Precursory regulatory cytotrophoblast cells and uses thereof

Abstract

To provide stem cell therapies for treating (curing) a variety of diseases or conditions, as an alternative to overcome certain shortcomings of existing embryonic stem cells and iPS cells.SOLUTION: Provided herein are: precursory regulatory cytotrophoblast cells produced in vitro and expressing specific cell surface proteins and combinations thereof; compositions thereof; and methods of treating disorders (e.g., cancers, inflammations, or autoimmune diseases) or conditions (e.g., skin conditions) by using the cells and compositions.SELECTED DRAWING: None

Description

[Technical field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 843,925, filed May 6, 2019, which is incorporated herein by reference in its entirety. [Background technology]

[0002]

[0002] There is a need for novel stem cell therapies for treating various diseases or conditions as alternatives that overcome certain drawbacks of current embryonic stem cells and iPS cells. Incorporation by Reference

[0003] All publications, patents, and patent applications herein are incorporated by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term in this specification and a term in an incorporated reference, the term in this specification shall control. Summary of the Invention [Means for solving the problem]

[0003]

[0004] The embodiments of the invention provided in this Summary are for illustrative purposes only and are meant to provide an overview of selective embodiments disclosed herein. The Summary of the Invention is illustrative and selective, does not limit the scope of any claims, does not provide the entire scope of the embodiments of the invention disclosed or contemplated herein, and should not be construed as limiting or restricting the scope of the disclosed or claimed embodiments of the invention.

[0004]

[0005] Disclosed herein in some embodiments is an isolated progenitor regulatory cytotrophoblast (prCTB), (i) the prCTB is capable of expressing the following receptors: beta-hormone human chorionic gonadotropin (β-hCG), human leukocyte antigen G (HLA-G), CD56, insulin, heat shock protein 90 (HSP90), CD4, CD16, CD56, CD107a, CD8, interleukin 15 (IL-15), leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), T cell receptor (TCR), killer cell immunoglobulin-like receptor 2 (DL4) (KCR), and / or IL-16. (ii) the prCTB expresses p53, Ki67, glutamate decarboxylase (GAD65), heat shock protein 70 (HSP70), soluble CD40-ligand (sCD40L), B-cell leukemia / lymphoma 2-associated protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), or any combination thereof. In some examples, the prCTB expresses CD4, CD16, CD56, CD107a, CD8, or any combination thereof.

[0005]

[0006] Disclosed herein in some embodiments is an isolated population of cells comprising progenitor regulatory cytotrophoblasts (prCTB), (i) the population of cells is a subset of the population of cells that is capable of expressing a specific marker of beta-hormone human chorionic gonadotropin (β-hCG), human leukocyte antigen G (HLA-G), CD56, insulin, heat shock protein 90 (HSP90), CD4, CD16, CD56, CD107a, CD8, interleukin 15 (IL-15), leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), T cell receptor (TCR), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), programmed death-ligand 1 (PD-L1), and / or IL-16. L1), apoptosis signaling receptor (Fas), Fas ligand (FasL), CD335 (NKp46), CD11b, CD49f, CD3, CD19, CD34, or any combination thereof, and (ii) the population of cells express p53, Ki67, glutamate decarboxylase (GAD65), heat shock protein 70 (HSP70), soluble CD40-ligand (sCD40L), B-cell leukemia / lymphoma 2-associated protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), or any combination thereof.

[0006]

[0007] In some examples, at least about 10% of the population is prCTB that expresses CD16 and CD56. In some examples, at least about 2% of the population is prCTB that expresses CD4. In some examples, at least about 2% of the population is prCTB that expresses CD8. In some examples, at least about 5% of the population is prCTB that expresses CD107.

[0007]

[0008] Disclosed herein in some embodiments is an isolated population of cells comprising progenitor regulatory cytotrophoblasts (prCTB), wherein (i) at least about 10% of the population are prCTB that express CD16 and CD56, (ii) at least about 2% of the population are prCTB that express CD4, (iii) at least about 2% of the population are prCTB that express CD8, or (iv) at least about 5% of the population are prCTB that express CD107, or any combination thereof.

[0008]

[0009] In some examples, (i) at least about 10% of the population is prCTB expressing CD16 and CD56, (ii) at least about 2% of the population is prCTB expressing CD4, (iii) at least about 2% of the population is prCTB expressing CD8, and (iv) at least about 5% of the population is prCTB expressing CD107. In some examples, the population of cells includes a population in which at least about 2% of the population is prCTB expressing CD16, CD56, and CD107. In some examples, the prCDB or prCDBs express interleukin 15 (IL-15). In some examples, the prCTB or prCTBs express leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), T cell receptor (TCR), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), programmed death-ligand 1 (PD-L1), apoptosis signaling receptor (Fas), Fas ligand (FasL), CD335 (NKp46), B cell leukemia / lymphoma 2 associated protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), or any combination thereof. In some examples, the prCTB or prCTBs further express beta-hormone human chorionic gonadotropin (β-hCG), soluble human leukocyte antigen G (sHLA-G), transforming growth factor β1 (TGF-β1), plasminogen activator inhibitor-1 (PAI-1), interleukin 10 (IL-10), CD105, CD146, or any combination thereof. In some examples, the prCTB or prCTBs lack expression of syncytin, programmed cell death protein 1 (PD-1), or a combination thereof. In some examples, the prCTB or prCTBs secrete a chemokine, a cytokine, a growth factor, or any combination thereof, or an exosome carrying a chemokine, a cytokine, a growth factor, or any combination thereof.In some instances, the cytokine is chemokine (C-C motif) ligand 5 (CCL5), monocyte chemoattractant protein-1 (MCP-1), monocyte chemoattractant protein-1 (MCP-3), chemokine (C-X-C motif) ligand 1 (CCL1), chemokine (C-X-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 11 (CCL11), chemokine (C-C motif) ligand 24 (CCL24), chemokine (C-C motif) ligand 26 (C-C motif) ligand 27 (C-C motif) ligand 29 (C-C motif) ligand 30 (C-C motif) ligand 31 (C-C motif) ligand 32 (C-C motif) ligand 33 (C-C motif) ligand 34 (C-C motif) ligand 35 (C-C motif) ligand 36 (C-C motif) ligand 37 (C-C motif) ligand 38 (C-C motif) ligand 39 (C-C motif) ligand 40 (C-C motif) ligand 41 (C-C motif) ligand 42 (C-C motif) ligand 43 (C-C motif) ligand 44 (C-C motif) ligand 45 (C-C motif) ligand 46 (C-C motif) ligand 47 (C-C motif) ligand 48 (C-C motif) ligand 49 (C-C motif) ligand 50 (C-C motif) ligand 51 (C-C motif) ligand 52 (C-C motif) ligand 53 (C-C motif) ligand 54 (C-C motif) ligand 55 (C-C motif) ligand 56 (C-C motif) ligand 57 (C-C motif) ligand 59 (C-C motif) ligand 60 (C-C motif) ligand 61 (C-C motif) ligand 62 (C-C motif) ligand 63 (C-C motif) CL26), chemokine (C-C motif) ligand 22 (CCL22), chemokine (C-X-C motif) ligand 10 (CCL10), fractalkine, and chemokine (C-C motif) ligand 4 (CCL4), or any combination thereof. In some examples, the cytokine comprises interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), interleukin (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12p40 (IL-12p40), interleukin 13 (IL-13), interleukin 15 (IL-15), or any combination thereof. In some examples, the cytokine comprises interferon alpha (IFN-α) or interferon gamma (IFN-γ). In some examples, the growth factor comprises platelet-derived growth factor homodimer AA (PDGF-AA), PDGF homodimer BB (PDGF-BB), PDGF heterodimer (PDGF-AB), vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF), fibroblast growth factor (FGF) family proteins, FMS-like tyrosine kinase 3 ligand (Flt3L), soluble CD40 ligand (sCD40L), tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1β), or any combination thereof. In some examples, the prCTB or prCTBs have a higher level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN as measured by immunoblotting than the progenitor cell from which the isolated prCTB was differentiated in vitro. In some examples, the prCTB has at least about 1.1, 1.2, 1.5, 1.5, 2, 2.2, 2.5, 2.8, 3, 3.5, 4, 5, 8, 10 fold higher level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN as measured by immunoblotting than the progenitor cell from which the isolated prCTB was differentiated in vitro.In some examples, the prCTB expresses at least about 1.1, 1.2, 1.5, 1.5, 2, 2.2, 2.5, 2.8, 3, 3.5, 4, 5, 8, 10 times higher levels of SOX2 protein as measured by immunoblotting than the progenitor cells from which the isolated prCTB was differentiated in vitro. In some examples, the isolated prCTB is differentiated in vitro from progenitor cells derived from chorionic villi that lack expression of glutamate decarboxylase (GAD65), Ki67, heat shock protein 70 (HSP70), p53, soluble CD40-ligand (sCD40L), or any combination thereof. In some examples, the isolated prCTB is differentiated in vitro from progenitor cells derived from chorionic villi, and both the progenitor cells derived from chorionic villi and the isolated prCTB express heat shock protein 90 (HSP90). In some examples, the isolated prCTB is a human cell. In some examples, the isolated prCTB is of rodent, rabbit, bovine, ovine, porcine, canine, feline, monkey, or ape origin. In some examples, the isolated prCTB is genetically engineered. In some examples, the isolated prCTB comprises an exogenous polynucleotide encoding a cell receptor, an immune checkpoint protein, a cytokine, or any combination thereof. In some examples, the isolated prCTB comprises an exogenous polynucleotide encoding a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof.

[0009]

[0010] Disclosed herein in some embodiments is a pharmaceutical composition comprising a pharma- ceutically acceptable excipient or carrier and a prCTB or population of cells described herein.

[0011] Disclosed herein in some embodiments is a method of treating a disease or condition comprising administering to a subject in need of treatment of the disease or condition a prCTB or population of cells described herein.

[0010]

[0012] In some examples, the method kills antigen-bearing target cells. In some examples, the antigen-bearing cells are not antigen-presenting cells, e.g., not dendritic cells, macrophages, or B cells. In some examples, the antigen-bearing target cells are cancer cells. In some examples, the cancer cells include bladder cancer cells, bone cancer cells, brain cancer cells, breast cancer cells, cervical carcinoma, colorectal cancer cells, esophageal cancer cells, gastrointestinal cancer cells, hematopoietic malignancies, head and neck squamous cell carcinoma, leukemia, liver cancer cells, lung cancer cells, lymphoma, myeloma, nasal cancer cells, nasopharyngeal cancer cells, oral cancer cells, oropharyngeal cancer cells, ovarian cancer cells, prostate cancer cells, sarcoma, gastric cancer cells, melanoma, thyroid cancer cells, or any combination thereof. In some examples, the antigen-bearing cells are pathogens. In some examples, the pathogens include viruses, bacteria, protozoa, prions, fungi, or any combination thereof. In some examples, the method kills at least about 5%, at least about 10%, at least about 20%, at least about 50%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100% of a population of antigen-bearing target cells. In some examples, the method downregulates an inflammatory pathway. In some examples, the method is used to treat a variety of conditions, including transplant rejection, infection, endotoxin shock associated with infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic onset juvenile idiopathic arthritis (JIA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), asthma, pelvic inflammatory disease, Alzheimer's disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, ankylosing spondylitis, dermatomyositis, uveitis. In some examples, the method treats diseases or conditions including inflammatory bowel disease, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, vasculitis, post-surgical adhesions, stroke, type I diabetes, Lyme arthritis, meningoencephalitis, immune-mediated inflammatory disorders of the central and peripheral nervous system, pancreatitis, post-surgical disorders, graft versus host disease, cardiac disease, bone resorption, burn patients, myocardial infarction, Paget's disease, osteoporosis, sepsis, hepatic or pulmonary fibrosis, periodontitis, or hypochlorhydria. In some examples, the method treats an autoimmune disease.In some examples, the method treats type I diabetes, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, polymyositis, chronic active hepatitis, mixed connective tissue disease, primary biliary cirrhosis, pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease, vitiligo, gluten-sensitive enteropathy, Graves' disease, myasthenia gravis, autoimmune neutropenia, idiopathic thrombocytopenic purpura, rheumatoid arthritis, liver cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's disease, bullous pemphigoid, discoid lupus, ulcerative colitis, dense deposit disease, inflammatory bowel disease, or psoriasis.In some examples, the method treats type 1 diabetes.In some examples, the method improves transplant rejection.

[0011]

[0013] Disclosed herein are compositions comprising secretomes, including exosomes, where the secretomes comprise or the exosomes carry chemokines, cytokines, such as interleukins, growth factors, or any combination thereof, and pharma- ceutically or cosmetically acceptable excipients, and the compositions do not comprise cells.

[0012]

[0014] In some examples, the secretome comprises or the exosome carries (i) chemokines including CXCL2, MCP-1, fractalkine, IP-10, MCP-3, eotaxin, MIP-1β, or any combination thereof, (ii) interleukins including IL-6, IL-8, IL-4, IL-1RA, IL-10, IL-12P40, IL-15, IL-1α, IL-17A, or any combination thereof, and (iii) growth factors including PDGF-AA, VEGF, bFGF, G-CSF, Flt-3L, GM-CSF, or any combination thereof. In some examples, the composition comprises MCP-1 and one, two, three, or all of CXCL2, IL-6, IL-8, and VEGF proteins. In some examples, the MCP-1 and CXCL2 in the composition have a weight ratio of about 1:1 to about 2:1. In some examples, the MCP-1 and CXCL2 in the composition have a weight ratio of about 3:1 to about 4:1. In some examples, the MCP-1 and IL-6 in the composition have a weight ratio of about 2:1 to about 3:1. In some examples, the MCP-1 and IL-6 in the composition have a weight ratio of about 3:1 to about 4:1. In some examples, MCP-1 and IL-8 in the composition have a weight ratio of about 4:1 to about 6:1. In some examples, MCP-1 and VEGF in the composition have a weight ratio of about 4:1 to about 6:1. In some examples, MCP-1 and VEGF in the composition have a weight ratio of about 7:1 to about 9:1. In some examples, the composition further comprises PDGF-AA, and MCP-1 and PDGF-AA are present in a weight ratio of about 3:1 to about 5:1. In some examples, the composition further comprises PDGF-AA, and MCP-1 and PDGF-AA are present in a weight ratio of about 6:1 to about 9:1. In some examples, the composition further comprises PDGF-AA and G-CSF. In some examples, the composition further comprises PDGF-AA and FGF-2 (bFGF). In some examples, the composition further comprises one or more proteins: IP-10, Eotaxin, Flt-3L, GM-CSF, MIP-1a, MIP-1b, IL-1a, IL-1RA, IL-4, IL-7, IL-10, IL-12P40, IL-13, IL-15, IL-17A, CCL5 (RANTES), MDC, MCP-3, IL-12P70, IFNa, IFNr, PDGF-AB / BB, or EGF.

[0013]

[0015] Disclosed herein in some aspects is a method of regulating skin condition, comprising administering to a subject in need thereof a composition described herein.

[0014]

[0016] In some examples, the method treats a disease or provides a cosmetic application. In some examples, the method tightens the skin. In some examples, the method hydrates the skin. In some examples, the method rejuvenates the skin. In some examples, the composition does not include stem cells. In some examples, the composition is administered to the subject topically, subcutaneously, transdermally, intramuscularly, or intratumorally. In some examples, the composition is in the dosage form of a lotion, cream, liquid, gel, emulsion, suspension, paste, stick, aerosol, foam, patch, powder, ointment, beads, mask, pad, sheet, wound dressing, bandage, or any combination thereof. In some examples, the subject is a mammal. In some examples, the subject is a primate. In some examples, the subject is a human.

[0015]

[0017] Disclosed herein in some embodiments is a method for obtaining progenitor regulatory cytotrophoblasts (prCTBs), comprising differentiating pluripotent stem cells in vitro by contacting the stem cells with a culture medium comprising a fibroblast growth factor and a nucleoside, L-glutamine, a dipeptide containing L-glutamine, a platelet lysate, or a combination thereof.

[0016]

[0018] In some examples, the culture medium comprises a nucleoside, a dipeptide, and a platelet lysate. In some examples, the culture medium comprises about 2 mM to about 200 mM L-glutamine. In some examples, the contacting step lasts for about 24 hours to about 48 hours. In some examples, the stem cells are progenitor cells derived from chorionic villi. In some examples, the method includes culturing the stem cells with the culture medium before contacting the stem cells with a fibroblast growth factor to differentiate into prCTB. In some examples, the culture medium does not contain an antibiotic. In some examples, the antibiotic is penicillin, streptomycin, or any combination thereof. In some examples, the culture medium does not contain retinoic acid. In some examples, the culture medium does not contain mercaptoethanol, nicotinamide, or any combination thereof. In some examples, the culture medium does not contain dexamethasone, recombinant human oncostatin M, BMP4, HGF, or any combination thereof. In some examples, the culture medium does not contain animal components. In some examples, the culture medium does not contain components derived from humans. In some examples, the culture medium does not contain serum. In some examples, the culture medium does not contain fetal bovine serum. In some examples, the fibroblast growth factor is basic fibroblast growth factor (bFGF).

[0017]

[0019] In some embodiments, the isolated precursor regulatory cytotrophoblast cells ( The present invention relates to a method for the treatment of cancer, comprising administering to a patient a therapeutically effective amount of prCTB (prCTB), the method comprising administering to a patient a therapeutically effective amount of prCTB, the method comprising administering to a patient a therapeutically effective amount of prCTB, the method comprising administering to a patient a therapeutically effective amount of prCTB, the method comprising administering to a patient a therapeutically effective amount of prCTB, the method comprising administering to a patient a therapeutically effective amount of prCTB, the method comprising administering to a patient a therapeutically effective amount of prCTB, the method comprising administering to a patient a therapeutically effective amount of prCTB, the method comprising administering to a patient a therapeutically effective amount of prCTB (ii) the prCTB expresses p53, Ki67, glutamate decarboxylase (GAD65), heat shock protein 70 (HSP70), soluble CD40-ligand (sCD40L), B-cell leukemia / lymphoma 2-associated protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), or any combination thereof. In some examples, the prCTB kills cancer cells or pathogens. In some examples, the prCTB induces apoptosis of cancer cells, for example, by infiltrating a colony of cancer cells, and optionally the prCTB itself does not undergo apoptosis upon contact with the cancer cells. In some examples, the cancer cells are solid tumor cells. In some examples, the cancer cells are pancreatic cancer cells, breast cancer cells, liver tumor cells, ovarian tumor cells, lung tumor cells, gastric tumor cells, melanoma cells, or any combination thereof. In some examples, the prCTB downregulates inflammatory pathways. In some examples, the prCTB expresses CD4, CD16, CD56, CD107a, CD8, or any combination thereof. In some examples, the prCTB lacks syncytin, programmed cell death protein 1 (PD-1), or any combination thereof.In some instances, prCTBs are expressed as interleukin 15 (IL-15), leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), T cell receptor (TCR), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), programmed death-ligand 1 (PD-L1), apoptosis signaling receptor (Fas), Fas ligand (FasL), CD335 (NKp46), B-cell leukemia / lymphoma 2-associated protein (B-LCP) and / or B-cell leukemia / lymphoma 2-associated protein (B-LCP). A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), beta-hormone human chorionic gonadotropin (β-hCG), soluble human leukocyte antigen G (sHLA-G), transforming growth factor beta 1 (TGF-β1), plasminogen activator inhibitor-1 (PAI-1), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10), CD105, CD146, or any combination thereof. In some examples, the prCTB is a human cell. In some examples, the prCTB is genetically engineered. In some examples, the genetically engineered prCTB comprises a polynucleotide encoding an exogenous protein, including a cell receptor, an immune checkpoint protein, a cytokine, a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof. In some examples, the prCTB secretes a chemokine, a cytokine, a growth factor, or any combination thereof, or an exosome carrying any of the above. In some embodiments, disclosed herein is a pharmaceutical composition comprising the prCTB disclosed herein and a pharma- ceutically acceptable excipient. In some embodiments, disclosed herein is a method of treating a disease or condition, comprising administering to a subject in need of such treatment a pharmaceutical composition described herein. In some embodiments, disclosed herein is a population of cells comprising a plurality of the prCTB disclosed herein. In some examples, at least about 10% of the population of prCTB expresses CD16 and CD56.In some examples, at least about 2% of the population of prCTB expresses CD4, at least about 2% of the population of prCTB expresses CD8, at least about 5% of the population of prCTB expresses CD107, or any combination thereof. In some examples, at least about 10% of the population of prCTB expresses C. In some instances, at least about 2% of the prCTB population expresses CD16, CD56, and CD107, at least about 2% of the prCTB population expresses CD4, at least about 2% of the prCTB population expresses CD8, and at least about 5% of the prCTB population expresses CD107. In some instances, at least about 2% of the prCTB population expresses CD16, CD56, and CD107.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Various aspects of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0019] [Figure 1A]

[0021] Figures 1A-1D show the characteristics of insulin expression in human trophoblast stem (hTS) cells. Figure 1A is a schematic illustration of the molecular mechanism of insulin synthesis and secretion in hTS cells. Figure 1B shows by ChIP-qPCR analysis that CREB1 and FABA antibodies inhibited insulin (INS) gene transcription by reducing CREB1 and MAFA levels. Data represent mean ± SD. n=5, Input: positive control (as 100%), IgG: negative control. Figure 1C shows by RIA of secretome analysis that glucose (Gluc; 20 mM) and sulfonylurea (Gli, gliclazide; 10 μM) promoted insulin secretion in hTS cells (1 × 106 cells), while the VDCC inhibitor nifedipine (Nif; 10 μM) inhibited insulin secretion. Data represent mean ± SD. n=3, Student's t-test, *p<0.05 significant compared to control. FIG ID shows representative imaging of immunoreactive molecules characterized biological roles in hTS cells. Scale bars are as indicated. [Figure 1B] Same as description for Figure 1A. [Figure 1C] Same as description for Figure 1A. [Figure 1D] Same as description for Figure 1A. [Figure 2A]

[0022] Figures 2A-2M illustrate the Gαq / 11 / PIP2 / IP3 / IP3R / CaMKII / CREB1 and Gβ / PI3K / AKT / GSK3β / MAFA signaling pathways in insulin-expressing hTS cells. Figures 2A-2C show that glucose (20 mM) stimulated rapid transient activation of sweet taste receptor T1R2 / T1R3 at the plasma membrane in hTS cells by qPCR analysis (Figure 2A), resulting in activation of G protein signals including Gαq / 11 and Gβ (Figure 2B) in 15 min by blotting analysis (Figure 2C). This effect was inhibited by the T1R2 / T1R3 inhibitor 2,4-dichlorophenoxyacetic acid (2,4-D; 100 μg) (Figure 2B). Data represent the mean ± SD. n=5, Student's t-test, *p<0.05: statistical significance. Figures 2D-2F illustrate the establishment of the Gαq / 11 / PIP2 / IP3 / IP3R / CaMKII / CREB1 pathway by blotting analysis. Active Gαq / 11 induces inositol triphosphate (IP3) to act on its receptor IP3R in the membrane of the endoplasmic reticulum (ER) to increase intracellular calcium levels, which in turn activates CaMKII. This action was inhibited by shGα, which links Gαq / 11 and CaMKII molecules. CaMKII then activates downstream CREB1 by phosphorylation (p), which was further confirmed by using KN93, a CaMKII inhibitor. Figures 2G-2I illustrate the establishment of the Gβ / PI3K / AKT / GSK3α / β pathway. Active Gβ rapidly induced PI3K / AKT signaling by phosphorylation of AKT at ser473 (p) and downstream inhibitory GSK3α / β by phosphorylation at ser21 / 9. These effects were inhibited by LY294002, a PI3K inhibitor, and MK2206, an AKT inhibitor. Inhibitory pGSKα / β then promoted pMAFA to nuclear localization confirmed by its presence in the nucleus, and expression of pMAFA was inhibited by SB216763, an inhibitor of GSK3. Figure 2J shows that both pCREB1 and pMAFA entered the nucleus as evidenced by nuclear-cytoplasmic fractionation. β-actin: loading control.α-Tubulin and H3 represent the cytoplasmic and nuclear compartments, respectively. Figure 2K shows that pretreatment with CREB1 shRNA, but not PDX1 shRNA, significantly reduced insulin expression in response to glucose stimulation (Glu; 20 mM) in hTS cells by qPCR analysis. C: control, shGFP: positive control. Student's t-test, *p<0.05, n=4. Figure 2L shows insulin expression in two hTS cell lines with two different anti-insulin antibodies by flow cytometry. Isotype: control. Figure 2M shows by immunocytochemistry that naive hTS does not express the stress protein or proliferation marker Ki67, but does express HSP90. Scale bar: 50 μm. [Figure 2B] Same as description for Figure 2A. [Figure 2C] Same as description for Figure 2A. [Figure 2D] Same as description for Figure 2A. [Figure 2E] Same as description for Figure 2A. [Figure 2F] Same as description for Figure 2A. [Figure 2G] Same as description for Figure 2A. [Figure 2H] Same as description for Figure 2A. [Figure 2I] Same as description for Figure 2A. [Figure 2J] Same as description for Figure 2A. [Figure 2K] Same as description for Figure 2A. [Figure 2L] Same as description for Figure 2A. [Figure 2M] Same as description for Figure 2A. [Diagram 3]

[0023] Figure 3 shows representative images of insulin-related immune response molecules, including PDX1, HNF-1β, NGN3, SOX9, NKX6.1, and insulin in the prCTB. Scale bar: 50 μm. [Figure 4A]

[0024] Figures 4A-4F are described herein. Figure 4A shows by immunocytochemistry that hTSCs express TGF-β1. Figure 4B shows by Western blot assay that bFGF (10 ng / ml) upregulated TGF-β1 and vimentin but downregulated E-cadherin. Figure 4D shows that anti-TGF-β1 antibody neutralized these effects. Data represent mean ± SD. n=3, Student's t-test, statistical significance: *p<0.05, **p<0.001. Figure 4C shows by immunocytochemistry that bFGF (10 ng / ml) upregulated TGF-β1 and vimentin but downregulated E-cadherin. Figure 4E shows that bFGF (10 ng / ml) induced morphological changes in hTSCs after 1 day of incubation, where the cell shape changed from long spindle to short and enlarged, and the nuclear shape changed from oval to more round. Figure 4F shows confirmation of bFGF-induced FOXA2 activation, which is attenuated in the presence of shRNA against β-catenin. Cells transfected with shGFP (non-specific shRNA) were used as a control, and β-actin was used as a loading control. [Figure 4B] Same as explanation for Figure 4A. [Figure 4C] Same as explanation for Figure 4A. [Figure 4D] Same as explanation for Figure 4A. [Figure 4E] Same as explanation for Figure 4A. [Figure 4F] Same as explanation for Figure 4A. [Figure 5A]

[0025] Figures 5A-5C illustrate the biological characteristics of precursor regulatory cytotrophoblasts (prCTB). Figure 5A shows representative images of immunoreactive molecules and oxidative stress proteins accompanied by insulin expression in chorionic villi of a tubal ectopic pregnancy (7-8 weeks gestation). Scale bar: 50 μm. Figure 5B shows the distribution of insulin and stress proteins in chorionic villi of a normal pregnancy (7-8 weeks gestation) by imaging. Scale bar: 200 μm. Figure 5C is a schematic illustration of the differences in the expression of stress proteins of prCTB and hTS cells in the pathway of trophoblast differentiation, also compared to cells of a normal uterine pregnancy. [Figure 5B] Same as explanation for Figure 5A. [Figure 5C] Same as explanation for Figure 5A. [Figure 6A]

[0026] Figures 6A and 6B show a comparison of syncytial nodules expressing stress proteins p53 and insulin in chorionic villi between normal pregnancies and damaged oocytes. Stressed syncytial nodules are defined by expressing insulin and p53 in areas containing more than 15 positive cells counted in a single chorionic villi at 8 weeks of normal pregnancy (Figure 6A). Meanwhile, chorionic villi from a patient with damaged oocytes at 7-8 weeks of pregnancy (Figure 6B) show that the frequency of stressed syncytial nodules is 1.96 times higher in (Figure 6B) than in (Figure 6A). The number of syncytial nodules was counted independently in 80 fields (Figure 6A) and 50 fields (Figure 6B) by two assistants. Meanwhile, another 25 fields are not shown. Figure 6C shows the change in the expression of pluripotency transcription factors, immunoreactive OCT4 imaging (but not CDX2 imaging), during cell transformation at the DE stage by 4 hours of induction. [Figure 6B] Same as explanation for Figure 6A. [Figure 6C] FIG. 6C shows the change in expression of pluripotency transcription factors, immunoreactive OCT4 imaging (but not CDX2 imaging), during cell transformation at the DE stage induced for 4 hours. [Figure 7A]

[0027] Figures 7A-7Q are described herein. Figure 7A shows Luminex analysis of exosomes secreted from hTS cells (left dark column) and prCTB cells (right light column) in culture medium containing PLUS (upper panel), as well as exosomes presented by naive hTS cells (left dark column) and naive prCTB (right light column) after excluding the influence of components in the PLUS medium used (lower panel). Figure 7B shows the results of an immunoblot assay demonstrating the presence of numerous receptor molecules in hTS cells (left) and prCTB (right). Figure 7C shows the results of a secretome assay of proteins released from hTS cells and prCTB by immunoblot analysis. sHLA-G: soluble HLA-G. n=3. Figure 7D shows by FACS analysis that hTSCs express the angiogenic factors CD105 and CD146. Figure 7E shows the results of immunoblot assays demonstrating the presence of numerous receptor molecules in hTS cells (left) and prCTB (right). Figure 7F shows by immunoblot assays that bFGF (10 ng / ml) induced upregulation of STAT3, c-JUN, and Fas ligand (FasL), but downregulation of Fas. This effect was inhibited by shFGFR1. shGFP was used as a negative control. Figures 7G-7J show by Western blot assays that bFGF (10 ng / ml) induced activation of CREB1 signaling. Figure 7G shows that bFGF activated its receptor FGFR1 and downstream PI3K signaling, which was inhibited by the FGFR inhibitor PD166866. Figure 7H shows that PI3K phosphorylates (p)AKT to form PI3K / pAKT signaling, which was inhibited by PI3K shRNA. Figure 7I shows that pAKT activated its downstream CREB1, which was neutralized by using AKT antibodies, including AKT1 shRNA, AKT2 shRNA, and AKT3 shRNA. Figure 7J shows that pAKT activated its downstream CREB1 by direct interaction, as shown by immunoprecipitation (IP) assay.FIG. 7K shows that bFGF (10 ng / ml) induced IL-6 (left panel) and IL-8 (right panel) in a time-dependent manner. Data represent the mean ± SD. n=3 independent cell lines. Student's t-test, statistical significance: *p<0.05, **p<0.01, ***p<0.001. FIG. 7L shows RT-qPCR analysis showing that IL-6 (10 ng / ml) up-regulated IL-6R mRNA, CREB1 mRNA, and β-hCG mRNA in hTSCs (left panel), and IL-8 (30 ng / ml) up-regulated CREB1 mRNA and CD56 mRNA but not the classical receptor IL-8R (right panel). Data represent the mean ± SD. n=4. Student's t-test, statistical significance: **p<0.01, ***p<0.001. Figures 7M-7N show by Western blot that IL-6 generated β-hCG(+)CD56(+)prCTB. Figure 7M shows that IL-6 (10 ng / ml) induced β-hCG through GnRHR and IL-6R, which was inhibited by GnRHR inhibitor Elagolix sodium and CREB1 inhibitor 666-15. Figure 7N shows that IL-8 (30 ng / ml) induced CD56 through CXCR2 / STAT3 signaling, which was inhibited by CXCR2 inhibitor SB225002 and STAT3 inhibitor Stattic. Figure 7O shows that IL-8 (30 ng / ml) generated CD4(+)prCTB through both CXCR2 / CREB1 and CXCR2 / STAT3 signaling pathways, which was inhibited by CREB1 inhibitor 666-15, CRCR2 inhibitor SB225002, and STAT3 inhibitor Stattic. Figure 7P shows that IL-8 (30 ng / ml) induced Foxp3 via the CXCR2 / STAT3 signaling pathway and was inhibited by the CRCR2 inhibitor SB225002 and the STAT3 inhibitor Stattic (E). Data represent the mean ± SD. n=3 independent cell lines. Student's t-test, statistical significance: *p<0.05, **p<0.01, ***p<0.001. Figure 7Q shows by immunocytochemistry that bFGF induced co-expression of CD4 and Foxp3 in prCTB. Bar scale: 50 μm. [Figure 7B]Same as explanation for Figure 7A. [Figure 7C] Same as explanation for Figure 7A. [Figure 7D] Same as explanation for Figure 7A. [Figure 7E] Same as explanation for Figure 7A. [Figure 7F] Same as explanation for Figure 7A. [Figure 7G] Same as explanation for Figure 7A. [Figure 7H] Same as explanation for Figure 7A. [Figure 7I] Same as explanation for Figure 7A. [Figure 7J] Same as explanation for Figure 7A. [Figure 7K] Same as explanation for Figure 7A. [Figure 7L] Same as explanation for Figure 7A. [Figure 7M] Same as explanation for Figure 7A. [Figure 7N] Same as explanation for Figure 7A. [Figure 7O] Same as explanation for Figure 7A. [Figure 7P] Same as explanation for Figure 7A. [Figure 7Q] Same as explanation for Figure 7A. [Figure 8]

[0028] FIG. 8 shows two tables listing CD molecules in hTS cells and prCTB measured by flow cytometry, as well as markers of T cell and NK cell subtypes detected in hTS cells and prCTB measured by flow cytometry. [Figure 9A]

[0029] 9A-9N show representative flow cytometry analyses of the distribution of CD biomarkers in hTS cells and prCTB. Figures 9A-9K show FACS plot analysis in a total of eight representative samples, showing undetectable CD3 and CD45 (Figure 9A), CD34 (Figure 9B), and CD3 and gdTCR (Figure 9C), but detectable CD(16+56) and CD107a (Figure 9D), CD(16+56) (Figure 9E), detectable CD(16+56) but faint CD4 (Figure 9F), detectable CD(16+56) and CD8 (Figure 9G), detectable CD(16+56) but no CD19 (Figure 9H), detectable CD107a but faint CD4 (Figure 9I), detectable CD107a and CD8 (Figure 9J), and detectable CD107a but no CD19 (Figure 9K) in hTS cells (left panel) and prCTB (right panel). Figures 9L and 9M show the distribution of CD molecules (Figure 9L) and NK and T cell subtypes (Figure 9M) in populations of hTS cells (black columns) and prCTB (gray columns). Data represent the mean ± SD. n=8 independent samples. Figure 9N shows the expression of CD11b and CD49f in prCTB by immunostaining. [Figure 9B] Same as explanation for Figure 9A. [Figure 9C] Same as explanation for Figure 9A. [Figure 9D] Same as explanation for Figure 9A. [Figure 9E] Same as explanation for Figure 9A. [Figure 9F] Same as explanation for Figure 9A. [Figure 9G] Same as explanation for Figure 9A. [Figure 9H] Same as explanation for Figure 9A. [Figure 9I] Same as explanation for Figure 9A. [Figure 9J] Same as explanation for Figure 9A. [Figure 9K] Same as explanation for Figure 9A. [Figure 9L] Same as explanation for Figure 9A. [Figure 9M] Same as explanation for Figure 9A. [Figure 9N] Same as explanation for Figure 9A. [Figure 10A]

[0030] Figures 10A-10K are described herein. Figures 10A-10D show the results of transwell invasion and migration assays. MCP-1 (Figures 10A and 10B) and CXCL2 (Figures 10C and 10D) markedly induced the migration of both hTSCs and prCTB in a dose- and time-dependent manner. Figure 10E shows immunohistochemistry in normal chorionic villi at 7-8 weeks of gestation, revealing that the invading EVT expressed immunoreactive p53(+), syncytin, β-hCG, and HLA-G molecules. Figures 10F-10G show that CD56(+)prCTB (brown, upper and middle panels of Figure 10F) and β-hCG(+)prCTB (brown, upper panel of Figure 10G) migrate toward the EVT for implantation (red arrows). Many CD56(+) (lower panel in F) and β-hCG(+) (middle panel in Fig. 10G) prCTB under decidual epithelial cells. Replacement of arterial endothelial cells by β-hCG(+)prCTB in maternal decidua suggests SA remodeling (middle panel in Fig. 10G), and β-hCG(+)prCTB inside the lumen of decidual veins (lower panel in Fig. 10G) also suggests venous invasion. Fig. 10H-10K show the photographs of invasion and migration assays. Fig. 10H-10I show that MCP-1 promoted the motility of hTSC (4 × 103 cells / ml) in a dose-dependent manner (Fig. 10H) and in both hTSC and prCTB (4 × 103 cells / ml, respectively) in a time-dependent manner (Fig. 10I). Figures 10J-10K show that CXCL2 promoted the motility of hTSCs (4x103 cells / ml) in a time-dependent manner (Figure 10J) and in both hTSCs and prCTBs (4x103 cells / ml, respectively) in a dose-dependent manner (Figure 10K). Blue indicates cell staining with 0.2% crystal violet (Sigma-Aldrich, 115940). The number of migrated cells (blue) is counted using a hemocytometer or flow cytometer. n=3 independent samples in each experiment. [Figure 10B] Same as explanation for Figure 10A. [Figure 10C] Same as explanation for Figure 10A. [Figure 10D] Same as explanation for Figure 10A. [Figure 10E] Same as explanation for Figure 10A. [Figure 10F] Same as explanation for Figure 10A. [Figure 10G] Same as explanation for Figure 10A. [Figure 10H] Same as explanation for Figure 10A. [Figure 10I] Same as explanation for Figure 10A. [Figure 10J] Same as explanation for Figure 10A. [Figure 10K] Same as explanation for Figure 10A. [Figure 11A]

[0031] Figures 11A-11K are described herein. Figures 11A-11D show co-cultures of prCTB (arrowhead) and PANC-1 (arrow) at a ratio of 2:1 (3x104 cells / well) over time, and the apoptotic changes of PANC-1 engulfed by prCTB are shown by light microscopy (Figure 11A). Immunocytochemistry shows the interaction of two live cells, prCTB (blue, CytoCalcein450 staining) and PANC-1 (red, PKH26 staining) in the co-culture (Figure 11B). Apoptotic PANC-1 (green, apoxin staining, top) and intact prCTB (blue, center) are seen upon interaction (merged, arrow, bottom) (Figure 11C). 3D fluorescence microscopy shows apoptotic PANC-1 (green) and intact prCTB (blue) upon interaction (Figure 11D). FIG. 11E shows immunocytochemistry revealing that prCTB expressed PD-L1 (top, left column) but not PD-1 (bottom, left column), while PANC-1 expressed both PD-1 (top, right column) and PD-L1 (bottom, right column). FIG. 11F shows that FasL appeared in prCTB (top, left column) but not in PANC-1 (bottom, right column), while Fas was expressed in both prCTB (bottom, left column) and PANC-1 (top, right column). Bar scales are as indicated. FIG. 11G-11K show that prCTB induced apoptosis of solid tumor cells upon interaction. FIG. 11G shows that prCTB (blue) induced apoptosis of breast MCF-7 cells (green) upon interaction by 3D Cell Explorer fluorescence microscopy (FIG. 11G). Figure 11H shows that prCTB induced PD-L1 but not PD-1 (left column), while PANC-1 induced both PD-1 and PD-L1 (right column). Figure 11I shows that prCTB expressed both FasL and Fas (left column), while PANC-1 expressed both (right column). Figure 11J shows that prCTB significantly upregulated anti-apoptotic Mcl-1 mRNA and Bfl-1 mRNA by RT-qPCR analysis. Data represent mean ± SD. n=4, Student's t-test, statistical significance: *p<0.05, **p<0.01.FIG 11K shows that prCTB induced apoptosis (apoxin, green) in Huh7 (liver), H1299 (lung), MKN45 (stomach), PA-1 (ovary), and A375 (melanoma) cells after 24 h of co-culture, as analyzed by 3D Cell Explorer fluorescence microscopy (Nanolive, Swiss). Bar scales are as indicated. [Figure 11B] Same as explanation for Figure 11A. [Figure 11C] Same as explanation for Figure 11A. [Figure 11D] Same as explanation for Figure 11A. [Figure 11E] Same as explanation for Figure 11A. [Figure 11F] Same as explanation for Figure 11A. [Figure 11G] Same as explanation for Figure 11A. [Figure 11H] Same as explanation for Figure 11A. [Figure 11I] Same as explanation for Figure 11A. [Figure 11J] Same as explanation for Figure 11A. [Figure 11K] Same as explanation for Figure 11A. [Figure 12A]

[0032] 12A-12B show the cellular processes of CD56(+) β-hCG(+) cells in migration from EVT to maternal decidua. In fallopian tube ectopic chorionic villi, Left panel: Immunocytochemistry of CD56 expressing cells. Middle panel: Scattered distribution of CD56(+) cells among the chorionic stroma, inner layer of the chorionic CTB (black arrow), enriched in the EVT area. Right panel: Distribution of β-hCG expressing trophoblast cells in the villous surface and EVT area. In normal placental villi, Left panel: Histological identification of anchored villi, EVT, and maternal decidual tissue by H&E staining. Middle panel: Appearance of CD56(+) cells in the EVT area and adjacent decidual tissue. Right panel: Prominent CD56(+) cells appear in maternal decidua. Distribution of β-hCG(+) trophoblast cells similar to CD56(+) cells. [Figure 12B] Same as explanation for Figure 12A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020]

[0033] The details of one or more embodiments of the invention are set forth in the accompanying drawings, claims and the appended claims. and described herein. All other features, objects, and advantages of the inventive embodiments disclosed and contemplated herein may be combined with any other embodiment, unless expressly excluded.

[0021]

[0034] Disclosed herein are unique pioneer regulatory cytotrophoblast cells produced in vitro, compositions thereof, and their use in treating disorders (e.g., cancer, inflammation, or autoimmune diseases) or improving conditions (e.g., skin conditions). Pioneer regulatory cytotrophoblast cells are distinct from trophoblast stem cells. Pioneer regulatory cytotrophoblast cells are also distinct from embryonic stem cells. Pioneer regulatory cytotrophoblast cells are also distinct from primitive cytotrophoblast cells and cells derived from primitive cytotrophoblast cells, including villous cytotrophoblast cells (villous CTB), primitive syncytiotrophoblast cells (pSTB), syncytiotrophoblast cells (STB), and extravillous cytotrophoblast cells (EVT). See FIG. 5C. Pioneer regulatory cytotrophoblast cells are also distinct from placental cytotrophoblast cells. In some instances, pioneer regulatory cytotrophoblast cells are not pioneer villous cytotrophoblast cells.

[0022]

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that the above general description and the following detailed description are for purposes of illustration and explanation only and are not intended to limit any of the claimed subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. It should be noted that the singular forms "a," "an," and "the," as used in the specification and the appended claims, include plural references unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Additionally, the use of the term "including," as well as other forms such as "include," "includes," and "included," is not limiting.

[0023]

[0036] As used herein, ranges and amounts can be expressed as "about" a particular value or range, for example, ±15% of the cited numerical value. "About" also includes the exact amount. Thus, "about 5 μL" also means "about 5 μL" and "5 μL". In general, the term "about" includes amounts that are expected to be within experimental error.

[0024]

[0037] The terms "treating," "treatment," and the like are used herein as connotations of , as used herein to mean to obtain a desired pharmaceutical and / or physiological effect. In some examples, an individual (e.g., an individual suspected of suffering from and / or genetically predisposed to a liver-related disease or disorder) is prophylactically treated with a preparation of cells described herein, such prophylactic treatment completely or partially prevents the liver-related disease or disorder or its signs or symptoms. In some examples, an individual (e.g., when the individual suffers from a liver-related disease or disorder) is therapeutically treated, such therapeutic treatment causes a partial or complete cure of the disease or disorder, and / or reverses adverse events attributable to the disease or disorder, and / or stabilizes the disease or disorder, and / or delays the progression of the disease or disorder, and / or causes regression of the disease or disorder.

[0025]

[0038] Administration (e.g., transplantation) of the cells disclosed herein to an area in need of treatment can be accomplished by, for example, but not limited to, local infusion during surgery, injection, by catheter means, by implant means, etc., where the implant is a porous, non-porous, or gel-like material, such as a membrane, e.g., a silastic membrane or fiber.

[0026]

[0039] "Implanting" a composition into a mammal means introducing the composition into the body of a mammal by any method established in the art. The subject is the "subject" and the mammal is the "recipient." The transplant and recipient may be syngeneic, allogeneic, or xenogeneic. Additionally, the transplant may be an autologous transplant.

[0027]

[0040] The term "isolated," when used in reference to a cell or population of cells, refers to the state of a cell or population of cells separated from the host organism from which the cell or population of cells originates. In some examples, the isolated cell is in contact with other cells isolated from the same host organism. In some examples, the isolated cell is separated from any other cells. In some examples, the isolated prCTB is derived in vitro from a progenitor cell. In some examples, the isolated prCTB is obtained from and separated from a host organism.

[0028]

[0041] An "effective amount" is an amount of a therapeutic agent sufficient to achieve its intended purpose. An effective amount of a composition for treating or ameliorating a disorder is an amount of the composition sufficient to reduce or eliminate the symptoms of the disorder.

[0029]

[0042] The headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. Cells and compositions

[0043] In some cases, in vitro isolated pioneer regulatory cytotrophoblast cells ( Disclosed herein is a prCTB that is an isolated prCTB that is capable of binding to a variety of receptors, including HSP90, insulin, CD4, CD16, CD56, CD107a, CD8, interleukin 15 (IL-15), leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), T cell receptor (TCR), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), programmed death-ligand 1 (PD-L1), apoptosis signaling receptor (Fas), Fas ligand (FasL), CD335 (NKp46), CD11b, CD49f, CD3, CD19, CD34, B cell leukemia / Lymphoma 2 associated protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), or any combination thereof, or the prCTB secretes a chemokine, a cytokine, a growth factor, or any combination thereof, or secretes exosomes carrying a chemokine, a cytokine, a growth factor, or any combination thereof, and / or the prCTB expresses p53, Ki67, glutamate decarboxylase (GAD65), heat shock protein 70 (HSP70), soluble CD40-ligand (sCD40L), or any combination thereof. In some examples, the isolated prCTB expresses CD4, CD16, CD56, CD107a, CD8, or any combination thereof. In some examples, the prCTB induces apoptosis of cancer cells, for example, by infiltrating a colony of cancer cells, and optionally the prCTB itself does not undergo apoptosis upon contact with the cancer cells. In some examples, the cancer cells are solid tumor cells. In some examples, the cancer cells are pancreatic cancer cells, breast cancer cells, liver tumor cells, ovarian tumor cells, lung tumor cells, gastric tumor cells, melanoma cells, or any combination thereof. In some examples, the cancer cells express PD-1 and PD-L1, while the prCTB expresses PD-L1 but does not express PD-1. In some examples, the cancer cells express Fas but does not express FasL, while the prCTB expresses Fas and FasL.In some instances, the prCTB expresses Bfl-1 and Mcl-1.

[0030]

[0044] In some examples, disclosed herein is an isolated population of cells that comprises progenitor regulatory cytotrophoblasts (prCTB), the population of prCTB being characterized by the expression of HSP90, insulin, CD4, CD16, CD56, CD107a, CD8, interleukin-15 (IL-15), leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), leukocyte Immunoglobulin-like receptor subfamily B member 2 (LILRB2), T cell receptor (TCR), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), programmed death-ligand 1 (PD-L1), apoptosis signaling receptor (Fas), Fas ligand (FasL), CD335 (NKp46), B cell leukemia / lymphoma 2-associated protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), CD11b, CD49f, CD3, CD19, CD34, or any of their related proteins. The prCTB expresses one or more proteins in any combination, or the prCTB secretes a chemokine, a cytokine, a growth factor, or any combination thereof, or secretes exosomes carrying a chemokine, a cytokine, a growth factor, or any combination thereof, and / or the population of prCTB expresses p53, Ki67, glutamate decarboxylase (GAD65), heat shock protein 70 (HSP70), soluble CD40-ligand (sCD40L), or any combination thereof. In some examples, at least about 10% of the population is prCTB that expresses CD16 and CD56. In some examples, at least about 2% of the population is prCTB that expresses CD4. In some examples, at least about 2% of the population is prCTB that expresses CD8. In some examples, at least about 5% of the population is prCTB that expresses CD107.

[0031]

[0045] In some examples, an isolated population of cells comprising progenitor regulatory cytotrophoblast cells (prCTB) is disclosed herein, wherein (i) at least about 10% of the population is prCTB expressing CD16 and CD56, (ii) at least about 2% of the population is prCTB expressing CD4, (iii) at least about 2% of the population is prCTB expressing CD8, or (iv) at least about 5% of the population is prCTB expressing CD107, or any combination thereof. In some examples, an isolated population of cells comprising progenitor regulatory cytotrophoblast cells (prCTB) is disclosed herein, wherein (i) at least about 10% of the population is prCTB expressing CD16 and CD56, (ii) at least about 2% of the population is prCTB expressing CD4, (iii) at least about 2% of the population is prCTB expressing CD8, and (iv) at least about 5% of the population is prCTB expressing CD107, or any combination thereof. In some examples, the population of cells includes a population where at least about 2% of the population are prCTB that express CD16, CD56, and CD107.

[0032]

[0046] In some examples, the isolated prCTB expresses interleukin 15 (IL-15). In some examples, the isolated prCTB expresses leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), T cell receptor (TCR), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), programmed death-ligand 1 (PD-L1), apoptosis signal receptor (Fas), Fas ligand (FasL), CD335 (NKp46), B cell leukemia / lymphoma 2 associated protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), CD11b, CD49f, CD3, CD19, CD34, or any combination thereof. In some examples, the isolated prCTB further expresses the beta-hormone human chorionic gonadotropin (β-hCG or hCG-β), soluble human leukocyte antigen G (sHLA-G), transforming growth factor β1 (TGF-β1), plasminogen activator inhibitor-1 (PAI-1), interleukin 10 (IL-10), CD105, CD146, or any combination thereof. In some examples, the isolated prCTB lacks syncytin, programmed cell death protein 1 (PD-1), or a combination thereof. In some examples, the isolated prCTB secretes a chemokine, a cytokine, a growth factor, or any combination thereof, or an exosome carrying a chemokine, a cytokine, a growth factor, or any combination thereof. In some examples, the cytokine is chemokine (CC motif) ligand 5 (CCL5), monocyte ketone kinase (K-K) kinase, or any combination thereof. chemokine (C-C motif) ligand 1 (CCL1), chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 11 (CCL11), chemokine (C-C motif) ligand 24 (CCL24), chemokine (C-C motif) ligand 26 (CCL26), chemokine (C-C motif) ligand 22 (CCL22), chemokine (C-C motif) ligand 10 (CCL10), fractalkine, and chemokine (C-C motif) ligand 4 (CCL4), or any combination thereof. In some examples, the cytokine comprises interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), interleukin (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12p40 (IL-12p40), interleukin 13 (IL-13), interleukin 15 (I-15), or any combination thereof. In some examples, the cytokine comprises interferon alpha (IFN-alpha) or interferon gamma (IFN-gamma). In some examples, the growth factor comprises platelet-derived growth factor homodimer AA (PDGF-AA), PDGF homodimer BB (PDGF-BB), PDGF heterodimer (PDGF-AB), vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF), fibroblast growth factor (FGF) family proteins, FMS-like tyrosine kinase 3 ligand (Flt3L), soluble CD40 ligand (sCD40L), tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1β), or any combination thereof. In some examples, the isolated prCTB has a higher level of activated signal transducer / activator of transcription 3 (STAT3) or transcription factor c-JUN than the progenitor cells from which the isolated prCTB was differentiated in vitro, as measured by immunoblotting.In some examples, the isolated prCTB has at least about 1.1, 1.2, 1.5, 1.5, 2, 2.2, 2.5, 2.8, 3, 3.5, 4, 5, 8, 10 fold higher levels of activated signal transducer / activator of transcription 3 (STAT3) or transcription factor c-JUN as measured by immunoblot than the progenitor cells from which the isolated prCTB was differentiated in vitro. In some examples, the isolated prCTB expresses at least about 1.1, 1.2, 1.5, 1.5, 2, 2.2, 2.5, 2.8, 3, 3.5, 4, 5, 8, 10 fold higher levels of SOX2 protein as measured by immunoblot than the progenitor cells from which the isolated prCTB was differentiated in vitro. In some examples, the chorionic villi-derived progenitor cells lack expression of glutamate decarboxylase (GAD65), Ki67, heat shock protein 70 (HSP70), p53, soluble CD40-ligand (sCD40L), or any combination thereof. In some examples, both the chorionic villi-derived progenitor cells and the isolated prCTB express heat shock protein 90 (HSP90). In some examples, the isolated prCTB is a human cell. In some examples, the isolated prCTB is of rodent, rabbit, bovine, ovine, porcine, canine, feline, monkey, or ape origin. In some examples, the isolated prCTB is present in a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0033]

[0047] In some examples, the cells provided herein, such as prCTB, are genetically modified. In some examples, the cells are genetically modified to express an exogenous gene, such as a transgene. As used herein, the term "transgene" and its grammatical equivalents can refer to a gene or genetic material that is transferred to an organism. For example, a transgene can be a stretch or segment of DNA that contains a gene that is introduced into an organism. Once a transgene is transferred to an organism, the organism is referred to as a transgenic organism. A transgene is an RNA or polypeptide that is expressed in a transgenic organism. The transgene may retain its ability to produce a peptide (e.g., a protein). The transgene may be comprised of different nucleic acids, e.g., RNA or DNA. The transgene may encode an engineered T cell receptor, e.g., a TCR transgene. The transgene may include a TCR sequence. The transgene may include an oncogene. The transgene may include an immuno-oncogene. The transgene may include a recombination arm. The transgene may include an engineered site. In some examples, the transgene is an oncogene. In some examples, the transgene is an immuno-oncogene. In some examples, the transgene is a tumor suppressor gene. In some examples, the transgene encodes a protein that directly or indirectly promotes protein degradation. In some examples, the transgene is an oncolytic gene. In some examples, the transgene can aid lymphocytes in targeting tumor cells. In some examples, the transgene is a T cell enhancer gene. In some examples, the transgene is an oncolytic virus gene. In some examples, the transgene inhibits tumor cell growth. In some examples, the transgene is an anti-cancer receptor. In some instances, the transgene is an anti-angiogenic factor. In some instances, the transgene is a cytotoxic gene. Exemplary transgenes include, but are not limited to, one or more genes encoding CD28, inducible costimulator (ICOS), CD27, 4-1BB (CD137), ICOS-L, CD70, 4-1BBL, signal 3, a cytokine such as IL-2, IL-7, IL-12, IL-15, IL-21, ICAM-1 (CD54), LFA-3 (CD58), HLA class I genes, B7, CD80, CD83, CD86, CD32, CD64, 4-1BBL, CD3, CD1d, CD2, membrane-bound IL-15, membrane-bound IL-17, membrane-bound IL-21, membrane-bound IL-2, cleaved CD19, VEGF, a caspase, a chemokine, or an antibody (e.g., a monoclonal antibody) against any of the above, or any combination thereof.In some examples, the transgene encodes a protein involved in cell or tissue repair, such as DNA repair, proteins associated with immune responses (e.g., interferons and interleukins), and structural proteins. In some examples, the transgene encodes a growth factor receptor. In some examples, the prCTB described herein includes a transgene encoding a TCR, a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof. In some examples, the CAR may include an antigen recognition domain, a hinge region, a transmembrane domain, and an intracellular signaling domain. In some examples, the antigen recognition domain may be exposed to the outside of the cell and may interact with a possible target molecule. The antigen recognition domain may include a variable region of a monoclonal antibody, which may be linked as a single chain variable fragment. In some examples, the single chain variable fragment may include a variable light chain and a variable heavy chain of an immunoglobulin, which may be linked with a linker peptide. In some examples, a ligand or receptor system may be used as an alternative to an antibody-based antigen recognition domain. A hinge domain may be designed to connect the transmembrane domain to the antigen recognition domain. In some examples, the hinge region may be a peptide that can be designed to increase the flexibility of the antigen recognition domain. The transmembrane domain may include a hydrophobic alpha helix that spans the cell membrane. For example, the CD28 transmembrane domain can be used as the transmembrane domain in the CAR. In some examples, the transmembrane domain can anchor the CAR to the plasma membrane. The intracellular T cell signaling domain can be linked to the transmembrane domain and can be inside the cell. In some examples, when a target protein binds to the antigen recognition domain, the intracellular signaling domain can transmit an activation signal. In some examples, the activation of the signaling domain can include phosphorylation of an immunoreceptor tyrosine-based activation motif (ITAM). The intramolecular signaling domain can include a modified ITAM. In some examples, the modified ITAM can include a signaling domain, such as a CD3-zeta domain.In some examples, the modified ITAM signaling domain comprises CD3-zeta, CD3-epsilon, CD3-gamma, CD3-delta, derivatives thereof, or any combination thereof. In some examples, the intracellular signaling domain may include one or more costimulatory domains. In some examples, the intracellular signaling domain may include one or more signaling domains and one or more costimulatory signaling domains. In some examples, the costimulatory domain may include a signaling domain from CD28, CD27, CD40, CD134, CD137, inducible costimulatory (ICOS), DAP10, derivatives thereof, or any combination thereof. In some examples, the prCTB described herein includes a transgene encoding an oncogenic receptor.

[0034]

[0048] In some examples, the compositions comprising the cells disclosed herein are formulated as pharmaceutical compositions for intravenous administration to mammals, including humans. In some examples, the compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the compositions also include a local anesthetic to ameliorate any pain at the injection site. If the composition is administered by infusion, the composition may be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0035]

[0049] In one aspect, disclosed herein is a composition (e.g., a pharmaceutical composition) comprising the cells disclosed herein. In some examples, the composition further comprises a pharma- ceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, and combinations thereof. In other examples, a colloidal dispersion system is used. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and oil-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0036] Secretome and composition

[0050] In some embodiments, the trophoblast stem cells or prCTB sections described herein The present specification discloses compositions that include secretomes. In some examples, the secretomes include exosomes secreted by trophoblast stem cells or prCTB and other soluble molecules (e.g., proteins, nucleic acids, and lipids) secreted by trophoblast stem cells or prCTB.

[0037]

[0051] In some examples, compositions comprising chemokines, cytokines, growth factors, or any combination thereof are disclosed herein. In some examples, the compositions comprise exosomes, and the exosomes carry chemokines, interleukins, growth factors, or any combination thereof, and pharma- ceutically or cosmetically acceptable excipients. In some examples, the compositions do not comprise cells. In some examples, the compositions comprise or the exosomes carry (i) chemokines, including CXCL2, MCP-1, fractalkine, IP-10, MCP-3, eotaxin, MIP-1β, or any combination thereof, (ii) interleukins, including IL-6, IL-8, IL-4, IL-1RA, IL-10, IL-12P40, IL-15, IL-1α, IL-17A, or any combination thereof, and (iii) growth factors, including PDGF-AA, VEGF, bFGF, G-CSF, Flt-3L, GM-CSF, or any combination thereof.

[0038]

[0052] In some examples, the composition includes MCP-1 and one, two, three, or all of CXCL2, IL-6, IL-8, and VEGF proteins. In some examples, the MCP-1 and CXCL2 in the composition have a weight ratio of about 1:1 to about 2.5:1. For example, the MCP-1 and CXCL2 in the composition have a weight ratio of about 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:, 2.2:1, 2.3:1, 2.4:1, or 2.5:1. In some examples, the MCP-1 and CXCL2 in the composition have a weight ratio of about 2.0:1 to about 2.5:1. In some examples, the MCP-1 and CXCL2 in the composition have a weight ratio of about 3:1 to about 4:1 or about 3:1 to about 5:1. For example, the MCP-1 and CXCL2 in the composition have a weight ratio of about 3:1, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.5, 5, or 4.0. In some examples, the MCP-1 and CXCL2 in the composition have a weight ratio of about 3.2.

[0039]

[0053] In some examples, the composition includes MCP-1 and one, two, three, or all of CXCL2, IL-6, IL-8, and VEGF proteins. In some examples, the MCP-1 and CXCL2 in the composition have a weight ratio of about 1:7 to about 1:4. For example, the MCP-1 and CXCL2 in the composition have a weight ratio of 1:7.0, 1:6.8, 1:6.6, 1:6.4, 1:6.2, 1:6, 1:5.8, 1:5.6, 1:5.4, 1:5.2, 1:5.0, 1:4.8, 1:4.6, 1:4.4, 1:4.2, or 1:4.0. In some examples, the MCP-1 and CXCL2 in the composition have a weight ratio of about 1:5.0. In some examples, the MCP-1 and CXCL2 in the composition have a weight ratio of about 1:4 to about 1:1.5. For example, MCP-1 and CXCL2 in the composition have a weight ratio of about 1:4.0, 1:3.8, 1:3.6, 1:3.4, 1:3.2, 1:3.0, 1:2.8, 1:2.6, 1:2.4, 1:2.2, 1:2.0, 1:1.8, 1:1.7, 1:1.6, or 1:1.5. In some examples, MCP-1 and CXCL2 in the composition have a weight ratio of about 1:4 to about 1:2.5.

[0040]

[0054] In some examples, MCP-1 and IL-6 in the composition have a weight ratio of about 2:1 to about 3:1. For example, MCP-1 and IL-6 in the composition have a weight ratio of about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0. In some examples, MCP-1 and IL-6 in the composition have a weight ratio of about 2.3. In some examples, MCP-1 and IL-6 in the composition have a weight ratio of about 2.5. In some examples, MCP-1 and IL-6 in the composition have a weight ratio of about 3:1 to about 4:1. For example, MCP-1 and IL-6 in the composition have a weight ratio of about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0. In some examples, the MCP-1 and IL-6 in the composition have a weight ratio of about 3.6. In some examples, the MCP-1 and IL-6 in the composition have a weight ratio of about 3.8.

[0041]

[0055] In some examples, the MCP-1 and IL-8 in the composition have a weight ratio of about 4:1 to about 6:1. For example, the MCP-1 and IL-8 in the composition have a weight ratio of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. In some examples, the MCP-1 and IL-8 in the composition have a weight ratio of about 4.6. In some examples, the MCP-1 and IL-8 in the composition have a weight ratio of about 4.4. In some examples, the MCP-1 and IL-8 in the composition have a weight ratio of about 4.9. In some examples, the MCP-1 and IL-8 in the composition have a weight ratio of about 4.5.

[0042]

[0056] In some examples, the MCP-1 and VEGF in the composition have a weight ratio of about 5:1 to about 7:1. For example, the MCP-1 and VEGF in the composition have a weight ratio of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In some examples, the MCP-1 and VEGF in the composition have a weight ratio of about 5.6. In some examples, the MCP-1 and VEGF in the composition have a weight ratio of about 6.0. In some examples, the MCP-1 and VEGF in the composition have a weight ratio of about 7:1 to about 9:1, for example, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7. In some examples, the MCP-1 and VEGF in the composition have a weight ratio of about 7.6:1. In some examples, the MCP-1 and VEGF in the composition have a weight ratio of about 7.3:1.

[0043]

[0057] In some examples, the composition further comprises PDGF-AA. In some examples, MCP-1 and PDGF-AA are present in a weight ratio of about 3:1 to about 5:1, for example, about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0. In some examples, MCP-1 and PDGF-AA are present in a weight ratio of about 3.5. In some examples, MCP-1 and PDGF-AA are present in a weight ratio of about 6:1 to about 9:1, for example, about 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, or 9.0. In some instances, the MCP-1 and PDGF-AA are present in a weight ratio of about 7.8.

[0044]

[0058] In some examples, the composition further comprises PDGF-AA. In some examples, MCP-1 and PDGF-AA are present in a weight ratio of about 1:2.5 to about 1:1.5, such as about 1:2.5, 1:2.4, 1:2.3, 1:2.2, 1:2.1, 1:2.0, 1:1.9, 1:1.8, 1:1.7, 1:1.6, or 1:1.5. In some examples, MCP-1 and PDGF-AA are present in a weight ratio of about 0.6. In some examples, MCP-1 and PDGF-AA are present in a weight ratio of about 1:1.5 to about 1.5:1, such as about 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1.0, 1.1:1, 1.2:1, 1.3:1, 1.4:1, or 1.5:1. In some instances, the MCP-1 and PDGF-AA are present in a weight ratio of about 1.2.

[0045]

[0059] In some examples, the composition further comprises PDGF-AA. In some examples, MCP-1 and PDGF-AA are present in a weight ratio of about 3:1 to about 5:1, for example, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0. In some examples, MCP-1 and PDGF-AA are present in a weight ratio of about 3.5. In some examples, MCP-1 and PDGF-AA are present in a weight ratio of about 6:1 to about 9:1, for example, about 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, or 9.0. In some instances, the MCP-1 and PDGF-AA are present in a weight ratio of about 7.8.

[0046]

[0060] In some examples, the composition further comprises PDGF-AA and G-CSF. In some examples, the composition further comprises PDGF-AA and FGF-2 (bFGF). In some examples, the composition further comprises one or more of the following proteins: IP-10, Eotaxin, Flt-3L, GM-CSF, MIP-1a, MIP-1b, IL-1a, IL-1RA, IL-4, IL-7, IL-10, IL-12P40, IL-13, IL-15, IL-17A, CCL5 (RANTES), MDC, MCP-3, IL-12P70, IFNa, IFNr, PDGF-AB / BB, or EGF.

[0047]

[0061] In some examples, the composition further comprises a nucleic acid, such as an mRNA, siRNA, shRNA, or DNA. In some examples, the composition further comprises a lipid molecule secreted from the prCTB or trophoblast stem cells.

[0048]

[0062] In some examples, the compositions disclosed herein may be sterile. In some examples, the compositions may include indigenous microorganisms. The microorganisms may be viruses, bacteria, eukaryotic cells, or any combination thereof. In some examples, the microorganisms may not be pathogenic. In some examples, the composition may include bacteria at a concentration of less than about 10 colony forming units (CFU) / gram (g), 50 CFU / g, 100 CFU / g, 150 CFU / g, 200 CFU / g, 300 CFU / g, 400 CFU / g, 500 CFU / g, 600 CFU / g, 700 CFU / g, 800 CFU / g, 900 CFU / g, or 1000 CFU / g. In some examples, the composition may include bacteria at a concentration of about 10 CFU / g to about 1000 CFU / g, 10 CFU / g to about 50 CFU / g, 20 CFU / g to about 100 CFU / g, 50 CFU / g to about 200 CFU / g, 100 CFU / g to about 250 CFU / g, 200 CFU / g to about 500 CFU / g, 500 CFU / g to about 700 CFU / g, or 600 CFU / g to about 1000 CFU / g. In some examples, the composition is substantially free (e.g., at least 95% free) or free of Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Pseudomonas species, Klebsiella pneumoniae, or any combination thereof.

[0049]

[0063] In some examples, the compositions disclosed herein may be free of heavy metals such as lead, bithionol, chlorofluorocarbon propellants, nitrosamines, chloroform, halogenated salicylanilides, hexachlorophene, mercury compounds, 1,4-dioxane, methylene chloride, prohibited bovine materials, sunscreen compounds, vinyl chloride, zirconium-containing complexes, or any combination thereof. In some examples, prohibited bovine materials may include the brain, skull, eyeballs, trigeminal ganglion, spinal cord, vertebral column, dorsal root ganglion, tonsils, distal ileum of the small intestine, or any combination thereof. In some examples, the compositions may include lead at levels of 10 parts per million or less.

[0050]

[0064] In some examples, the compositions herein do not include colorants, fragrances, parabens, phthalates, alcohols, or any combination thereof. In some examples, colorants, fragrances, parabens, phthalates, or alcohols are present in the composition at insignificant levels, such as less than 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%. In some examples, incidental ingredients have no technical / structural, functional, or any combination thereof effect in the composition, such as being not active ingredients. In some examples, the compositions do not include.

[0051]

[0065] In some examples, excipients disclosed herein may include water, glycerol, saline, vegetable oils (e.g., seed oils), fruit oils, floral extracts, mineral oils, synthetic oils, sugar compounds, silicates, calcium salts, magnesium salts, sodium chloride, potassium chloride, lactic acid, starch, sugar alcohols, cellulose, activated charcoal, glycerin, butter, amine acids, paraffin, honey, wax, beeswax, agar, calcium carbonate, citric acid, tartaric acid, stearic acid, xanthan gum, benzoic acid, polyethylene glycol, silicones, derivatives thereof, salts thereof, or any combination thereof.

[0052] How to use

[0066] In some instances, the isolated prCTB is administered to a subject intravenously, subcutaneously, transdermally, by inhalation, or the like. In some examples, the subject is administered intravenously, orally, intramuscularly, or intratumorally. In some examples, the subject is a mammal. In some examples, the subject is a primate. In some examples, the subject is a human. In some examples, the isolated prCTB expresses interleukin 15 (IL-15). In some examples, the isolated prCTB expresses CD4, CD16, CD56, CD107a, CD8, or any combination thereof. In some examples, the isolated prCTB expresses leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), T cell receptor (TCR), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), programmed death-ligand 1 (PD-L1), apoptosis signaling receptor (Fas), , Fas Ligand (FasL), CD335 (NKp46), CD11b, CD49f, CD3, CD19, CD34, or any combination thereof. In some examples, the isolated prCTB further expresses beta-hormone human chorionic gonadotropin (β-hCG), soluble human leukocyte antigen G (sHLA-G), transforming growth factor β1 (TGF-β1), plasminogen activator inhibitor-1 (PAI-1), interleukin 10 (IL-10), CD105, CD146, or any combination thereof. In some examples, the isolated prCTB lacks syncytin, programmed cell death protein 1 (PD-1), or a combination thereof. In some examples, the isolated prCTB secretes a chemokine, a cytokine, a growth factor, or any combination thereof, or an exosome carrying a chemokine, a cytokine, a growth factor, or any combination thereof. In some examples, the cytokine comprises chemokine (C-C motif) ligand 5 (CCL5), monocyte chemoattractant protein-1 (MCP-1), monocyte chemoattractant protein-1 (MCP-3), chemokine (C-X-C motif) ligand 1 (CCL1), chemokine (C-X-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 11 (CCL11), chemokine (C-C motif) ligand 24 (CCL24), chemokine (C-C motif) ligand 26 (CCL26), chemokine (C-C motif) ligand 22 (CCL22), chemokine (C-X-C motif) ligand 10 (CCL10), fractalkine, chemokine (C-C motif) ligand 4 (CCL4), or any combination thereof.In some examples, the cytokine comprises interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), interleukin (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12p40 (IL-12p40), interleukin 13 (IL-13), interleukin 15 (I-15), or any combination thereof. In some examples, the cytokine comprises interferon alpha (IFN-alpha) or interferon gamma (IFN-gamma). In some examples, the growth factor comprises platelet-derived growth factor homodimer AA (PDGF-AA), PDGF homodimer BB (PDGF-BB), PDGF heterodimer (PDGF-AB), vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF), fibroblast growth factor (FGF) family proteins, FMS-like tyrosine kinase 3 ligand (Flt3L), soluble CD40 ligand (sCD40L), tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1β), or any combination thereof. In some examples, the isolated prCTB has a higher level of activated signal transducer / activator of transcription 3 (STAT3) or transcription factor c-JUN than the progenitor cells from which the isolated prCTB was differentiated in vitro, as measured by immunoblotting. In some examples, the isolated prCTB has at least about 1.1, 1.2, 1.5, 1.5, 2, 2.2, 2.5, 2.8, 3, 3.5, 4, 5, 8, 10 fold higher levels of activated signal transducer and activator of transcription 3 (STAT3) or the transcription factor c-JUN than the progenitor cell from which the isolated prCTB was differentiated in vitro, as measured by immunoblotting.In some examples, the isolated prCTB expresses at least about 1.1, 1.2, 1.5, 1.5, 2, 2.2, 2.5, 2.8, 3, 3.5, 4, 5, 8, 10 times higher levels of SOX2 protein than the progenitor cells from which the isolated prCTB differentiated in vitro, as measured by immunoblotting. In some examples, the progenitor cells lack expression of p53, glutamate decarboxylase (GAD65), Ki67, heat shock protein 70 (HSP70), soluble CD40-ligand (sCD40L), or any combination thereof. In some examples, the isolated prCTB is a human cell. In some examples, the isolated prCTB is of rodent, rabbit, bovine, ovine, porcine, canine, feline, monkey, or ape origin.

[0053]

[0067] In some examples, disclosed herein are methods of killing antigen-bearing target cells, comprising administering progenitor regulatory cytotrophoblast (prCTB) to a subject in need of killing the antigen-bearing target cells, wherein the isolated prCTB is capable of binding to a number of receptors, including HSP90, insulin, CD4, CD16, CD56, CD107a, CD8, interleukin-15 (IL-15), leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), T cell receptor (TCR), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), progesterone-binding protein 1 (PGP1), and cytochrome P450 (CYP450). The antigen-bearing target cell expresses one or more proteins including death-ligand 1 (PD-L1), apoptosis signal receptor (Fas), Fas ligand (FasL), CD335 (NKp46), B-cell leukemia / lymphoma 2 associated protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), CD11b, CD49f, CD3, CD19, CD34, or any combination thereof, and glutamate decarboxylase (GAD65), Ki67, heat shock protein 70 (HSP70), p53, soluble CD40-ligand (sCD40L), or any combination thereof. In some examples, the antigen-bearing target cell is not an antigen-presenting cell, e.g., not a dendritic cell, macrophage, or B cell. In some examples, the antigen-bearing target cell is a cancer cell. In some examples, the cancer cell is a solid tumor cell. In some examples, the cancer cell is a blood cancer cell. In some examples, the cancer cells include bladder cancer cells, bone cancer cells, brain cancer cells, breast cancer cells, cervical carcinoma, colorectal cancer cells, esophageal cancer cells, gastrointestinal cancer cells, hematopoietic malignancies, head and neck squamous cell carcinoma, leukemia, liver cancer cells, lung cancer cells, lymphoma, myeloma, nasal cancer cells, nasopharyngeal cancer cells, oral cancer cells, oropharyngeal cancer cells, ovarian cancer cells, prostate cancer cells, sarcoma, gastric cancer cells, melanoma, thyroid cancer cells, or any combination thereof. In some examples, the antigen-bearing target cells are pathogens. In some examples, the pathogens include viruses, bacteria, protozoa, prions, fungi, or any combination thereof.In some examples, the method kills at least about 5%, at least about 10%, at least about 20%, at least about 50%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100% of a population of antigen-bearing target cells.

[0054]

[0068] Disclosed herein, in some examples, is a method of downregulating an inflammatory pathway, comprising administering progenitor regulatory cytotrophoblast (prCTB) to a subject in need of downregulating an inflammatory pathway, wherein (i) the isolated prCTB is capable of downregulating an inflammatory pathway that ... L), CD335 (NKp46), B-cell leukemia / lymphoma 2 associated protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), CD11b, CD49f, CD3, CD19, CD34, or any combination thereof, and (b) glutamate decarboxylase (GAD65), Ki67, heat shock protein 70 (HSP70), p53, soluble CD40-ligand (sCD40L), or any combination thereof, and (ii) the isolated prCTB secretes a chemokine, a cytokine, a growth factor, or any combination thereof, or secretes exosomes that carry a chemokine, a cytokine, a growth factor, or any combination thereof.

[0055]

[0069] In some examples, the method includes treating a disease, such as transplant rejection, infection, endotoxic shock associated with infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic onset juvenile idiopathic arthritis (JIA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), asthma, pelvic inflammatory disease, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, ...rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, juvenile idiopathic arthritis, rheumatoid arthritis, rheumatoid In one embodiment, the present invention treats diseases or conditions including endometrial inflammatory disease, Alzheimer's disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, ankylosing spondylitis, dermatomyositis, uveitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, vasculitis, post-surgical adhesions, stroke, type I diabetes, Lyme arthritis, meningoencephalitis, immune-mediated inflammatory disorders of the central and peripheral nervous system, pancreatitis, post-surgical injuries, graft versus host disease, cardiac disease, bone resorption, burn patients, myocardial infarction, Paget's disease, osteoporosis, sepsis, hepatic or pulmonary fibrosis, periodontitis, or hypochlorhydria. In some examples, the method treats autoimmune diseases including type I diabetes, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, polymyositis, chronic active hepatitis, mixed connective tissue disease, primary biliary cirrhosis, pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease, vitiligo, gluten-sensitive enteropathy, Graves' disease, myasthenia gravis, autoimmune neutropenia, idiopathic thrombocytopenic purpura, rheumatoid arthritis, liver cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's disease, bullous pemphigoid, discoid lupus, ulcerative colitis, dense deposit disease, inflammatory bowel disease, or psoriasis.In some examples, the method treats type 1 diabetes.In some examples, the method improves transplant rejection.

[0056]

[0001] In some examples, disclosed herein is a method of regulating a skin condition, comprising administering to a subject in need thereof a composition (e.g., a pharmaceutical composition) comprising a cytokine, such as a chemokine, an interleukin, a growth factor, or any combination thereof, or an exosome carrying a cytokine, such as a chemokine, an interleukin, a growth factor, or any combination thereof. In some examples, the method improves the condition of the skin, such that the condition of the skin has one or more better characteristics after application of the method compared to before application of the method. In some examples, the chemokine comprises GRO, MCP-1, fractalkine, IP-10, MCP-3, eotaxin, MIP-1β, or any combination thereof. In some examples, the composition comprises an interleukin, including IL-6, IL-8, IL-4, IL-1RA, IL-10, IL-12P40, IL-15, IL-1α, IL-17A, or any combination thereof. In some examples, the growth factor comprises PDGF-AA, VEGF, bFGF, G-CSF, Flt-3L, GM-CSF, or any combination thereof. In some examples, the method provides a cosmetic application. In some examples, the method tightens the skin. In some examples, the method hydrates the skin. In some examples, the method rejuvenates the skin. In some examples, the composition is a medium after passaging stem cells. In some examples, the stem cells are isolated progenitor regulatory cytotrophoblast (prCTB) cells as described herein. In some examples, the passage number is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, the stem cells are cultured in the medium for at least about 1-3 days, e.g., about 1-2 days, e.g., before the medium is harvested. In some examples, the stem cells are cultured in the medium for at least about 12-24 hours, e.g., before the medium is harvested. In some examples, the passage number is 5-10. In some examples, the passaging is performed about every 1, 2, or 3 days. In some examples, the passaging occurs about every 2, 4, 6, 8, 12, 16, 20, or 24 hours. In some examples, the medium does not contain stem cells. In some examples, the composition is in the form of a cream, liquid, gel, lotion, mist, capsule, or mask.In some examples, the method can be used to treat skin disorders. In some examples, the skin disorder can be eczema, psoriasis, acne, rosacea, ichthyosis, vitiligo, urticaria, seborrheic dermatitis, shingles, sunburn, burns, contact dermatitis, rashes, or any combination thereof. In some examples, the method can reduce the appearance of skin aging, photoaging, or any combination thereof. In some examples, the method can reduce the appearance of scars. In some examples, the method can improve wound healing. In some examples, the method can prevent, reduce, or remove scars, benign tumors, age spots, cancerous growths, ulcers, infections, or any combination thereof. In some examples, the method can prevent, reduce, or remove skin lines, wrinkles, or any combination thereof. In some examples, the lines or wrinkles are crow's feet, smile lines, frown lines, forehead lines, tear troughs. The wrinkles may be bunny lines, nasolabial folds, marionette lines, psychological furrows, necklines, aging wrinkles, wrinkle lines, elastic wrinkles, expression lines, gravitational sagging, dynamic wrinkles, static wrinkles, atrophic wrinkles, atrophic crinkling rhytids, or any combination thereof. In some examples, the method can prevent, reduce, or eliminate loss of skin volume, elasticity, or any combination thereof. In some examples, the method can prevent, reduce, or eliminate sagging skin, dull skin tone, mottled discoloration, rough skin, dry skin, itchy skin, thinning skin, or any combination thereof. In some examples, the method can improve or ameliorate a skin condition, a skin disorder, or any combination thereof. In some examples, the method can moisturize, firm, lift, or rejuvenate the skin. In some examples, the method can restore or sustain skin health, smoothness, blemish-free, translucent, tough, or any combination thereof. In some examples, the method can heal, treat, repair, or any combination thereof, of the glycosaminoglycans, dermis, collagen, and elastin of the skin. In some examples, the improved skin health can be measured by a wrinkle severity rating scale, transepidermal water loss measurement, skin color measurement, skin surface topography measurement, viscoelasticity measurement by Cutometer®, histological examination, or any combination thereof. In some examples, the improved skin health can be measured by diagnostic imaging, such as magnetic resonance imaging (MRI). In some examples, the measurements can be compared before and after administration of the composition. In some examples, the measurements can be compared to a standard.

[0057]

[0070] In another aspect, disclosed herein is a method of treating a condition in a subject, comprising administering to the subject a pharmaceutical composition comprising the cells of the present invention in an amount effective to engraft the cells into the subject (e.g., into the liver of the subject). In some examples, the cells are administered in a pharmaceutically acceptable carrier. In some examples, the pharmaceutically acceptable carrier comprises saline, such as phosphate buffered saline, or fetal bovine serum. In some examples, the cells are administered at a concentration of about 1×10 per ml. 6 ~About 100×10 6 Cells, approximately 1 x 10 per ml 6 ~Approx. 250×10 6 Cells, approximately 1 x 10 per ml 6 ~About 500×10 6 cells, or approximately 10 x 10 per ml 6 ~About 40×10 6 The cells are administered in a suspension comprising the cells. In some examples, the cells are administered in a volume of about 1-5 ml, 1-10 ml, 1-50 ml, 1-100 ml, or 10-150 ml. In some examples, the subject is a human. In some examples, the administration comprises an injection, e.g., an intravenous injection. In some examples, the injection is administered into a hepatic vein. In some examples, the injection is administered into a hepatic artery. In some examples, the condition is a liver-related disease or disorder. In some examples, the condition is liver failure. In some examples, the liver-related disease or disorder comprises Alagille syndrome, alpha 1 antitrypsin deficiency, autoimmune hepatitis, benign liver tumors, biliary atresia, cirrhosis, cystic disease of the liver, fatty liver disease including alcohol-related liver disease and non-alcoholic fatty liver disease (NAFLD), galactosemia, gallstones, Gilbert's syndrome, hemochromatosis, liver cysts, liver cancer, liver disease of pregnancy (optionally acute fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, preeclampsia, or HELLP syndrome (hemolysis, elevated liver lab values, decreased platelets)), neonatal hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, porphyria, Reye's syndrome, sarcoidosis, toxic hepatitis, glycogen storage disease type 1, tyrosinemia, viral hepatitis, Wilson's disease, or any combination thereof.

[0058]

[0071] The mode of administration of the cells disclosed herein includes, but is not limited to, systemic intravenous injection and direct injection (e.g., endoscopic retrograde injection) into the intended site of activity. The formulation can be administered by any convenient route, such as infusion or bolus injection, and can be administered together with other bioactive agents. In some examples, administration is systemic localized administration.

[0059]

[0072] In some embodiments, compositions and methods for transplanting the cells disclosed herein into a subject are provided. Methods and methods are provided herein. In some examples, the subject is injected with the cells (e.g., intravenously, intramuscularly, percutaneously, endoscopic retrograde injection, or intraperitoneally). In some examples, the subject has not been treated with an immunosuppressant prior to transplantation. In some examples, the method further includes treating the patient with an immunosuppressant, such as FK-506, cyclosporine, or a GAD65 antibody.

[0060]

[0073] In some examples, the cells described herein are delivered to target site (e.g., liver defect) by a delivery system suitable for targeting cells to specific tissue. For example, cells are encapsulated in a delivery vehicle that allows slow release of cells at target site. The delivery vehicle is modified so that it is specifically targeted to specific tissue. The surface of the targeted delivery system is modified by various methods. In the case of liposomal targeted delivery system, lipid groups are incorporated into the lipid bilayer of liposome to maintain targeting ligand in stable association with the liposomal bilayer.

[0061]

[0074] Administration of the cells described herein is optionally tailored to the individual by (1) increasing or decreasing the amount of cells injected, (2) varying the number of injections, or (3) varying the method of delivery of the cells.

[0062] Detection Method

[0075] Methods for determining the expression or presence of the above-mentioned biomarkers are known in the art, For example, it can be measured by flow cytometry, immunohistochemistry, Western blot, immunoprecipitation, magnetic bead selection, and quantification of cells expressing any of these cell surface markers. The level of expression of biomarker RNA can be measured by RT-PCR, Qt-PCR, microarray, Northern blot, or other similar techniques.

[0063]

[0076] "Detecting expression" or "detecting expression levels" is intended to determine the expression level or presence of a biomarker protein or gene in a biological sample. That is, "detecting expression" encompasses cases where the biomarker is not expressed, not detectably expressed, expressed at low levels, expressed at normal levels, or overexpressed.

[0064]

[0077] In some examples, the expression or presence of the biomarkers described herein is determined at the nucleic acid level, for example, using immunohistochemistry or nucleic acid-based techniques such as in situ hybridization and RT-PCR. In some examples, the expression or presence of one or more biomarkers is determined by means for nucleic acid amplification, nucleic acid sequencing, utilizing nucleic acid microarrays (DNA and RNA), or by means for in situ hybridization using specifically labeled probes.

[0065]

[0078] In some examples, the determination of the expression or presence of the biomarker is performed by gel electrophoresis. In some examples, the determination is performed by transfer to a membrane and hybridization with a specific probe. In some examples, the determination of the expression or presence of the biomarker is performed by a diagnostic imaging technique. In some examples, the determination of the expression or presence of the biomarker is performed by a detectable solid substrate. In some examples, the detectable solid substrate is a paramagnetic nanoparticle functionalized with an antibody.

[0066]

[0079] In some examples, the expression or presence of a biomarker is at the RNA (e.g., mRNA) level. In some examples, techniques for detecting RNA (e.g., mRNA) levels include, but are not limited to, Southern or Northern analysis, polymerase chain reaction analysis, and probe arrays.

[0067]

[0080] One method for detecting mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that hybridizes to the mRNA encoded by the gene to be detected. The nucleic acid probe consists of an oligonucleotide of at least 7, 15, 30, 50, 100, 250, or 500 nucleotides in length sufficient to specifically hybridize under stringent conditions with, for example, a full-length cDNA or a portion thereof, such as the mRNA or genomic DNA encoding a biomarker described herein. Hybridization of the mRNA with the probe indicates that the biomarker or other protein of interest is expressed.

[0068]

[0081] In some examples, the mRNA is immobilized on a solid surface and contacted with a probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane such as nitrocellulose. In some examples, the probe is immobilized on a solid surface and the mRNA is contacted with the probe, for example, in a gene chip array. Those skilled in the art can easily adapt known mRNA detection methods for use in detecting the level of mRNA encoding a biomarker or other protein of interest.

[0069]

[0082] Alternative methods for determining the level of the target mRNA in a sample include the process of nucleic acid amplification, for example by RT-PCR, ligase chain reaction, self-sustained sequence replication, transcription amplification system, Q-beta replicase, rolling circle replication, or any other nucleic acid amplification method, and then detection of the amplified molecules using techniques known to those skilled in the art. These detection schemes are particularly useful for the detection of nucleic acid molecules when they are present in very low numbers. In some examples, the expression of biomarkers is evaluated by quantitative fluorescent RT-PCR (e.g., TAQMAN system).

[0070]

[0083] Expression levels of the RNA of interest are monitored using membrane blots (e.g., as used in Northern, dot, and other hybridization analyses), or microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acid). Detection of expression also includes the use of nucleic acid probes in solution.

[0071]

[0084] In some examples, microarrays are used to determine the expression or presence of one or more biomarkers. Nucleic acid microarrays provide a method for simultaneous measurement of the expression levels of a large number of genes. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning. The hybridization intensity of each probe on the array is determined and converted into a quantitative value that represents the relative gene expression level. High-density oligonucleotide arrays are particularly useful for determining the gene expression profile for a large number of RNAs in a sample.

[0072]

[0085] In some instances, the array is fabricated on a surface of virtually any shape or even multiple surfaces. In some instances, the array is a flat array surface. In some instances, the array comprises peptides or nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass, or any other suitable substrate. In some instances, the array is packaged to allow for diagnostic or other all-inclusive device operation.

[0073]

[0086] In some instances, the expression or presence of a biomarker described herein is determined at the protein level, for example, using antibodies directed to a particular biomarker protein. These antibodies are used in a variety of methods, such as Western blots, ELISA, multiplexing techniques, immunoprecipitation, or immunohistochemistry. In some instances, detection of the biomarkers is accomplished by ELISA. In some instances, detection of the biomarkers is accomplished by electrochemiluminescence (ECL).

[0074]

[0087] Any means for specifically identifying and quantifying a biomarker in a biological sample is contemplated. That is, in some examples, the expression level of a biomarker protein of interest in a biological sample is detected by a binding protein or a biologically active variant thereof that can specifically interact with the biomarker protein. In some examples, a labeled antibody, binding portion thereof, or other binding partner is used. The word "label" as used herein means a detectable compound or composition that is directly or indirectly conjugated to an antibody, thereby producing a "labeled" antibody. In some examples, the label is itself detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzyme label, catalyzes a chemical change of a substrate compound or composition that is detectable.

[0075]

[0088] Antibodies for detection of biomarker proteins can be monoclonal or polyclonal in origin, or synthetically or recombinantly produced. The amount of complexed protein, e.g., the amount of biomarker protein associated with a binding protein, e.g., an antibody that specifically binds to the biomarker protein, is determined using standard protein detection methods known to those of skill in the art. Detailed reviews of immunological assay design, theory, and protocols can be found in numerous texts in the art.

[0076]

[0089] The choice of marker used to label the antibody will vary depending on the application. However, the choice of marker can be easily determined by one skilled in the art. These labeled antibodies are used in immunoassays as well as histological applications to detect the presence of any biomarker or protein of interest. The labeled antibodies are polyclonal or monoclonal. Furthermore, antibodies for use in detecting proteins of interest are labeled with radioactive atoms, enzymes, chromogenic or fluorescent moieties, or colorimetric tags as described elsewhere herein. The choice of tagging label will also depend on the desired detection limit. Enzyme assays (e.g., ELISA) typically allow for the detection of colored products formed by the interaction of enzyme-tagged complexes with enzyme substrates. Radionuclides that serve as detectable labels include, for example, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, and Pd-109. Examples of enzymes that act as detectable labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and glucose-6-phosphate dehydrogenase. Chromogenic moieties include, but are not limited to, fluorescein and rhodamine. Antibodies are conjugated to these labels by methods known in the art. For example, enzymes and chromogenic moieties are conjugated to antibodies by dialdehydes, carbodiimides, dimaleimides, and other coupling agents. Alternatively, conjugation occurs through ligand-receptor pairs. Examples of suitable ligand-receptor pairs include biotin-avidin or biotin-streptavidin, and antibody-antigen.

[0077]

[0090] In some examples, the expression or presence of one or more biomarkers or proteins of interest in a biological sample is determined by radioimmunoassay or enzyme-linked immunoassay (ELISA), competitive binding enzyme-linked immunoassay, chromatography such as dot blot, Western blot, high performance liquid chromatography (HPLC), or other assays known in the art. That is, detection assays include steps such as, but are not limited to, immunoblotting, immunodiffusion, immunoelectrophoresis, or immunoprecipitation. can be.

[0078] Methods for obtaining pioneer regulatory cytotrophoblast cells

[0091] In some embodiments, the prCTB (e.g., human prCTB) ) are derived in vitro from pluripotent stem cells, e.g., chorionic villi-derived progenitor cells. In some examples, the prCTB is differentiated from the pluripotent stem cells in culture medium supplemented with one or more differentiation factors. In some examples, the stem cells are chorionic villi-derived progenitor cells. In some examples, the chorionic villi-derived progenitor cells include mammalian trophoblast stem cells, e.g., human trophoblast stem cells.

[0079]

[0092] In some examples, disclosed herein is a method for obtaining progenitor regulatory cytotrophoblast (prCTB), comprising differentiating stem cells in vitro by contacting pluripotent stem cells with fibroblast growth factors in a culture medium. In some examples, the culture medium comprises a nucleoside, L-glutamine, a dipeptide containing L-glutamine, a platelet lysate, or a combination thereof. In some examples, the culture medium comprises a nucleoside, a dipeptide, and a platelet lysate. In some examples, the culture medium comprises about 2 mM to about 200 mM L-glutamine.

[0080]

[0093] In some examples, pluripotent stem cells, such as human trophoblast stem cells, are contacted with fibroblast growth factors for about 24 hours to 48 hours, thereby producing prCTB. In some examples, the contact is for at least about 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 40 hours, or 44 hours. In some examples, the contact is for at most about 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 40 hours, 44 hours, or 48 hours.

[0081]

[0094] In some examples, the pluripotent stem cells, such as human trophoblast stem cells, are contacted with a fibroblast growth factor when the stem cells are at passages 5 to 10. In some examples, the pluripotent stem cells are contacted with a fibroblast growth factor when the stem cells are at passages 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0082]

[0095] In some examples, the method includes culturing the stem cells with a culture medium prior to contacting the stem cells with a fibroblast growth factor to differentiate into prCTB.

[0096] In some examples, the culture medium for obtaining and / or maintaining prCTB does not contain an antibiotic, such as penicillin, streptomycin, or any combination thereof. In some examples, the culture medium for obtaining and / or maintaining prCTB does not contain retinoic acid. In some examples, the culture medium for obtaining and / or maintaining prCTB does not contain mercaptoethanol, nicotinamide, or any combination thereof. In some examples, the culture medium for obtaining and / or maintaining prCTB does not contain dexamethasone, recombinant human oncostatin M, BMP4, HGF, or any combination thereof. In some examples, the culture medium for obtaining and / or maintaining prCTB is xenogeneic, e.g., animal component-free. In some examples, the culture medium for obtaining and / or maintaining prCTB is human- and animal-derived, e.g., chemically defined. In some examples, the culture medium for obtaining and / or maintaining prCTB does not contain serum. In some examples, the culture medium for obtaining and / or maintaining prCTB does not contain fetal bovine serum.

[0083]

[0097] In some examples, the fibroblast growth factor is basic fibroblast growth factor (bFGF). In some examples, the stem cells are cultured at about 1 ng / ml to about 100 ng / ml of bFGF. In some examples, the stem cells are contacted with about 2ng / ml to about 50ng / ml, about 4ng / ml to about 30ng / ml, about 6ng / ml to about 15ng / ml, or about 8ng / ml to about 12ng / ml of bFGF. In some examples, the stem cells are contacted with about 6ng / ml, 7ng / ml, 8ng / ml, 9ng / ml, 10ng / ml, 11ng / ml, 12ng / ml, 13ng / ml, 14ng / ml, or 15ng / ml of bFGF. In some examples, the stem cells are contacted with about 10ng / ml of bFGF.

[0084] Methods for obtaining mammalian trophoblast stem cells

[0098] In some embodiments, the mammalian trophoblast stem cells (e.g., human trophoblast stem cells) described herein Human trophoblast stem (hTS) cells can be isolated from umbilical cord, amniotic fluid, amniotic membrane, Wharton's jelly, chorionic villi, placenta, or ectopic pregnancies in a manner that does not disturb or destroy the embryo.

[0085]

[0099] In some examples, the mammalian trophoblast stem cells described herein are cultured in a culture medium that does not contain an antibiotic, such as penicillin, streptomycin, or any combination thereof. In some examples, the culture medium for obtaining mammalian trophoblast stem cells does not contain retinoic acid. In some examples, the culture medium for obtaining and / or passaging mammalian trophoblast stem cells does not contain mercaptoethanol, nicotinamide, or any combination thereof. In some examples, the culture medium for obtaining and / or passaging mammalian trophoblast stem cells does not contain dexamethasone, recombinant human oncostatin M, BMP4, HGF, or any combination thereof. In some examples, the culture medium for obtaining and / or passaging mammalian trophoblast stem cells is xenogeneic, e.g., animal component-free. In some examples, the culture medium for obtaining and / or passaging mammalian trophoblast stem cells is human- and animal-derived, e.g., chemically defined, medium. In some examples, the culture medium for obtaining and / or passaging mammalian trophoblast stem cells is serum-free. In some instances, the culture medium for obtaining and / or passaging mammalian trophoblast stem cells does not contain fetal bovine serum.

[0086]

[0100] In one example, the mammalian trophoblast stem cells (e.g., hTS cells) herein are , amniocentesis biopsy, or amniotic fluid can be isolated from the amniotic fluid. In one example, amniocentesis can be a procedure used to obtain a small sample of the amniotic fluid that surrounds the fetus during pregnancy. In one example, amniocentesis can be offered to women 15-20 weeks pregnant who are at increased risk of chromosomal abnormalities, such as women who are over 35 years old at the time of delivery, or women who have had abnormal maternal serum (blood) screening tests indicating an increased risk of chromosomal abnormalities or neural tube defects. In one example, a needle, such as a long, thin, hollow needle, can be used with ultrasound guidance through the abdomen into the uterus and amniotic sac. A predetermined amount of amniotic fluid, such as about 30 ml (1 ounce), can be withdrawn into a syringe.

[0087]

[0101] In another example, the mammalian trophoblast stem cells (e.g., hTS cells) herein can be obtained from a blastomere biopsy, for example during preimplantation genetic diagnosis (PGD) in conjunction with a reproductive treatment such as in vitro insemination (IVF). In one example, the cells herein can be produced by a method for biopsy of a placenta cyst, the remainder of which implants, resulting in pregnancy and subsequent live birth, e.g., removing the zona pellucida from the placenta cyst and biopsying the placenta cyst.

[0088]

[0102] In another example, the mammalian trophoblast cells (e.g., hTS cells) herein This can be obtained from prenatal chorionic villus sampling (CVS). In one example, CVS is done to extract chromosomal samples from the placenta to test for chromosomal abnormalities and certain other genetic problems. It may be a prenatal test that involves taking a tissue sample. In one example, CVS may be performed between 10 and 12 weeks of pregnancy. In one example, the CVS procedure is transcervical, e.g., by inserting a catheter through the cervix and into the placenta to obtain a tissue sample. In one example, the CVS procedure is transabdominal, e.g., by inserting a needle through the abdomen and uterus into the placenta to obtain a tissue sample.

[0089]

[0103] In one example, the mammalian trophoblast stem cells (e.g., hTS cells) herein are , can be obtained from a placenta biopsy after a full-term pregnancy. In one example, mammalian trophoblast stem cells (e.g., hTS cells) herein can be isolated from the placenta after vaginal or Caesarean delivery.

[0090]

[0104] In some embodiments, the mammalian trophoblast stem cells described herein (e.g. hTS cells have been isolated from first trimester chorionic villus sampling (e.g., gestational age 8). +3 ~12 +0 Chorionic tissue can be isolated from term placentas from 3-week (12-week) or cesarean section deliveries. Chorionic tissue can be separated from the amniotic membrane, minced, and / or enzymatically digested (e.g., with 0.05% trypsin EDTA, for example, for 20 minutes). Cells can then be centrifuged (e.g., at 1500 rpm, for example, for 5 minutes), counted, and / or replated (e.g., at 1000 rpm, for example, for 5 minutes) in medium (e.g., Dulbecco's modified Eagle's medium + 10% fetal bovine serum) (e.g., at 1000 rpm, for example, for 5 minutes). 2 10 per 4 In one example, the isolated cells can be plastic adherent. In one example, the cells can be used at passages 4-8.

[0091]

[0105] In one example, the mammalian trophoblast stem cells (e.g., hTS cells) herein are can be isolated from term (e.g., 38-40 weeks gestation) placentas according to the following procedure: Umbilical cord blood is drained from the placenta, which is then carefully dissected open. The collected tissue pieces are washed several times (e.g., with phosphate-buffered saline), then minced (e.g., mechanically) and enzymatically digested (e.g., with 0.25% trypsin-EDTA). The homogenate is then pelleted by centrifugation and suspended in complete medium (e.g., Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and / or 100 g / ml streptomycin). Culture in a well-ventilated room temperature for 24 hours at room temperature. The homogenates are then incubated at 4° C. for 24 hours at room temperature ... 2Maintain cell cultures at 37 °C. Change medium regularly, e.g., 1-2 times per week. Once cells reach a desired level of confluence, e.g., above 80% confluence, harvest cells, e.g., with 0.25% trypsin / EDTA, and re-plate, e.g., at a dilution of 1:3.

[0092]

[0106] In another example, the mammalian trophoblast stem cells (e.g., hTS cells) herein can be isolated from human placenta after birth according to the following procedure: The chorion is separated by peeling it off from the amniotic membrane. The decidual tissue is minced (e.g. mechanically), washed (e.g. in Dulbecco's phosphate-buffered saline), and cut into small pieces (e.g. about 2 x 2 cm). The chorion is minced into small pieces, treated with enzymes (e.g. 0.5% trypsin-EDTA for e.g. 5 min), and digested with collagenase I (e.g. 0.3% for 20-30 min in a 37°C incubator). The mobilized cells are then collected and passed through a cell strainer (e.g. 100 μm). The filtered cells are collected by centrifugation (e.g. 2,500 rpm, e.g. 5 min). The cells are resuspended in medium (e.g. alpha modified minimum essential medium supplemented with 10% fetal bovine serum and / or 1% penicillin-streptomycin) and incubated under suitable conditions (e.g. 37°C and / or 5% CO). 2 ) in a container (e.g., a T25 flask). The medium is replaced periodically, e.g., every 3 days, until the chorionic MSCs reach a desired confluency, e.g., 70% confluency.

[0093]

[0107] In another example, chorionic villi are the site of unruptured preimplantation embryos from women with ectopic pregnancies. The chorionic tissue can be obtained from the oviduct of a female fetus (e.g., 5-7 weeks of gestation). Microscopic chorionic tissue is thoroughly dissected in a suitable medium (e.g., serum-free α-MEM), identified under a microscope, and incubated for a certain period of time (e.g., 1 Trypsinize with trypsin / EDTA (e.g., 0.025% trypsin / EDTA) for 5 min and stop the reaction by adding medium (e.g., α-MEM with 10% FBS). Obtain adherent cells and culture them under suitable conditions (e.g., conditioned α-MEM, 10% FBS, and 1% penicillin-streptomycin, 37°C, 5% CO). 2 After two passages, levels of hCG may become undetectable as measured by commercially available kits (e.g., Dako, Carpinteria, Calif.).

[0094] Kits / Manufacturing Articles

[0108] Disclosed herein are kits and articles of manufacture for use with one or more of the methods and compositions described herein.Such kits include carriers, packages, or containers that are partitioned to accommodate one or more vials, tubes, or other containers, each of which contains one of the individual elements used in the methods described herein.Suitable containers include, for example, bottles, vials, syringes, and test tubes.In some examples, containers are formed from various materials, such as glass or plastic.

[0095]

[0109] The articles of manufacture provided herein include packaging materials. Examples of pharmaceutical packaging materials include: Included are, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material appropriate for the selected dosage form and intended mode of use.

[0096]

[0110] For example, the container may contain hTS cells, which may be included in the compositions disclosed herein. Such kits optionally include an identifying description or a label or instructions for use in the methods described herein.

[0097]

[0111] Kits typically include a label listing the contents and / or instructions for use. The pharmaceutical composition includes a package insert containing instructions for use. A set of instructions is also typically included.

[0112] In some instances, the label is on or associated with the container. In some instances, a label is on a container when letters, numbers, or other characters forming the label are attached, molded, or etched into the container itself, and a label is associated with a container when the label is present in a receptacle or carrier that holds the container, e.g., as a package insert. In some instances, a label is used to indicate that the contents are to be used for a particular therapeutic application. The label also indicates directions for using the contents, e.g., in the methods described herein.

[0098] Working Example

[0113] The following examples are non-limiting and merely representative of various aspects and features of the present invention. EXAMPLES

[0099] Transformation properties of human trophoblast stem (hTS) cells upon stress

[0114] How do blastocyst trophoblasts respond to the threat of maternal hyperglycemia during embryo transport in the oviduct? To explore how this mechanism plays a role in insulin resistance, the effects of high glucose (20 mM) were tested in hTS cells. It was found that glucose rapidly induced the transient activation of sweet taste receptors T1R2 / T1R3 in the cell membrane, which resulted in the activation of G protein signaling in the cells, resulting in the Gαq / 11 / CaMKII / CREB1 and Gβ / GSK3β / MAFA signaling pathways (Figure 1A). The regulatory molecular mechanisms are shown in the supporting information (Figures 2A-2J). In the nucleus, the two G protein pathways cooperated to target the promoter of the insulin gene for insulin-producing transcription (Figure 1A). ChIP-qPCR analysis showed that knockdown of transcription factors CREB1 and MAFA by specific shRNA reduced insulin expression (Figure 1B). Meanwhile, the activated T1R2 / T1R3 signaling induced the glucose sensor and transporter GLUT2 to promote glucose entry into the cells. Glucose enhanced ATP production and opened L-type voltage-gated Ca2+ channels (VGCCs), leading to the entry of extracellular calcium into the cells, which promoted insulin secretion into the culture medium as detected by radioimmunoassay (RIA). This effect was confirmed by sulfonylureas promoting insulin secretion, whereas the VGCC inhibitor nifedipine blocked insulin secretion as measured by RIA (Figure 1C). These results suggested that glucose allows the control of insulin synthesis and secretion in hTS cells. This insulin expression was attributed to CREB1 signaling, but not to conventional pancreatic and duodenal homeobox 1 (PDX1), as measured by qPCR analysis (Figure 2K).

[0100]

[0115] Furthermore, immunofluorescence imaging studies revealed that insulin, CREB1, Ca We found that not only MKII, GLUT2, and MAFA, but also β-hCG, histocompatibility antigen HLA-G, and pluripotency transcription factor CDX2, but not OCT4 (octamer-binding transcription factor 4), were expressed in hTS cells (Figure 1D). The stress protein heat shock protein 90 (HSP90) was also expressed. However, hTS cells did not express the proliferation marker Ki67, the protein folding activator HSP70, the tumor suppressor p53, the autoantigen GAD65, and the cell-cell fusion protein syncytin (Figure 2M), supporting the notion that hTSCs are at the initial position of TE-differentiated trophoblasts.

[0101]

[0116] The effect of glucose on hTS cells was further examined morphologically, and Positive staining of zinc chelator dithizone (DTZ), a specific stain in in vitro culture of cells, showed that high glucose rapidly induced cell changes from adherent fibroblast characteristics to 3D aggregated cell clusters. By stopping high glucose, the cells were rapidly released from the clusters and reattached to the culture dish, reverting to the original fibroblast characteristics. The cellular processes were recorded by a computer microscope video system (Olympus, IX-81, DP30, MIU-IBC-IF-2) and shown in the online video in the appendix (Video S1). Furthermore, ultrastructural studies of the cell clusters revealed a large cytoplasm-to-nucleus ratio, with desmosomal connections between the two cells. Similar to those in pancreatic β cells, various empty vesicles, abundant mitochondria, and immature granules in the vesicles were observed within the cytoplasmic compartment. To this end, it was demonstrated that while hTS cells have the ability to express insulin and the stress protein HSP90 to cope with external threats, they are highly susceptible to glucose stimulation, which can initiate reversible cellular transformation of hTS cells. EXAMPLES

[0102] In vitro induction of pioneer regulatory cytotrophoblast (prCTB) cells

[0117] In this example, we investigated the effect of bFGF on trophectoderm-derived hTS cells. In one experiment, prCTB was induced in vitro from hTS cells.

[0103]

[0118] bFGF induces epithelial-mesenchymal transition (EMT) via TGF-β1.

[0119] bFGF induced the expression of TGF-β1 in hTSCs (Figure 4A), which led to the development of mesenchymal It was found by Western blot assay (Figure 4B) and immunocytochemistry (Figure 4C) that bFGF upregulated the cell marker vimentin but downregulated the cell-cell adhesion protein E-cadherin. These effects were neutralized by pretreatment with TGF-β1 antibody (Figure 4D). Light microscopy revealed that the cell shape changed from elongated spindle-like to thick and short features with a rounder nucleus (Figure 4E). These phenomena suggested that bFGF induced EMT to gain the ability of migration and invasion.

[0104]

[0120] bFGF promotes definitive endoderm signature via mRNA-124α do.

[0121] bFGF targets the receptor FGFR1 on the cell membrane and activates PI3K / AKT / It was found that bFGF induced activation of the CREB1 signaling pathway. Computational survey of DIANA-mirGen 2.0 confirmed that bFGF-induced CREB1 targets the consensus CREB binding sequence (TGACGTCA) in the promoter of microRNA-124a (miR-124a) by qPCR analysis, while knockdown of CREB1 reduced the expression of miR-124a. A positive correlation between CREB1 and miR-124a was observed.

[0105]

[0122] To gain insight into the downstream effectors of miR-124a, we Plasmids of SMAD4, GSK3β, and CDX2 using pGL4.51 vector (Promega, Madison, WI) were constructed for the reporter gene assay. First, miR-124a was shown to target the SMAD4 gene to produce inhibitory SMAD4. As SMAD4 can interact with SMAD2 / 3 and bind to sequences in the central promoter of the MIXL1 gene for transcription, inhibitory SMAD4 elicited the inhibitory homeodomain protein MIXL1. This was confirmed by using SMAD4 shRNA and supported the imaging study (bFGF (10 ng / ml) was found to induce apparent expression of MIXL1 at 15 min compared to the control, and the intensity of MIXL1 expression was reduced at 4 h of induction, indicating that it is a transient expression). Second, miR-124a was shown to suppress glycogen synthase kinase 3β (GSK3β), which was confirmed by pretreatment of miR-124a and anti-miR-124a antibody. As a result, inhibitory GSK3β triggered downstream β-catenin accumulation, resulting in nuclear translocation targeting FOXA2 gene for transcription, which was proven by using shRNA against β-catenin assay (Figure 4F). And also, forkhead box protein A2 (FOXA2) controls the expression of PDX1, and proendocrine transcription factor neurogenin 3 (NGN3) contributes to participate in the decision of endocrine cell differentiation. Third, miR-124a targeted CDX2 gene to inhibit CDX2 synthesis, which was confirmed by pretreatment of miR-124a and anti-miR-124a antibody. Downregulated CDX2 resulted in upregulation of OCT4. Then, OCT4 targeted SOX17 gene for transcription to produce SOX17 (SRY-box protein 17), which was supported by imaging study. Taken together, we demonstrated that bFGF could promote miR-124a to upregulate SOX17, FOXA2, and OCT4, and downregulate MIXL1 at 8 h induction, resulting in the acquisition of a definitive endoderm (DE) signature, which may contribute to further insulin expression.

[0106]

[0123] bFGF induces the production of prCTB from hTS cells.

[0124] PDX1, pancreatic transcription factor 1 protein (PTF1a), SOX9, and homeo A significant upregulation of pancreatic progenitor biomarkers, including the box 1 protein NKX (NKX6.1), was noted at 8 h of bFGF induction. The appearance of these molecular signals initiated the expression of NGN3, proinsulin C-peptide, and insulin at 20 h of induction. Besides insulin, immunofluorescence imaging confirmed the presence of various pancreatic progenitor and endocrine cell markers, including PDX1, hepatocyte nuclear factor 1-β (HNF1B), NGN3, SOX9, NKX6.1, and insulin, as well as NANOG, SOX2, glucagon, somatostatin, GLUT2, and polypeptide (PP) in bFGF-treated hTS cells (Figure 3).

[0107]

[0125] Next, hTS cells induced by bFGF initially expressed CDX2, but , but not expressing OCT4 (Figure 5C). As differentiation proceeded, OCT4 was upregulated, whereas CDX2 was downregulated, controlling pluripotency at the DE stage at 8 h of induction. As OCT4 was gradually downregulated, NANOG was upregulated to a peak at 12 h of induction, similar to the mesendoderm stage. HSP90 maintained the levels of OCT4 and NANOG in the differentiating trophoblast. As differentiation entered the progenitor stage, toward the end of the day after 12 h of induction, NANOG was downregulated, whereas SOX2 sustained upregulation, resulting in the formation of insulin-expressing prCTB. Of note, these molecular processes are similar to β-cell development in the pancreas, but distinct from the glucose-induced insulin expression in hTS cells described above. While the spatiotemporal changes in pluripotency transcription factors are mainly attributed to mutual negative autoregulatory mechanisms, SOX2 plays a major role in maintaining the stemness of prCTB. To this end, our experiments have demonstrated that bFGF can efficiently induce differentiation of isolated hTS cells into a novel extrapancreatic tissue-specific phenotype that expresses insulin, which we refer to herein as pioneer regulatory cytotrophoblast (prCTB). As demonstrated here, prCTB can be maintained primarily by SOX2 during trophoblast differentiation in vitro. EXAMPLES

[0108] Differences between prCTB derived from hTS cells and primitive trophoblast cells

[0126] Immunohistochemistry revealed that the majority of prCTB cells express insulin, GLUT2, and C Staining by aMKII, Ki67 (growth factor), β-hCG, and HLA-G was evident, suggesting that prCTB was proliferative (Figure 5A). Stress proteins, including GAD65, HSP70, HSP90, and p53, but excluding syncytin, were also expressed in prCTB, distinct from the findings in hTS cells (Figure 2M). These results implied that the effects of extracellular stressors could drive the conversion of hTS cells to prCTB in vitro.

[0109]

[0127] In normal uterine pregnancy, primitive cytotrophoblasts (pCTBs) are responsible for 1) trophoblast growth and ) syncytiotrophoblasts, 2) primitive syncytiotrophoblasts (pSTB) and late syncytiotrophoblasts (STB), and 3) extravillous cytotrophoblasts (EVT). Villous cytotrophoblasts were found to express HSP90 and insulin, but not p53 and syncytin (Figure 5B). STB expressed insulin, p53, and syncytin, but not HSP90. EVT expressed clear insulin and p53, but not HSP90 and syncytin, suggesting that they are invasive but not proliferative. Figure 5C illustrates such a developmental process. EXAMPLES

[0110] Secretomes and exosomes from hTS cells and prCTB

[0128] Chemokine, cytokine, and growth factor levels were measured in hTS cells and p in the culture medium of rCTB, measured by Milliplex assay based on Luminex technology. The results revealed that both hTS cells and prCTB could release obvious amounts of various secretomes or exosomes (Figure 7A, upper panel).

[0111]

[0129] 1) Chemokines

[0130] This group includes RANTES (also CCL5), MCP-1 (CCL2, monocyte cell lineage also moiractant protein-1), GROα (also CXCL1, CXCL2, macrophage inflammatory protein 2-α or MIP2-α), MCP-3 (also CCL7), IL-8, eotaxin (also CCL11, CCL24, and CCL26), MDC (also CCL22), IP-10 (also IFNγ-induced CXCL10), fractalkine (also CX3CL1), MIP-1β (also CCL4), and soluble CD40-IL-1. The tumor necrosis factor (CD154) was also involved.

[0112]

[0131] 2) Cytokines and growth factors

[0132] This group includes IL-6, IL-10, IL-4, IL-7, IL-15, IL The secreted growth factors included PDGF-AA and PDGF-AB / BB (platelet-derived growth factor family), VEGF, EGF (epidermal growth factor), bFGF, GM-CSF (granulocyte-macrophage colony-stimulating factor), Flt3L (FMS-like tyrosine kinase 3 ligand), and IL-1β.

[0113]

[0133] 3) Other proteins

[0134] Furthermore, secretome analysis revealed that both hTS cells and prCTB express soluble They were able to release human leukocyte antigen G (sHLA-G), transforming growth factor beta 1 (TGF-β1), plasminogen activator inhibitor-1 (PAI-1), and IL-10 (Figure 7B).

[0114]

[0135] Nevertheless, the commercially available human platelet lysate used (i.e., PLUS, mpass) also secreted exosomes into the culture medium. By excluding the basal effect of PLUS, it was found that both naive hTS cells and prCTB were indeed able to secrete exosomes containing mainly chemokines such as GRO, MCP-1, fractalkine, IP-10, MCP-3, eotaxin, and small amounts of MIP-1β, as well as cytokines such as IL-6 and IL-8 with small amounts of IL-4, IL-1RA, IL-10, IL-12P40, IL-15, IL-1α, and IL-17A (Fig. 7A, lower panel). Meanwhile, growth factors included PDGF-AA, VEGF, bFGF, and G-CSF, as well as smaller amounts of Flt-3L and GM-CSF (Fig. 7A, lower panel). These data suggested that both naive hTS cells and prCTB may have the ability to release various secretomes or exosomes for functionality.

[0115]

[0136] Figure 7C shows the expression of cytokines IL-6 and IL-6 in both hTSCs and prCTBs. Another summary of the production of IL-8, chemokines MCP-1 and CXCL2, and angiogenic factors PDGF-AA and VEGF is shown. prCTB expressed the angiogenic molecules CD105 (endoglin, a receptor for TGF-β that functions in angiogenesis) and CD146 (vascular endothelial cadherin) (Figure 7D). These results suggested that prCTB has the ability to function in the decidual tissue at the fetal-maternal interface. EXAMPLES

[0116] prCTB exhibits immune cell-associated biomarkers.

[0137] Expression of immune cell-associated biomarkers was analyzed in prCTB by FACS analysis. Eight independent cell lines were examined for NK and T cell biomarkers, showing similar patterns for NK and T cell biomarkers. Results revealed that both NK and T cells expressed CD biomarkers including CD4+, CD8+, CD107a+, and (CD16+CD56)+, sharing similar biomarkers between T and NK cells. Representative cytometric analyses are shown in Figure 8 and tables in Figures 9A-9M. (CD16+CD56) + hTSC and CD107a(+) cells showed the highest expression in both hTSC and prCTB (Figs. 8 and 9L). Their combined panels also accounted for the majority of cell populations in distribution (Figs. 8 and 9M). These results suggested that prCTB has the ability to exert immune cell-like functions at the fetal-maternal interface. It was also found by immunostaining that prCTB expressed CD11b and CD49f (Fig. 9N).

[0117]

[0138] Furthermore, immunoblot assays demonstrated that both hTS cells and prCTB We identified that they expressed ILT-2 (leukocyte Ig-like receptor 1, also LILRB1), ILT-4 (also LILRB2), TCR (T cell receptor), and specifically, KIR2DL4 (killer cell Ig-like receptor), which is expressed by a subset of NK cells and CD8+ T cells and inhibits cytolytic NK cell function (Figure 7E). They also expressed PD-L1 (programmed death-ligand 1), Fas (apoptosis signal receptor, also APO-1), FasL (Fas ligand), which induces apoptosis of infiltrating lymphocytes, and NKp46 (the major NK cell activating receptor) (Figure 7E), which are involved in the elimination of target cells. Interestingly, bFGF activated signal transducer and activator of transcription 3 (STAT3) and the transcription factor c-JUN, which consequently promoted the expression of FasL in the differentiation of prCTB (Figure 7F). This effect was confirmed by knockdown of FGFR1 by shRNA. As a result, the (CD16+CD56) molecules of prCTB produced IFN-γ and stimulated PD-L1 production, thereby recognizing the related receptor PD-1 in tumor cells and downregulating the immune response against malignant diseases.

[0118]

[0139] bFGF mediates the FGFR1 / CREB1 signaling pathway in the prCTB. This induces IL-6 and IL-8.

[0140] How prCTB produces the cytokines IL-6 and IL-8 To explore, hTSCs were incubated with bFGF for 1 day to mimic their transient residence in the oviduct. Mechanistically, bFGF activated its receptor FGFR1 in the cell membrane to induce PI3K / phosphorylated (p)AKT signaling. Then, pAKT interacted with and phosphorylated downstream pCREB1 (cAMP-responsive element-binding protein 1), activating pCREB1 signaling. These molecular processes were confirmed by using the FGFR1 inhibitor PD166866, as well as specific shRNAs for PI3K and pAKT (Figure 7G-7J). In the nucleus, CREB1 targeted genes to produce IL-6 in a time-dependent manner (Figure 7K, left panel) and IL-8 in a dose-dependent manner (Figure 7K, right panel), which was confirmed by ELISA assay (Figure 7C). These molecular processes occurred in a time- and spatial-consistent manner with EMT. In conclusion, bFGF induces the conversion of hTSCs to prCTBs and the production of IL-6 and IL-8 in prCTBs in an autocrine / paracrine manner. In prCTB, IL-6 induces the trophoblast marker β-hCG and IL-8 induces the NK cell marker CD56.

[0119]

[0141] RT-qPCR assay revealed that IL-6 was expressed on the cell membrane in prCTB. In the IL-6R-IL-8 complex, IL-6 bound to the receptor IL-6R and activated CREB1 signaling, resulting in the production of β-hCG (Fig. 7L, left panel). Interestingly, Western blot showed that IL-6 could bind to another receptor GnRHR and simultaneously activate CREB1 signaling, resulting in the production of β-hCG (Fig. 7M). Meanwhile, RT-qPCR analysis showed that IL-8 induced the production of CD56 (also known as NCAM), but not through IL-8R (Fig. 7L, right panel). However, Western blot assay found that IL-8 could instead bind to another receptor CXCR2 and activate STAT3 (signal transducer / activator of transcription 3) signaling, and also target transcriptional genes to produce CD56 (Fig. 7N). These molecular processes were confirmed by using the GnRHR inhibitor Elagolix and the CREB1 inhibitor 666-15 for the expression of β-hCG (Figure 7L), while the CXCR2 inhibitor SB225002 and the STAT3 inhibitor Stattic for the expression of CD56 (Figure 7N). These results suggested that IL-6 induces the trophoblast biomarker β-hCG in prCTB, while IL-8 simultaneously induces the NK cell biomarker CD56 in an autocrine / paracrine manner. To this end, we investigated whether bFGF induces the conversion of hTSCs to prCTB and induces the unique β-hCG ( It was demonstrated that CD56(+)prCTB results.

[0120]

[0142] IL-8 mediates CXCR2 / CREB1 signaling in prCTB Induce the T cell marker CD4.

[0143] At the same time, IL-8 is expressed in an autocrine / paracrine manner at the cell membrane of prCTB. It bound to and activated the receptor CXCR2, which induced CREB1 signaling and allowed its nuclear translocation. In the nucleus, CREB1 targeted the CD4 gene for transcription to produce CD4 molecules by Western blot assay (Figure 7O). However, it was discovered that IL-8 can also bind to the receptor CXCR2, which resulted in activating STAT3 signaling and leading to the nuclear translocation of STAT3. In the nucleus, STAT3 targeted various sites in the CD4 gene for transcription to produce CD4 (Figure 7P). These molecular processes were confirmed by using the CXCR2 inhibitor SB225002, the CREB1 inhibitor 666-15, and the STAT3 inhibitor Stattic (Figures 7O and 7P).

[0121]

[0144] IL-8 induces CD4(+)Foxp3(+)Treg cell-like prCTB Induce Foxp3 to promote cell death.

[0145] IL-8-induced CXCR2 signaling binds and results in S It was found that IL-8 activated STAT3 signaling for the nuclear translocation of TAT3. Then, according to Western blot analysis, STAT3 targeted the Foxp3 gene for transcription to produce Foxp3 protein (also known as scarfin, which is involved in immune system response) (Figure 7P). These molecular processes were confirmed by using the CXCR2 inhibitor SB225002 and the STAT3 inhibitor Stattic (Figure 7P). These results suggested that IL-8 produced CD4(+) and Foxp3(+) molecules in prCTB, mimicking CD4(+)Foxp3(+) Treg cells. Immunocytochemical co-staining of CD4(+)Foxp3(+) biomarkers was achieved in prCTB (Figure 7Q). EXAMPLES

[0122] prCTB barrier at the fetal-maternal interface

[0146] prCTB expresses factors that promote angiogenesis in decidual tissue.

[0147] prCTB inhibited VEGF and They could secrete PDGF-AA and plasminogen activator inhibitor-1 (PAI-1) and IL-10 by ELISA assay (Figure 7B), and express CD105(+) and CD146(+) markers by FACS analysis (Figure 7D). The expression of all these molecules suggested that prCTB has the ability to promote angiogenesis and vasculogenesis in SA remodeling of decidual tissue, for example, at the fetal-maternal interface.

[0123]

[0148] MCP-1 and CXCL2 synergistically promote prCTB translocation.

[0149] Transwell invasion and migration assays revealed that hTSC and prCTB It was revealed that MCP-1 significantly induced the invasion and migration of prCTB in both dose-dependent (Fig. 10A) and time-dependent (Fig. 10B) manners. However, CXCL2 promoted cell migration in a dose-dependent manner in prCTB (Fig. 10C) and in a time-dependent manner in hTSC and prCTB (Fig. 10D). These results suggested that MCP-1 and CXCL2 have the ability to synergistically promote the migration of prCTB in a time- and dose-dependent manner.

[0124]

[0150] Formation of a novel prCTB barrier at the fetal-maternal interface

[0151] Immunohistochemical imaging revealed that prCTB inhibited syncytin, p53, and β-h It was revealed that CD56(+)prCTB migrated into the EVT with the expression of syncytin, p53, β-hCG, and HLA-G, while HSP90 appeared in the inner CTB layer (Fig. 10E). Immunohistochemistry revealed that at the fetal-maternal interface, CD56(+)prCTB was sporadically distributed in the villous stromal tissue with a tendency to migrate into aggregation in the invasive EVT (Fig. 10F, upper and middle panels). These results suggested that prCTB differentiated into EVT expressing syncytin, p53, β-hCG, and HLA-G to synergistically invade the decidual epithelial cell layer of the maternal decidua for implantation.

[0125]

[0152] Both CD56(+) and β-hCG(+) prCTB are anchored in the maternal decidua It was found that CD56(+)prCTB could be invaded by the decidual vein (middle panel of Fig. 10F, upper panel of Fig. 10G). Through invasion and migration, CD56(+)prCTB occupied a large amount of cellular components similar to conventional decidual natural killer (dNK) cells in the decidual tissue (lower panel of Fig. 10F), while β-hCG(+)prCTB showed a similar behavior (middle panel of Fig. 10G). It was found that β-hCG(+)prCTB replaced the position of endothelial cells in the ductus arteriosus (middle panel of Fig. 10G) and also appeared in the vascular lumen of the vein (lower panel of Fig. 10G), suggesting the phenomenon of SA remodeling and the invasion of prCTB into the decidual vein. Finally, many CD56(+)β-hCG(+)prCTB could accumulate on the decidual side to form a cell barrier, which is referred to herein as the "prCTB barrier" at the fetal-maternal interface. EXAMPLES

[0126] Upon interaction, prCTB induces apoptosis of solid tumor cells.

[0153] Pancreatic cancer cells (PANC-1, CRL-1469)

[0154] Investigating how pancreatic cancer cells (PANV-1) interact with prCTB Optical microscopy revealed that in the co-culture of prCTB and PANC-1, prCTB was able to migrate, engulf and invade the cell colonies of PANC-1, resulting in the apoptosis of PANC-1 (Fig. 11A). This apoptosis phenomenon was further demonstrated by using the Apoptosis / Necrosis Detection Kit (blue, green, red) according to the manufacturer's instructions (ab176749, Abcam). Figure 11B showed the interaction of the two live cells, and Figure 11C revealed the apoptosis of PANC-1 upon the interaction. This interaction was further demonstrated by 3D fluorescence microscopy, which showed the apoptosis of PANC-1 (Fig. 11D).

[0127]

[0155] Mechanistically, prCTB binds to the protein PD-L1 (programmed cell death ligand 1). We found that prCTB expressed PD-L1 (PD-1) but not PD-1 (programmed cell death protein 1) (Fig. 11E, left column), whereas PANC-1 expressed both PD-L1 and PD-1 (Fig. 11E, right column). This means that prCTB could deliver PD-L1 / PD-1 cell death signaling to target PANC-1 cells to trigger apoptosis of PANC-1 cells. However, since prCTB lacks PD-1, PD-L-1 in PANC-1 cells could not deliver cell death signaling to prCTB, explaining the failure of apoptosis with prCTB. While addressing the Fas / FasL cell death signaling pathway, we found that prCTB expressed both Fas ligand (FasL) and Fas (as a receptor for FasL), whereas PANC-1 expressed Fas but not FasL (Figure ​(Figure11F),11F), suggesting that prCTB delivers apoptotic FasL / Fas signaling to the target PANC-1, initiating PANC-1 apoptosis. In contrast, prCTB expresses Fas, but PANC-1 lacks FasL, suggesting that FasL / Fas cell death signaling does not occur in prCTB upon interaction.

[0128]

[0156] Breast cancer cells (MCF-7, HTB22)

[0157] 3D fluorescence microscopy of prCTB co-cultured with breast cancer cell line (MCF-7) The attraction and interaction leading to the apoptosis of MCF-7 observed in prCTB was revealed (Figure 11G). PD-L1 expressed by prCTB is thought to send cell death signaling to its receptor PD-1 on MCF-7 cells, resulting in an apoptotic response (Figure 11H, upper panel). Although MCF-7 also expressed PD-L1, apoptosis was prevented because prCTB lacks the PD-1 receptor (Figure 11H, lower panel). However, examining the other FasL / Fas cell death axis, it was discovered that prCTB expressed FasL, while MCF-7 expressed Fas, allowing the occurrence of FasL / Fas cell death signaling in MCF-7 (Figure 11I, upper panel).

[0129]

[0158] prCTB contains the anti-apoptotic proteins Bfl-1 and Mcl-1 .

[0159] Interestingly, prCTB expressed Fas, whereas MCF-7 expressed FasL. (Fig. 11I, lower panel). This fact suggests that apoptosis may occur in prCTB, but this was not the case. To explain this, we found by RT-qPCR analysis that prCTB significantly expressed high levels of anti-apoptotic Bfl-1 and Mcl-1 mRNA in prCTB (Fig. 11J). Both Bfl-1 and Mcl-1 are members of the Bcl-2 family of proteins and contain anti-apoptotic capabilities to circumvent cancer cell death signaling, thereby promoting cell survival.

[0130]

[0160] Other solid tumor cells

[0161] Therefore, we performed co-cultures of prCTB with a series of solid tumor cells and The apoptosis was detected using a combination of chemistry and 3D fluorescence microscopy. All solid tumor cells, including hepatic Huh7 cells, ovarian PA-1 (CRL-1572) cells, lung H1299 (CRL-5803) cells, gastric MNK45 (TCP-1008) cells, and melanoma A375 (CRL-1619) cells, exhibited apoptosis during co-culture (Figure 11K). These results suggested that prCTB contains the ability to eradicate various solid tumor cells, depending on which cell death signaling pathway is involved. EXAMPLES

[0131] Similarities between dNK cells and prCTB

[0162] Women who had abortions for medical reasons and women with ectopic pregnancies at the same gestational age of 8 weeks We obtained dNK cells in the villous tissue with the consent of the patient. First, we confirmed the presence of CD56 biomarker in dNK cells by immunocytochemistry (CD56 was also expressed in prCTB) (Fig. 12A, upper panel). Subsequently, immunohistochemistry of chorionic villi revealed that sporadic CD56(+) dNK cells were found among the inner villous CTB and villous stroma, while they were accumulated in the EVT zone (Fig. 12A, middle panel). β-hCG(+) dNK cells (β-hCG was also expressed in prCTB) were observed in the villous trophoblast and accumulated in the EVT zone (Fig. 12A, right panel). In normal implantation, accumulated CD56(+) dNK cells and β-hCG(+)CTB were found in the EVT zone and distributed sporadically in the nearby decidual tissue (Fig. 12B, left upper and lower panels, respectively). Interestingly, a large number of CD56(+) dNK cells and β-hCG(+) CTB were expressed in the maternal decidual tissue (Figure 12B, upper and lower panels, left, respectively). Through secreted exosomes, prCTB, like dNK cells, may have the ability to migrate into the decidual tissue and communicate with decidual stromal cells to control the maternal immune system and ultimately complete implantation. EXAMPLES

[0132] Materials and methods of the experiments described in Examples 1 to 8

[0163] Experimental model and subject details

[0164] Human trophoblast stem (hTS) cells were derived from trophoblast tissue. Women with ectopic pregnancies at 7–8 weeks of gestation were obtained with informed consent. The study was approved by the Institutional Review Board of KMUH. Naïve hTS cells were cultured and passaged in α-MEM (ThermoFisher Scientific) supplemented with 10% (v / v) fetal bovine serum (FBS, SAFC Bioscienses) at 37°C in humidified air containing 5% CO2. Cultures were manually passaged every 2–3 days at a split ratio of 1:3–1:6. Low seeding densities and new culture media were tested for the proliferation of hTS cells, including 1) MesenCult™-ACF Plus medium with MesenCult™-ACF PLUS 500x Supplement and L-glutamine, with and without substrates such as Cell Attachment Substrate, and 2) alpha-MEM with nucleosides, GlutaMAX™ Supplement, and 10% Stemulate™ Human Platelet Lysate Cell Culture Media Supplement, with and without substrates. FBS was replaced with CMP grade PLUS (Compass Biochemical) for flow cytometric analysis of CD molecules. Characteristic biomarkers including HLA-G, β-hCG, and CDX2 were stably expressed, along with undetectable CD34 and CD45. Induction of hTSC cells to prCTB was performed by treatment with 10ng / ml bFGF for 24 hours in hTS cells at passages 5–10. Several media were tested for induction, including 1) MesenCult™-ACF Plus medium with MesenCult™-ACF PLUS 500x Supplement and L-glutamine, with and without substrates such as Cell Attachment Substrate, and 2) alpha-MEM with nucleosides, GlutaMAX™ Supplement, and 10% Stemulate™ Human Platelet Lysate Cell Culture Media Supplement, with and without substrates. Seeding densities were approximately 10,000 cells / cm. 2The cultures were free of penicillin, streptomycin, mercaptoethanol, and / or nicotinamide. The cultures were also free of animal components, serum such as fetal bovine serum, antibiotics, retinoic acid, dexamethasone, recombinant human oncostatin M, BMP4, and / or HGF. Differentiation regimens were determined by experimental experience (data not shown). Stage-specific differentiation lineages were referenced to various cellular biomarkers as previously described. Cells were harvested at the indicated times for various analyses.

[0133]

[0165] Transfection experiments

[0166] hTS cells were transfected with TransIT-LT1 transfection reagent (Miru siRNA or shRNA, or 3'UTR reporter plasmids were transfected using a 50-mL PBS (Microfluidics Bio LLC). Transfections were performed with 2 μg of siRNA or shRNA and 4 μl of transfection reagent in 100 μl of OPTI-MEM (Gibco). After 10 min incubation at room temperature, the transfection mixture was gently added to the cells overnight. For further treatment, the transfected cells were re-incubated with α-MEM supplemented with 10% FBS.

[0134]

[0167] Plasmid construction and dual-luciferase reporter assay

[0168] To construct the luciferase-3'UTR reporter plasmid, 3'UTR fragments were amplified from genomic DNA extracts of cells. The 3'UTR regions were PCR amplified using a forward primer with a PsiI site and a reverse primer with an MfeI site. The primers for the 3'UTR reporter constructs are listed below: for Cdx2 3'UTR region: 5'-aaattataagctgtttgggttgttggtct-3' and 5'-aaacaattgcccccataatttctgactgc-3'; for Smad4 3'UTR region: 5'-aaattataactcccaaagtgctgggatta-3' and 5'-aaacaattgctg for Smad4 3'UTR region 1: 5'-aaattataacagttgtcccagtgctgcta-3' and 5'-aaacaattgatgacttgcccaaaggtcac-3'; for GSK3β 3'UTR region: 5'-aaattataacccacaactggggtaaaaga-3' and 5'-aaacaattgctgtggaaggggcaaagata-3'. After combined PsiI and MfeI digestion (NEB), the 3'UTR inserts were subcloned into pGL4.51 plasmid (Promega) by using T4 DNA ligase (Takara).

[0135]

[0169] For dual luciferase assays, firefly luciferase reporter (50 hTS cells were co-transfected with either an empty vector without a 3'UTR (1.5 × 10 per well) or the pGL4.74 vector (1.5 × 10 per well), Renilla luciferase plasmid (500 ng, Promega), and a nonspecific control miRNA (30 pmol) or miR-124a precursor (30 pmol, System Biosciences). 4Cells). After another 24 h of changing the transfection medium, luciferase activity was analyzed by Dual Luciferase Reporter Assay System (Promega) and Centro LB960 Microplate Luminometer (Berthold Technologies). For evaluation, Renilla luciferase values ​​were first normalized to firefly luciferase activity, and the calculated activity of each 3'UTR reporter was further normalized to the activity of the control vector. Data are presented as mean ± SD. n=8, p<0.05 is considered statistically significant. Total cell extracts prepared in cell lysis buffer were subjected to immunoblotting with antibodies for CDX2, SMAD4, GSK3β, and β-actin.

[0136]

[0170] Secretome analysis

[0171] Collect the hTS cell culture medium (10 ml) at 80–90% confluence and centrifuge. Centrifugation (3,000 rpm, 30 min, 4°C) was performed. The supernatant was further concentrated to 1 ml by using a 3 kDa Vivaspin concentrator (Sigma). The concentrated supernatant was further detected for TGF-β1, HLA-G, and PAI-1 by immunoblot assay. IL-10 levels were measured by OptEIA ELISA assay according to the supplier's instructions (BD Pharmingen, San Diego). The range of detectable IL-10 concentrations was 2-2,000 pg / ml. Aliquots of 100 μl of samples were measured in triplicate. Total protein in the supernatant was measured by using a Pierce BCA Protein Assay Kit (Thermo Scientific). To measure the levels of C-peptide and insulin in the glucose stimulation test, high glucose (20 mM) was added to α-MEM medium (5 ml) at >80% cell confluence after bFGF treatment. At various times (5, 10, 20, 30, 60, and 120 min), the medium was collected, lyophilized in a lyophilizer (VirTis, Warminster), and rehydrated with sterile water (400 μl) for radioimmunoassay (RIA). C-peptide and insulin levels were determined in quintuplicate assays by C-PEP II-RIA-CT (DIAsource ImmunoAssays SA) and Coat-A-Count Insulin (Siemens Healthcare Diagnostics), respectively.

[0137]

[0172] Exosome analysis

[0173] 1) hTS cell culture (1 x 1.86 cells / 10ml), 24 1) hTS cells treated with bFGF (10 ng / ml) for 24 hours (prCTB), and 2) hTS cells treated with bFGF (10 ng / ml) for 24 hours (prCTB). These supernatants were subjected to Milliplex analysis using a Luminex LX 200 instrument (R&D System, USA) at the National Experimental Research Laboratories, Taiwan, and the data were analyzed. The data were analyzed by Milliplex Analyst software (5.1.0.0).

[0138]

[0174] Transmission electron microscope

[0175] Cell classes formed on culture dishes by hTS cells after high glucose stimulation The culprits were excised using a Wolfram needle. For transmission electron microscopy, cell clumps were fixed in 0.1 M PBS (Merck, pH 7.4) containing 3% (w / v) paraformaldehyde (Merck), 1.5% (w / v) glutaraldehyde (Merck), and 2.5% (w / v) saccharose (Merck) for 1 h at room temperature and overnight at 4°C. Before and after osmification in Palade's fixative containing 1% (v / v) OsO4 (Sigma) for 2 h at 4°C, samples were washed in PBS, treated with uranyl acetate dihydrate (Merck), dehydrated in a graded series of ethanol solutions, and embedded using the EMBed-812 Embedding kit (Electron Microscopy Sciences). Ultrathin sections were stained with uranyl acetate dehydrate and lead citrate (Electron Microscopy Sciences) and examined using a JEM-2000 EXII (JEOL, Tokyo).

[0139]

[0176] Western blot

[0177] In cell culture, hTS cells were treated with bFGF and harvested at the indicated times. The lysates were loaded into RIPA lysis solution (Millipore) supplemented with protease (Thermo Scientific) and phosphatase inhibitors (Cell Signaling Technology). 30 μg of lysate was electrophoresed on a polyacrylamide gel followed by electroblotting on PDVF membranes (Millipore). Target proteins were incubated with primary antibodies by blocking with 5% nonfat dry milk in PBS at room temperature (1 h). All membranes were incubated with chemiluminescence (Millipore) and images were captured by a ChemiDoc XRS system (Bio-RAD). Antibodies used are listed in the Key Resource Table. Data were analyzed by AlphaEaseFC (version 4.0.0).

[0140]

[0178] Immunofluorescence imaging

[0179] Immunocytochemistry. Briefly, 9 slides containing cultured cells were The sections were fixed in 5% (v / v) ethanol for 30 min at room temperature, washed three times in PBS, and incubated with blocking buffer PBS containing 0.05% (v / v) Tween 20 (PBST, Sigma) and 5% (v / v) normal donkey serum (Millipore) for 60 min. Primary and secondary antibodies were diluted in blocking buffer as indicated. Primary antibodies were incubated overnight at 4°C or for 2 h at room temperature. After incubation with specific primary antibodies in blocking buffer, appropriate fluorescein isothiocyanate (FITC, Invitrogen) or Alexa Fluor 488, 594, 647 (Invitrogen) or Dylight 488, 594 (BioLegend) conjugated secondary antibodies were added for 1 h at room temperature. Slides were mounted with 50% glycerol by nuclear counterstaining with DAPI. Images were captured by a confocal laser scanning microscope (LSM700; Zeiss Z1 or Olympus FluoView 1000 confocal laser scanning microscope or Countess II FL (Invitrogen), or a 3D Explorer fluorescence microscope (Nanolive, Swiss), or a TissueFAXS system (TissueQnostics GmbH).

[0141]

[0180] All procedures were performed using a Leica Bond-III automated system. The staining was performed using a Bond polymer refinement kit (cat# DS9800, Leica). Diaminobenzidine and hematoxylin were utilized. Upon completion of the procedure and removal of the slide tray, the cover tile was removed by carefully lifting it up by its neck. Slides were dehydrated through two changes each of 95% and 100% alcohol and two changes of xylene before being coverslipped.

[0142]

[0181] TaqMan miRNA and quantitative real-time PCR assays

[0182] TRIZOL reagent (Invitrogen) and DNAase I on-column RNA was isolated from hTS cells in triplicate or quintuplicate samples using digestion (Qiagen) according to the manufacturer's protocol. Total RNA (500 ng) was used for reverse transcription using the iScript cDNA synthesis kit (Bio-Rad). PCR was performed with 1 / 40 cDNA per reaction and 400 nM forward and reverse primers. For miRNA stem-loop qPCR, single-tube TaqMan miRNA assays were used according to the manufacturer's instructions (Applied Biosystems). All RT reactions, including no template and no RT controls, were performed in a GeneAmp PCR 9700 Thermocycler (Applied Biosystems). Comparative real-time PCR, including no template controls, was performed in triplicate or quintuplicate using specific primers for miR-124 or RNU6B (Applied Biosystems). U6 snRNA (RNU6B, Applied Biosystems) served as an endogenous control. Relative expression was analyzed using SDS2.2.2 software (Applied The calculations were performed using the NMR spectroscopy (Digital Electron Microscopy, Inc., Biosystems).

[0143]

[0183] Immunoprecipitation (IP) assay

[0184] Cell lysates of hTS cells treated with bFGF were collected. Protein G-AgA was then analyzed. Total protein (100 μg) was treated with the listed specific primary antibodies overnight by incubation with protein G-agarose beads (Millipore) for 30 min. After treatment with protein G-agarose beads for 2 h, samples were washed three times with RIPA lysis buffer (Millipore) followed by addition of protein loading dye and boiling for 5 min. Samples were resolved on 8% SDS-PAGE and subjected to immunoblot analysis.

[0144]

[0185] Chromatin immunoprecipitation (ChIP) assay

[0186] ChIP assays were performed using ChIP-IT Express Chromatin Immunoprecipitation kits (Active Motif) were used according to the manufacturer's instructions. Briefly, the immunoprecipitated DNA fragments were immunotransfected into hTS cells (1 × 10 6 ) were extracted. Antibodies, anti-CREB1 or anti-CT4 or anti-β-catenin, were used. Specific primers listed below were used to amplify the conserved binding sites in the promoter regions of miR-124a or SOX17 or FOXA2. For the promoter of miR124-2, forward: 5'-tctgcggctctttggtttca-3' and reverse: 5'-tctgccttcagcacaagagg-3', forward: 5'-gcggctctttggtttcaagg-3' and reverse: 5'-ctgccttcagcacaagagga-3', for the promoter of miR124-3, 5'-cccgcagttctcaaggacac-3' and reverse: 5'-agaagggagccaggcaagtc-3', for the promoter of SOX17, 5'-ttgtagattgctctctctcctcc-3' and reverse: 5'-gtgaagccttggctagggg-3', for the promoter of FOXA2, 5'-cccatcattgattcctggat-3' and reverse: 5'-ttgggaggctgagatttgtc-3'.

[0145]

[0187] Exosome analysis

[0188] Cell culture supernatant was collected from 1) hTS cell culture (1×1.8 6 cells / 10ml), 24 1) hTS cells treated with bFGF (10 ng / ml) for 24 hours (prCTB), and 2) hTS cells treated with bFGF (10 ng / ml) for 24 hours (prCTB). These supernatants were subjected to Milliplex analysis using a Luminex LX 200 instrument (R&D System, USA) at the National Experimental Research Laboratories, Taiwan, and the data were analyzed by Milliplex Analyst software (5.1.0.0).

[0146]

[0189] Flow cytometry

[0190] For insulin analysis, strip or 1× TrypLE (ThermoFilm) hTS cells were harvested by trypsinization using a 5x10 PBS (Sherr Scientific) and washed with PBS. 6 Cells (1000 x 1000 cells / ml) were incubated in blocking buffer (PBST and 5% donkey serum) for 1 h on ice, followed by resuspension in blocking buffer containing Alexa Fluor® 647-conjugated anti-insulin antibody (9008s, Cell Signaling) or unconjugated anti-insulin antibody (sc-7839, Santa Cruz) for 30 min at 4°C. Cells were washed twice with blocking buffer, filtered with unconjugated antibody, and then incubated with Alexa Fluor® 647-conjugated secondary antibody in blocking buffer for 30 min on ice in the dark. After two washes, cells were passed through a polystyrene round-bottom tube with a cell filtration cap (BD Falcon) and subjected to flow cytometry (LSR-II flow cytometer, BD Biosciences). Results were analyzed by FlowJo software.

[0147]

[0191] For analysis of pluripotency transcription factors, CDX2 or OCT4 or SOX2 or Non-specific shRNA against NANOG was transfected into hTS cells. The cells (5 × 10 6Cells / ml) were incubated with specific primary antibodies for 30 min. Samples were washed and resuspended in PBS by incubation with appropriate fluorochrome-conjugated primary antibodies at adjusted dilutions for 1 h at 4°C, followed by passage of cells through polystyrene round-bottom tubes with cell filtration caps (BD Falcon) and flow cytometry (FACScan, BD Biosciences, San Jose, CA). Data were analyzed by Cell-Quest software (BD Biosciences).

[0148]

[0192] For analysis of CD biomarkers, hTS cells or prCTB (1 × 10 5 ~1×10 6 ) were suspended in 240 μl of 1× FCM buffer (Leinco, F1175). Cells (30 μl) were incubated with either 7-AAD (BD, 5599257), fluorescently labeled antibodies (BD Multitest 6-color TBNK (BD, 337166) + BV421-labeled anti-CD107a (BioLegend, 328625), or BV421-labeled anti-CD34 (BD, 562577) alone, or PerCP Cy5.5-labeled anti-CD45 (BD, 340952) + APC-labeled anti-CD3 (BD, 555342) + PE-labeled anti-γδTCR (BD, 340887), or fluorescently labeled isotype control antibodies (FITC-labeled IgG1κ (BD, 556649) + PE-labeled IgG1κ (BD, 556650) + PE-labeled IgG2bκ (BD, 556656) + PerCP-Cy™5.5-labeled IgG1κ (BD, 552834) + PE-Cy™7-labeled IgG1κ (BD, 557872) + APC-labeled IgG1 κ (BD, 550854) + APC-Cy7-labeled IgG1κ (BD, 557873) + BV421-labeled IgG1κ (BD, 562438), or BV421-labeled IgG1κ (BD, 562438) alone. After 15 min incubation at room temperature, cells were washed with 1 ml of 1×FCM buffer and resuspended in 200 μl of 1×FCM buffer. Finally, cell samples were analyzed using a FACSVerse flow cytometer (BD, 651155) and FACSuite software.

[0149]

[0193] Apoptosis assay

[0194] PANC-1 cells (pancreas), MCF-cells (mammary gland), H1299 cells (lung), M Various cancer cells (2,000 cells), including KN45 cells (stomach), HepG2 cells (liver), PA-1 cells (ovary), A375 cells (melanoma), and PC-3 cells (prostate), were seeded and cultured in 35 mm glass-bottom dishes (previously described, cat#81158) with culture medium and 5% CO2 at 37°C for incubation. After allowing the cells to adhere overnight, bFGF-induced hTS cells (2×10 4 Cells) were added for co-culture at 24 h. For apoptosis assay, Apoptosis / Necrosis kit (ab176749, Abcam, Cambridge, England) was used to stain the co-cultured cells according to the manufacturer's instructions. Briefly, after removing the medium, cells were washed twice using assay buffer. Apopxin green indicator (apoptotic cells / green) and CytoCalcein450 (healthy cells / blue) were added for staining, and cells were incubated at room temperature for approximately 40 min. After washing twice with buffer, cells were observed with 3D Cell Explorer fluorescence (Nanolive, Swiss).

[0150]

[0195] Coculture of prCTB and PANC-1 cells was performed at a 2:1 ratio (3 × 10 4 cell The apoptosis assay was performed at 100 μg / well in 12-well plates at 37°C for 6 days and observed under a light microscope. For apoptosis assay, the co-cultured cells were analyzed (at 24 h) using the Apoptosis / Necrosis kit (ab176749, Abcam, Cambridge, England) according to the manufacturer's instructions. After removing the medium, the cells were washed twice using assay buffer. Then, Apopxin Green indicator (apoptotic cells) and CytoCalcein450 (healthy cells) were added to each well and incubated for 60 min at room temperature. The cells were washed with assay buffer and analyzed under a fluorescent microscope.

[0151]

[0196] Transwell assay

[0197] Use a 6-well transwell insert (pore size 8 μm, Corning) prCTB(1×10 6 Cells / ml) were seeded and incubated at 37°C, 5% CO 2 Incubate in 37°C for 10 min to allow the cells to settle. For this, extracellular matrix (ECM) material is added on top of the transwell membrane and cells are added on top of the ECM. For example, Matrigel is thawed on ice to liquefy it, 30-50 μl of Matrigel is added to a 24-well transwell insert and allowed to solidify in a 37°C incubator for 15-30 min to form a thin gel layer. To mimic invasion through the extracellular matrix, the cell solution is added on top of the Matrigel coating. The transwell cell migration assay measures the chemotactic performance of cells towards chemoattractants. However, the transwell cell invasion assay measures both cell chemotaxis and cell invasion through the extracellular matrix, a process commonly seen in cancer metastasis or embryonic development.

[0152]

[0198] Using a pipette, dispense 600 µL of the solution into the bottom of the lower chamber of a 24-well plate. Add 1 ml of the desired chemoattractant very carefully. Add the chemoattractant without moving the transwell insert and avoid creating bubbles. Make sure the chemoattractant solution in the bottom well comes into contact with the membrane in the top well, forming a chemoattractant gradient. Incubation time depends on the cell type and chemoattractant used. Note: Further testing may be required to determine incubation time. Note: For adherent cells, migrated cells may attach to the other side of the membrane 1、8Quantification of migrated cells can be performed by following steps 2.4-2.8 (steps 2.4-2.8 do not need to be performed in a sterile environment). For non-adherent cells, the migrated cells will fall into the medium in the lower chamber. The number of migrated cells can be counted using a hemocytometer or flow cytometer. 5 .

[0153]

[0199] Remove the transwell insert from the plate. Repeat as many times as necessary. Using a cotton tip applicator, carefully remove the medium and any residual cells that have not migrated from above the membrane without damaging the membrane.

[0154]

[0200] Add 600 to 1,000 μl of 70% ethanol to the wells of a 24-well plate. Add 100 mM NaCl. Place the transwell insert in 70% ethanol for 10 min to fix the cells. Remove the transwell insert from the 24-well plate and use a cotton-tip applicator to remove any residual ethanol from the top of the membrane. Allow the transwell membrane to dry (typically 10-15 min).

[0155]

[0201] Add 600-1,000 μl of 0.2% crystallites to wells of a 24-well plate. Add violet and place the membrane in it for staining. Incubate at room temperature for 5-10 minutes.

[0156]

[0202] Clean the membrane with a pipette tip or cotton tip applicator. Gently remove the crystal violet. Remove excess crystal violet by dipping the membrane in distilled water as many times as necessary, very carefully to avoid washing away the fixed cells. Allow the transwell membrane to dry.

[0157]

[0203] Observe under an inverted microscope and count the number of cells in different fields to determine the chemoattractant concentration. Obtain the average total number of cells that migrated through the membrane towards the tunica media and attached to the underside of the membrane.

[0204] statistical analysis

[0205] Immunoblot assays, qPCR assays, reporter assays, and Experiments in insulin and IL-10 assays were performed in triplicate or quadruplicate and repeated twice as indicated, and p values ​​were calculated by two-tailed Student's t-test with p<0.05 being considered statistically significant.

[0158]

[0206] One or more of the embodiments, examples, or aspects disclosed herein may be suitable. Any of the above-described embodiments, examples, or aspects may be combined with any other embodiment, example, or aspect disclosed herein where applicable.

[0159]

[0207] Although several embodiments are shown and described herein, such implementations The embodiments are provided by way of example only. Numerous variations, modifications, and substitutions are possible without departing from the invention. It will be understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention.

Claims

1. 1. An isolated pioneer regulatory cytotrophoblast (prCTB) cell, comprising: (i) the prCTB expresses beta-hormone human chorionic gonadotropin (β-hCG), human leukocyte antigen G (HLA-G), CD56, insulin, heat shock protein 90 (HSP90), CD4, CD16, CD107a, CD8, interleukin 15 (IL-15), leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), T cell receptor (TCR), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), programmed death-ligand 1 (PD-L1), apoptosis signaling receptor (Fas), Fas ligand (FasL), CD335 (NKp46), CD11b, CD49f, CD3, CD19, CD34, or any combination thereof; and (ii) the prCTB expresses p53, Ki67, glutamate decarboxylase (GAD65), heat shock protein 70 (HSP70), soluble CD40-ligand (sCD40L), B-cell leukemia / lymphoma 2-associated protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), or any combination thereof; Isolated pioneer regulatory cytotrophoblast cells.

2. The prCTB of claim 1, which expresses CD4, CD16, CD56, CD107a, CD8, or any combination thereof.

3. 1. An isolated population of cells comprising pioneer regulatory cytotrophoblast (prCTB), comprising: (i) the population expresses the beta-hormone human chorionic gonadotropin (β-hCG), human leukocyte antigen G (HLA-G), CD56, insulin, heat shock protein 90 (HSP90), CD4, CD16, CD107a, CD8, interleukin 15 (IL-15), leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), T cell receptor (TCR), killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), programmed death-ligand 1 (PD-L1), apoptosis signal receptor (Fas), Fas ligand (FasL), CD335 (NKp46), CD11b, CD49f, CD3, CD19, CD34, or any combination thereof; and (ii) the population expresses p53, Ki67, glutamate decarboxylase (GAD65), heat shock protein 70 (HSP70), soluble CD40-ligand (sCD40L), B-cell leukemia / lymphoma 2-associated protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), or any combination thereof; An isolated population of cells.

4. The population of cells of claim 3, wherein at least about 10% of the population is prCTB that expresses CD16 and CD56.

5. 5. The population of cells of claim 3 or 4, wherein at least about 2% of the population is prCTB expressing CD4.

6. 6. The population of cells of any one of claims 3 to 5, wherein at least about 2% of the population is prCTB expressing CD8.

7. 7. The population of cells of any one of claims 3 to 6, wherein at least about 5% of the population is prCTB that expresses CD107.

8. 1. An isolated population of cells comprising pioneer regulatory cytotrophoblast (prCTB), comprising: (i) at least about 10% of the population is prCTB that expresses CD16 and CD56; (ii) at least about 2% of the population is prCTB that expresses CD4; (iii) at least about 2% of the population is prCTB that expresses CD8; or (iv) at least about 5% of the population is prCTB that expresses CD107; or An isolated population of cells, any combination thereof.

9. (i) at least about 10% of the population is prCTB that expresses CD16 and CD56; (ii) at least about 2% of the population is prCTB that expresses CD4; (iii) at least about 2% of the population is prCTB that expresses CD8; and (iv) at least about 5% of the population is prCTB that expresses CD107; A population of cells according to any one of claims 3 to 8.

10. 10. The population of cells of any one of claims 3 to 9, wherein at least about 2% of the population is prCTB that expresses CD16, CD56, and CD107.

11. 11. The prCTB or population of cells of any one of claims 1 to 10, wherein the prCTB or a plurality of the prCTB expresses interleukin 15 (IL-15).

12. 12. The prCTB or population of cells of any one of claims 1 to 11, wherein the prCTB or plurality of prCTBs expresses leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), T-cell receptor (TCR), killer cell immunoglobulin-like receptor 2 DL4 (KIR2DL4), programmed death-ligand 1 (PD-L1), apoptosis signal receptor (Fas), Fas ligand (FasL), CD335 (NKp46), B-cell leukemia / lymphoma 2 associated protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), or any combination thereof.

13. 13. The prCTB or population of cells of any one of claims 1 to 12, wherein the prCTB or plurality of prCTB further expresses beta-hormone human chorionic gonadotropin (β-hCG), soluble human leukocyte antigen G (sHLA-G), transforming growth factor β1 (TGF-β1), plasminogen activator inhibitor-1 (PAI-1), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10), CD105, CD146, or any combination thereof.

14. 14. The prCTB or population of cells of any one of claims 1 to 13, wherein the prCTB or plurality of the prCTBs lacks syncytin, programmed cell death protein 1 (PD-1), or a combination thereof.

15. 15. The prCTB or population of cells of any one of claims 1 to 14, wherein the prCTB or a plurality of the prCTBs secrete a chemokine, a cytokine, a growth factor, or any combination thereof, or an exosome carrying any of the above.

16. The cytokine is chemokine (C-C motif) ligand 5 (CCL5), monocyte ketone 16. The prCTB or population of cells of claim 15, comprising moiatractant protein-1 (MCP-1), monocyte chemoattractant protein-1 (MCP-3), chemokine (C-X-C motif) ligand 1 (CCL1), chemokine (C-X-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 11 (CCL11), chemokine (C-C motif) ligand 24 (CCL24), chemokine (C-C motif) ligand 26 (CCL26), chemokine (C-C motif) ligand 22 (CCL22), chemokine (C-X-C motif) ligand 10 (CCL10), fractalkine, and chemokine (C-C motif) ligand 4 (CCL4), or any combination thereof.

17. 17. The prCTB or population of cells of claim 15 or 16, wherein the cytokine comprises interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), interleukin (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12p40 (IL-12p40), interleukin 13 (IL-13), interleukin 15 (IL-15), or any combination thereof.

18. 18. The prCTB or population of cells of any one of claims 15 to 17, wherein the cytokine comprises interferon alpha (IFN-α) or interferon gamma (IFN-γ).

19. 19. The prCTB or population of cells of any one of claims 15 to 18, wherein the growth factors comprise platelet derived growth factor homodimer AA (PDGF-AA), PDGF homodimer BB (PDGF-BB), PDGF heterodimer (PDGF-AB), vascular endothelial growth factor (VEGF), granulocyte-macrophage colony stimulating factor (GM-CSF), epidermal growth factor (EGF), fibroblast growth factor (FGF) family proteins, FMS-like tyrosine kinase 3 ligand (Flt3L), soluble CD40 ligand (sCD40L), tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1β), or any combination thereof.

20. 20. The prCTB or population of cells of any one of claims 1 to 19, wherein the prCTB or plurality of prCTBs have higher levels of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN as measured by immunoblotting than the progenitor cell from which the isolated prCTB was differentiated in vitro.

21. 21. The prCTB or population of cells of any one of claims 1 to 20, wherein the prCTB has at least about 1.1, 1.2, 1.5, 1.5, 2, 2.2, 2.5, 2.8, 3, 3.5, 4, 5, 8, 10 fold higher levels of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN than the progenitor cell from which the isolated prCTB was differentiated in vitro, as measured by immunoblot.

22. 22. The prCTB or population of cells of any one of claims 1 to 21, wherein the prCTB expresses at least about 1.1, 1.2, 1.5, 1.5, 2, 2.2, 2.5, 2.8, 3, 3.5, 4, 5, 8, 10 times higher levels of SOX2 protein than the progenitor cell from which the isolated prCTB was differentiated in vitro, as measured by immunoblot.

23. 23. The prCTB or population of cells of any one of claims 1 to 22, wherein the isolated prCTB is differentiated in vitro from chorionic villi-derived progenitor cells that lack expression of glutamate decarboxylase (GAD65), Ki67, heat shock protein 70 (HSP70), p53, soluble CD40-ligand (sCD40L), or any combination thereof.

24. 24. The prCTB or population of cells of any one of claims 1 to 23, wherein the isolated prCTB is differentiated in vitro from chorionic villi-derived progenitor cells, and both the chorionic villi-derived progenitor cells and the isolated prCTB express heat shock protein 90 (HSP90).

25. 25. The prCTB or population of cells according to any one of claims 1 to 24, wherein the isolated prCTB is a human cell.

26. 25. The prCTB or population of cells of any one of claims 1 to 24, wherein the isolated prCTB is of rodent, rabbit, bovine, ovine, porcine, canine, feline, monkey, or ape origin.

27. 27. The prCTB or population of cells of any one of claims 1 to 26, wherein the isolated prCTB is genetically engineered.

28. 28. The prCTB or population of cells of claim 27, wherein the isolated prCTB comprises an exogenous polynucleotide encoding a cellular receptor, an immune checkpoint protein, a cytokine, or any combination thereof.

29. 28. The prCTB or population of cells of claim 27, wherein the isolated prCTB comprises an exogenous gene polynucleotide encoding a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof.

30. 30. A pharmaceutical composition comprising a pharma- ceutically acceptable excipient and a prCTB or population of cells according to any one of claims 1 to 29.

31. 30. A method of treating a disease or condition comprising administering to a subject in need of such treatment a prCTB or population of cells according to any one of claims 1 to 29.

32. 32. The method of claim 31, wherein a target cell bearing the antigen is killed.

33. 33. The method of claim 32, wherein the antigen-bearing cell is a cancer cell.

34. 34. The method of claim 33, wherein the cancer cells comprise bladder cancer cells, bone cancer cells, brain cancer cells, breast cancer cells, cervical carcinoma, colorectal cancer cells, esophageal cancer cells, gastrointestinal cancer cells, hematopoietic malignancies, head and neck squamous cell carcinoma, leukemia, liver cancer cells, lung cancer cells, lymphoma, myeloma, nasal cancer cells, nasopharyngeal cancer cells, oral cancer cells, oropharyngeal cancer cells, ovarian cancer cells, prostate cancer cells, sarcoma, gastric cancer cells, melanoma, thyroid cancer cells, or any combination thereof.

35. 33. The method of claim 32, wherein the antigen-bearing cell is a pathogen.

36. 36. The method of claim 35, wherein the pathogen comprises a virus, a bacterium, a protozoa, a prion, a fungus, or any combination thereof.

37. 37. The method of any one of claims 31 to 36, wherein at least about 5%, at least about 10%, at least about 20%, at least about 50%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100% of a population of antigen-bearing target cells are killed.

38. 32. The method of claim 31, which downregulates an inflammatory pathway.

39. Transplant rejection, infection, endotoxin shock associated with infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic onset juvenile idiopathic arthritis (JIA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), asthma, pelvic inflammatory disease, Alzheimer's disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, ankylosing spondylitis, dermatomyositis, uveitis, Peyronie's disease, celiac disease 39. The method of claim 38, wherein the method treats a disease or condition comprising: pulmonary arthritis, gallbladder disease, pilonidal disease, peritonitis, psoriasis, vasculitis, post-surgical adhesions, stroke, type I diabetes, Lyme arthritis, meningoencephalitis, immune-mediated inflammatory disorders of the central and peripheral nervous system, pancreatitis, post-surgical injury, graft versus host disease, heart disease, bone resorption, burn patients, myocardial infarction, Paget's disease, osteoporosis, sepsis, hepatic or pulmonary fibrosis, periodontitis, or hypochlorhydria.

40. 32. The method of claim 31 for treating an autoimmune disease.

41. 32. The method of claim 31, treating type I diabetes, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, polymyositis, chronic active hepatitis, mixed connective tissue disease, primary biliary cirrhosis, pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease, vitiligo, gluten-sensitive enteropathy, Graves' disease, myasthenia gravis, autoimmune neutropenia, idiopathic thrombocytopenic purpura, rheumatoid arthritis, cirrhosis of the liver, pemphigus vulgaris, autoimmune infertility, Goodpasture's disease, bullous pemphigoid, discoid lupus, ulcerative colitis, dense deposit disease, inflammatory bowel disease, or psoriasis.

42. 32. The method of claim 31 for treating type 1 diabetes.

43. 32. The method of claim 31, which ameliorates transplant rejection.

44. A cell-free composition comprising a chemokine, an interleukin, a growth factor, or any combination thereof, and a pharma- ceutically or cosmetically acceptable excipient.

45. (i) chemokines, including CXCL2, MCP-1, fractalkine, IP-10, MCP-3, eotaxin, MIP-1β, or any combination thereof; (ii) interleukins, including IL-6, IL-8, IL-4, IL-1RA, IL-10, IL-12P40, IL-15, IL-1α, IL-17A, or any combination thereof; and (iii) growth factors, including PDGF-AA, VEGF, bFGF, G-CSF, Flt-3L, GM-CSF, or any combination thereof; 45. The composition of claim 44, comprising:

46. 46. ​​The composition of claim 44 or 45, comprising MCP-1 and one, two, three or all of CXCL2, IL-6, IL-8 and VEGF proteins.

47. 47. The composition of claim 46, wherein MCP-1 and CXCL2 in said composition have a weight ratio of about 1:1 to about 2:

1.

48. 47. The composition of claim 46, wherein MCP-1 and CXCL2 in said composition have a weight ratio of about 3:1 to about 4:

1.

49. The composition of any one of claims 46 to 48, wherein MCP-1 and IL-6 in the composition have a weight ratio of about 2:1 to about 3:

1.

50. The composition of any one of claims 46 to 48, wherein MCP-1 and IL-6 in the composition have a weight ratio of about 3:1 to about 4:

1.

51. The composition of any one of claims 46 to 50, wherein MCP-1 and IL-8 in the composition have a weight ratio of about 4:1 to about 6:

1.

52. 52. The composition of any one of claims 46 to 51, wherein MCP-1 and VEGF in the composition have a weight ratio of about 4:1 to about 6:

1.

53. 52. The composition of any one of claims 46 to 51, wherein MCP-1 and VEGF in the composition have a weight ratio of about 7:1 to about 9:

1.

54. 54. The composition of any one of claims 46 to 53, further comprising PDGF-AA, wherein the MCP-1 and PDGF-AA are present in a weight ratio of about 3:1 to about 5:

1.

55. 54. The composition of any one of claims 46 to 53, further comprising PDGF-AA, wherein the MCP-1 and PDGF-AA are present in a weight ratio of about 6:1 to about 9:

1.

56. 56. The composition of any one of claims 46 to 55, further comprising PDGF-AA and G-CSF.

57. 57. The composition of any one of claims 46 to 56, further comprising PDGF-AA and FGF-2 (bFGF).

58. 58. The composition of any one of claims 46-57, further comprising one or more proteins: IP-10, eotaxin, Flt-3L, GM-CSF, MIP-1a, MIP-1b, IL-1a, IL-1RA, IL-4, IL-7, IL-10, IL-12P40, IL-13, IL-15, IL-17A, CCL5 (RANTES), MDC, MCP-3, IL-12P70, IFNa, IFNr, PDGF-AB / BB, or EGF.

59. 60. A method of regulating skin condition comprising administering to a subject in need thereof a composition according to any one of claims 44 to 58.

60. 60. The method of claim 59, which treats a disease or provides a cosmetic application.

61. 61. The method of claim 59 or 60, which tightens the skin.

62. 62. The method of any one of claims 59 to 61, wherein the method hydrates the skin.

63. 63. The method of any one of claims 59 to 62, for rejuvenating the skin.

64. 64. The method of any one of claims 59 to 63, wherein the composition does not comprise stem cells.

65. 65. The method of any one of claims 59 to 64, wherein the composition is in the dosage form of a lotion, cream, liquid, gel, emulsion, suspension, paste, stick, aerosol, foam, patch, powder, ointment, beads, mask, pad, sheet, wound dressing, bandage, or any combination thereof.

66. 66. The method of any one of claims 59 to 65, wherein the composition is administered to the subject topically, subcutaneously, transdermally, intramuscularly, or intratumorally.

67. 67. The method of any one of claims 59 to 66, wherein the subject is a mammal.

68. 68. The method of claim 67, wherein the subject is a primate.

69. 69. The method of claim 68, wherein the subject is a human.

70. 1. A method for obtaining progenitor regulatory cytotrophoblasts (prCTBs), comprising the step of differentiating pluripotent stem cells in vitro by contacting said stem cells with a culture medium comprising a fibroblast growth factor and a nucleoside, L-glutamine, a dipeptide containing L-glutamine, a platelet lysate, or a combination thereof.

71. 71. The method of claim 70, wherein the culture medium comprises a nucleoside, the dipeptide, and platelet lysate.

72. 72. The method of claim 70 or 71, wherein the culture medium comprises about 2 mM to about 200 mM L-glutamine.

73. 73. The method of any one of claims 70 to 72, wherein the contacting step lasts for about 24 hours to 48 hours.

74. 74. The method of any one of claims 70 to 73, wherein the stem cells are chorionic villi-derived progenitor cells.

75. 75. The method of any one of claims 70 to 74, comprising culturing the stem cells with the culture medium before contacting the stem cells with the fibroblast growth factor to differentiate into the prCTB.

76. 76. The method of any one of claims 70 to 75, wherein the culture medium does not contain antibiotics.

77. 77. The method of claim 76, wherein the antibiotic is penicillin, streptomycin, or any combination thereof.

78. 78. The method of any one of claims 70 to 77, wherein the culture medium does not contain retinoic acid.

79. 79. The method of any one of claims 70-78, wherein the culture medium does not contain mercaptoethanol, nicotinamide, or a combination thereof.

80. 80. The method of any one of claims 70-79, wherein the culture medium does not contain dexamethasone, recombinant human oncostatin M, BMP4, HGF, or any combination thereof.

81. 81. The method of any one of claims 70 to 80, wherein the culture medium is animal component free.

82. 82. The method of any one of claims 70 to 81, wherein the culture medium is free of human-derived components.

83. 83. The method of any one of claims 70 to 82, wherein the culture medium is serum-free.

84. 84. The method of claim 83, wherein the culture medium does not contain fetal bovine serum.

85. 85. The method of any one of claims 70 to 84, wherein the fibroblast growth factor is basic fibroblast growth factor (bFGF).

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