wherein the photothermal wavelength for inducing thermal damage penetrates at a tissue depth of about 1 mm to about 20 mm.

150. A method of treating a disease in a subject, comprising administering to an extrahepatic site of the subject a composition comprising an insulin producing cell, wherein the insulin producing cell comprises a nanoprobe, and wherein the insulin producing cell is characterized by reduced sensitivity to immune killing.

151. The method of claim 150, wherein prior to the administration, the subject received a daily infusion of insulin.

152. The method of claim 150 or 151, wherein prior to the administration, the subject has a medical history of severe hypoglycemic events.

153. The method of any one of claims 150-152, wherein prior to the administration, the subject is characterized by no residual endogenous islet cell function.

154. The method of any one of claims 150-153, wherein prior to the administration, the subject has undetectable blood C-peptide level when measured using a mixed meal tolerance test.

155. The method of any one of claims 150-154, wherein the disease is characterized by high blood sugar levels for a prolonged period of time.

156. The method of any one of claims 150-155, wherein the disease is diabetes.

157. The method of any one of claims 150-156, wherein the disease is Type 1 diabetes.

158. The method of any one of claims 150-156, wherein the disease is Type 2 diabetes.

159. The method of any one of claims 150-158, wherein the extrahepatic site is subcutaneous or intramuscular.

160. The method of any one of claims 150-159, wherein immune killing comprises an antibeta cell immune response.

161. The method of any one of claims 150-160, wherein the immune killing comprises a host versus graft immune response.

162. The method of any one of claims 150-161, wherein the insulin producing cell comprises a gene modification, and wherein the insulin producing cell is characterized by reduced sensitivity to immune killing as compared to a control cell lacking the gene modification.

163. The method of claim 162, wherein the gene modification comprises a disruption, inactivation, and / or loss of function of one or more genes.

164. The method of claim 162 or 163, wherein the one or more genes encodes a major histocompatibility complex (MHC) class I molecule and / or a regulator of transcription of an MHC class I molecule.

165. The method of claim 164, wherein the MHC class I molecule comprises a human leukocyte antigen (HLA) class I molecule.

166. The method of claim 165, wherein the HLA class I molecule is selected from the group consisting of beta-2 microglobulin (B2M), HLA-A, HLA-B, HLA-C, and a combination thereof.

167. The method of any one of claims 164-166, wherein the regulator of transcription of the MHC class I molecule is selected from the group consisting of RFX5, RFXAP, RFXANK, X2BP, NFY, NFY-A, NFY-B, NFY-C, IRF1, NLRC5, and a combination thereof.

168. The method of any one of claims 162-167, wherein the one or more genes encodes a MHC class II molecule and / or a regulator of transcription of an MHC class II molecule.

169. The method of any one of claims 162-168, wherein the MHC class II molecule comprises an HLA class II molecule.

170. The method of claim 169, wherein the HLA class II molecule is selected from the group consisting of HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, and a combination thereof.

171. The method of any one of claims 168-170, wherein the regulator of transcription of the MHC class II molecule is CIITA.

172. The method of any one of claims 162-171, wherein the one or more genes encodes a survival factor selected from the group consisting of: thioredoxin interacting protein (TXNTP);zinc finger protein 179 (ZNF143); forkhead box, class O (FOXOI); c-Jun N-terminal kinase (JNK) and a combination thereof.

173. The method of any one of claims 162-172, wherein the one or more genes encodes tet methylcytosine dioxygenase 2 (Tet2).

174. The method of any one of claims 162-173, wherein the gene modification comprises an insertion and / or amplification of one or more genes.

175. The method of claim 174, wherein the one or more genes encodes a protein selected from the group consisting of CD47; programmed death-ligand 1 (PDL1); A20; HLA-E; HLA- G; CTLA-4; mesencephalic astrocyte derived neurotrophic factor (MANF); and a combination thereof.

176. The method of any one of claims 150-175, wherein the composition comprises one or more tolerogenic factors.

177. The method of any one of claims 150-176, wherein the composition comprises a proangiogenic factor, a parathyroid gland (PTG) factor, an anti-inflammatory factor, or a combination thereof.

178. The method of claim 177, wherein the one or more factors is selected from vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, platelet derived growth factor (PDGF) AA, PDGF-BB, gamma-aminobutyric acid (GABA), leptin, serotonin, parathyroid hormone (PTH), hormone growth factor (HGF), osteopontin, parathyroid hormone-related protein (PTHrP), and a combination thereof.

179. The method of claim 177 or 178, wherein the one or more factors comprises or consists of VEGF, angiopoietin-1, and HGF.

180. The method of claim 177 or 178, wherein the one or more factors comprises or consists of osteopontin, PTH, and PTHrP.

181. The method of any one of claims 177-180, wherein one or more factors are operably linked to a surface of the insulin producing cell.

182. The method of any one of claims 177-180, wherein composition further comprises a polymer matrix, wherein the insulin producing cell and the one or more factors are encapsulated in the polymer matrix.

183. The method of any one of claims 177-180, wherein the composition comprises the one or more factors and insulin producing cell in a liquid suspension.

184. The method of any one of claims 177-183, wherein the insulin producing cell is differentiated from a stem cell.

185. The method of claim 184, wherein the stem cell is a pluripotent stem cell.

186. The method of claim 185, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.

187. The method of any one of claims 184-186, wherein the insulin producing cell is generated from a PDX1+ / NKX6.1+ progenitor cell, and wherein the PDX1+ / NKX6.1+ progenitor cell is generated from the stem cell.

188. The method of any one of claims 112-187, wherein the nanoprobe comprises a plasmonic active nanoparticle and a component attached to the surface thereof.

189. The method of claim 188, wherein the plasmonic-active nanoparticle is a nanosphere, a nanoshell, a nanorod, or a nanostar.

190. The method of claim 188 or 189, wherein the plasmonic-active nanoparticle comprises a single metal or more than one metal, and wherein the metal is selected from the groupconsisting of gold (Au), silver (Ag), platinum (Pt), palladium (Pd), iron (Fe), copper (Cu), aluminum (Al), and zinc (Zn).

191. The method of any one of claims 188-190, wherein the plasmonic-active nanoparticle is a silver nanosphere, gold nanosphere, silver nanoshell, gold nanoshell, silver nanorod, gold nanorod, silver nanostar, or gold nanostar.

192. The method of any one of claims 188-190, wherein the plasmonic-active nanoparticle is a gold nanostar.

193. The method of any one of claims 188-192, wherein the component comprises a synthetic polymer, a cell penetrating peptide, a membrane anchor, a bioreceptor, a nucleic acid, an optical reporter, or a combination thereof.

194. The method of claim 193, wherein the component comprises the synthetic polymer, and wherein the synthetic polymer is selected from a PEG, a N-isopropyl acrylamide (NIP AM), or a combination thereof.

195. The method of claim 193, wherein the component comprises the cell penetrating peptide, and wherein the cell penetrating peptide comprises a polycationic peptide, a human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription (TAT) peptide, a HIV- 1 cysteine-terminated TAT (cTAT) peptide, or a combination thereof.

196. The method of claim 193, wherein the component comprises the membrane anchor, and wherein the membrane anchor is selected from a cationic polymer, a lipid, cholesterol or a derivative thereof, porphyrin, tocopherol, or a combination thereof.

197. The method of claim 196, wherein the cationic polymer comprises poly -lysine.

198. The method of claim 193, wherein the component comprises the bioreceptor, and wherein the bioreceptor comprises and antibody or antigen-binding domain thereof.

199. The method of claim 193, wherein the component comprises the optical reporter.

200. The method of claim 193, wherein the component comprises the nucleic acid, optionally wherein the nucleic acid comprises a first end attached to the surface of the plasmonic-acid nanoparticle and a second end attached to an optical reporter.

201. The method of any one of claims 188-200, wherein the plasmonic-active nanoparticle is embedded in a silica shell.

202. A pharmaceutical composition comprising the nanoprobe of any one of claims 1-44, and a pharmaceutically acceptable carrier.

203. A kit comprising the nanoprobe of any one of claims 1-44, or the pharmaceutical composition of claim 202, and instructions for contacting a cell with the nanoprobe or the pharmaceutical composition.

204. A pharmaceutical composition comprising the cell of any one of claims 45-48, and a pharmaceutically acceptable carrier.

205. A kit comprising the cell of any one of claims 45-48, or the pharmaceutical composition of claim 204, and instructions for monitoring the cell following administration of the cell or the pharmaceutical composition to a subject.

206. A pharmaceutical composition comprising the composition of any one of claims 71- 111, and a pharmaceutically acceptable carrier.

207. A kit comprising the composition of any one of claims 71-111, or the pharmaceutical composition of claim 206, and instructions for administering the composition or the pharmaceutical composition to a subject.

208. Use of a composition comprising an insulin producing cell for treating a disease or disorder in a subject, wherein the insulin producing cell comprises a nanoprobe, and wherein the insulin producing cell is characterized by reduced sensitivity to immune killing.

209. Use of a composition comprising an insulin producing cell in the manufacture of a medicament for treating a disease or disorder in a subject, wherein the insulin producing cell comprises a nanoprobe, and wherein the insulin producing cell is characterized by reduced sensitivity to immune killing.

210. The use of claim 208 or 209, wherein the disease is diabetes.

211. The use of any one of claims 208 or 209, wherein the disease is Type 1 diabetes.

212. The use of any one of claims 208 or 209, wherein the disease is Type 2 diabetes.

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