Precursor regulatory cytotrophoblast cells and their uses

Abstract

The present disclosure relates to in vitro-generated precursor regulatory cytotrophoblast cells and compositions thereof. The present disclosure also relates to methods of treating disorders or conditions by utilizing the cells disclosed herein.

Description

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 843,925, filed on May 6, 2019, the entire content of which is incorporated herein by reference. BACKGROUND OF THE INVENTION

[0003] As an alternative to overcoming certain drawbacks of existing embryonic stem cells and iPS cells, there is a need for novel stem cell therapies for treating a variety of diseases or conditions.

[0004] Incorporated by reference

[0005] All publications, patents, and patent applications herein are incorporated by reference to the extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between the terms herein and the terms of the incorporated references, the terms herein shall control. SUMMARY OF THE INVENTION

[0006] The embodiments of the invention provided in the Summary of the Invention section are merely illustrative and provide an overview of alternative embodiments disclosed herein. The illustrative and alternative Summary of the Invention section 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 this disclosure or any claimed embodiment of the invention.

[0007] In some aspects, the present disclosure provides isolated precursor regulatory cytotrophoblasts (prCTBs), wherein: (i) the prCTBs express beta-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), apoptosis signal receptor (Fas), Fas ligand (FasL), CD335 (NKp46), CD11b, CD49f, CD3, CD19, CD34, or any combination thereof; and (ii) the prCTBs express p53, Ki67, glutamate decarboxylase (GAD65), heat shock protein 70 (HSP70), soluble CD40 ligand (sCD40L), B-cell leukemia / lymphoma 2-related protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), or any combination thereof. In some cases, the prCTBs express CD4, CD16, CD56, CD107a, CD8, or any combination thereof.

[0008] In some aspects, the present disclosure provides isolated cell populations comprising precursor regulatory cytotrophoblasts (prCTBs), wherein: (i) the cell populations express beta-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), apoptosis signal receptor (Fas), Fas ligand (FasL), CD335 (NKp46), CD11b, CD49f, CD3, CD19, CD34, or any combination thereof; and (ii) the cell populations express p53, Ki67, glutamate decarboxylase (GAD65), heat shock protein 70 (HSP70), soluble CD40 ligand (sCD40L), B-cell leukemia / lymphoma 2-related protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), or any combination thereof.

[0009] In some cases, at least about 10% of the population is prCTB expressing CD16 and CD56. In some cases, at least about 2% of the population is prCTB expressing CD4. In some cases, at least about 2% of the population is prCTB expressing CD8. In some cases, at least about 5% of the population is prCTB expressing CD107.

[0010] In some aspects, the present disclosure provides an isolated population of cells comprising precursor regulatory cytotrophoblasts (prCTB), 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.

[0011] In some cases, (i) at least about 10% of the population are prCTBs that express CD16 and CD56; (ii) at least about 2% of the population are prCTBs that express CD4; (iii) at least about 2% of the population are prCTBs that express CD8; and (iv) at least about 5% of the population are prCTBs that express CD107. In some cases, the cell population contains at least about 2% of the population that are prCTBs that express CD16, CD56, and CD107. In some cases, the prCT or multiple prCTBs express interleukin 15 (IL-15). In some cases, the prCTB or multiple 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 signal receptor (Fas), Fas ligand (FasL), CD335 (NKp46), B-cell leukemia / lymphoma 2-related protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), or any combination thereof. In some cases, the prCTB or multiple prCTBs further express beta-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 10 (IL-10), CD105, CD146, or any combination thereof. In some cases, the prCTB or multiple prCTBs lack the expression of syncytin, programmed cell death protein 1 (PD-1), or a combination thereof. In some cases, the prCTB or multiple prCTBs secrete chemokines, cytokines, growth factors, or any combination thereof, or carry exosomes containing chemokines, cytokines, growth factors, or any combination thereof. In some cases, the cytokines include 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 (CXCL1), chemokine (C-X-C motif) ligand 2 (CXCL2), 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 (CXCL10), fractalkine, and chemokine (C-C motif) ligand 4 (CCL4), or any combination thereof.In some cases, cytokines include interleukin 1α (IL-1α), interleukin 1β (IL-1β), 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 cases, cytokines include interferon α (IFN-α) or interferon γ (IFN-γ). In some cases, growth factors include 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 α (TNFα), interleukin 1β (IL-1β), or any combination thereof. In some cases, as measured by immunoblotting, prCTB or multiple prCTBs have a higher level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN in vitro from the progenitor cells from which they are differentiated than isolated prCTB. In some cases, as measured by immunoblotting, prCTB has a level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN that is 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 in vitro from the progenitor cells from which it is differentiated than isolated prCTB. In some cases, as measured by immunoblotting, the level of SOX2 protein expressed by prCTB is 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 in vitro from the progenitor cells from which it is differentiated than isolated prCTB. In some cases, isolated prCTB is differentiated in vitro from chorionic villus-derived progenitor cells that lack the expression of glutamate decarboxylase (GAD65), Ki67, heat shock protein 70 (HSP70), p53, soluble CD40 ligand (sCD40L), or any combination thereof. In some cases, isolated prCTB is differentiated in vitro from chorionic villus-derived progenitor cells, and wherein both the chorionic villus-derived progenitor cells and isolated prCTB express heat shock protein 90 (HSP90). In some cases, isolated prCTB is a human cell. In some cases, isolated prCTB is derived from a rodent, rabbit, cow, sheep, pig, dog, cat, monkey, or ape.In some cases, the isolated prCTB is genetically engineered. In some cases, the isolated prCTB comprises an exogenous polynucleotide encoding a cell receptor, an immune checkpoint protein, a cytokine, or any combination thereof. In some cases, 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.

[0012] In some aspects, the present disclosure provides pharmaceutical compositions comprising: a pharmaceutically acceptable excipient or carrier; and the prCTB or cell population described herein.

[0013] In some aspects, the present disclosure provides methods for treating a disease or disorder, comprising administering to a subject in need thereof the prCTB or cell population described herein.

[0014] In some cases, the method kills antigen-bearing target cells. In some cases, the antigen-bearing cells are not antigen-presenting cells, such as not dendritic cells, macrophages, or B cells. In some cases, the antigen-bearing target cells are cancer cells. In some cases, the cancer cells include bladder cancer cells, bone cancer cells, brain cancer cells, breast cancer cells, cervical cancer, 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, stomach cancer cells, melanoma, thyroid cancer cells, or any combination thereof. In some cases, the antigen-bearing target cells are pathogens. In some cases, the pathogens include viruses, bacteria, protozoa, prions, fungi, or any combination thereof. In some cases, 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 the population of antigen-bearing target cells. In some cases, the method downregulates inflammatory pathways. In some cases, the method treats diseases or conditions including: transplant rejection, infection, endotoxin shock associated with infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic 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, gallbladder disease, Pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, Lyme arthritis, meningoencephalitis, immune-mediated inflammatory disorders of the central and peripheral nervous systems, pancreatitis, surgical trauma, graft-versus-host disease, heart disease, bone resorption, burn patients, myocardial infarction, Paget's disease, osteoporosis, sepsis, liver or lung fibrosis, periodontitis, or achlorhydria. In some cases, the method treats autoimmune diseases. In some cases, 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, cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's disease, bullous pemphigoid, discoid lupus, ulcerative colitis, dense deposit disease, inflammatory bowel disease, or psoriasis. In some cases, the method treats type 1 diabetes. In some cases, the method improves transplant rejection.

[0015] The present disclosure relates to a composition comprising a secretome including exosomes, wherein the secretome comprises or the exosomes carry chemokines, cytokines such as interleukins, growth factors or any combination thereof, and a pharmaceutically or cosmetically acceptable excipient, and wherein the composition is cell-free.

[0016] In some cases, the secretome contains or exosomes carry: (i) chemokines, which include CXCL2, MCP-1, fractalkine, IP-10, MCP-3, Eotaxin, MIP-1β, or any combination thereof; (ii) interleukins, which include 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, which include PDGF-AA, VEGF, bFGF, G-CSF, Flt-3L, GM-CSF, or any combination thereof. In some cases, the composition contains MCP-1 and one, two, three, or all of CXCL2, IL-6, IL-8, and VEGF proteins. In some cases, the weight ratio of MCP-1 to CXCL2 in the composition is from about 1:1 to about 2:1. In some cases, the weight ratio of MCP-1 to CXCL2 in the composition is from about 3:1 to about 4:1. In some cases, the weight ratio of MCP-1 to IL-6 in the composition is from about 2:1 to about 3:1. In some cases, the weight ratio of MCP-1 to IL-6 in the composition is from about 3:1 to about 4:1. In some cases, the weight ratio of MCP-1 to IL-8 in the composition is from about 4:1 to about 6:1. In some cases, the weight ratio of MCP-1 to VEGF in the composition is from about 4:1 to about 6:1. In some cases, the weight ratio of MCP-1 to VEGF in the composition is from about 7:1 to about 9:1. In some cases, the composition further contains PDGF-AA, and wherein MCP-1 and PDGF-AA are present in a weight ratio of from about 3:1 to about 5:1. In some cases, the composition further contains PDGF-AA, and wherein MCP-1 and PDGF-AA are present in a weight ratio of from about 6:1 to about 9:1. In some cases, the composition further contains PDGF-AA and G-CSF. In some cases, the composition further contains PDGF-AA and FGF-2 (bFGF). In some cases, the composition further contains one or more proteins selected from 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, IFNα, IFNγ, PDGF-AB / BB, or EGF.

[0017] In some aspects, methods for modulating a skin condition are disclosed herein, including administering to a subject in need thereof a composition as described herein.

[0018] In some cases, the method treats a disease or provides a cosmetic application. In some cases, the method tightens the skin. In some cases, the method hydrates the skin. In some cases, the method rejuvenates the skin. In some cases, the composition does not contain stem cells. In some cases, the composition is administered locally, subcutaneously, transdermally, intramuscularly, or intratumorally to a subject. In some cases, the composition is in the form of a lotion, cream, liquid, gel, emulsion, suspension, paste, stick, aerosol, foam, patch, powder, ointment, bead, mask, pad, sheet, wound dressing, bandage, or any combination thereof. In some cases, the subject is a mammal. In some cases, the subject is a primate. In some cases, the subject is a human.

[0019] In some aspects, methods for obtaining precursor regulatory trophoblast cells (prCTBs) are disclosed herein, which comprise: differentiating pluripotent stem cells in vitro by contacting the stem cells with fibroblast growth factor and a culture medium comprising nucleosides, L-glutamine, a dipeptide comprising L-glutamine, platelet lysate, or a combination thereof.

[0020] In some cases, the culture medium comprises nucleosides, a dipeptide, and platelet lysate. In some cases, the culture medium comprises from about 2 mM to about 200 mM of L-glutamine. In some cases, the contact lasts for about 24 hours to 48 hours. In some cases, the stem cells are chorionic villus-derived progenitor cells. In some cases, the method comprises culturing the stem cells in the culture medium before contacting the stem cells with fibroblast growth factor to differentiate into prCTBs. In some cases, the culture medium does not contain antibiotics. In some cases, the antibiotics are penicillin, streptomycin, or any combination thereof. In some cases, the culture medium does not contain retinoic acid. In some cases, the culture medium does not contain mercaptoethanol, nicotinamide, or a combination thereof. In some cases, the culture medium does not contain dexamethasone, recombinant human oncostatin M, BMP4, HGF, or any combination thereof. In some cases, the culture medium does not contain animal components. In some cases, the culture medium does not contain human-derived components. In some cases, the culture medium does not contain serum. In some cases, the culture medium does not contain fetal bovine serum. In some cases, the fibroblast growth factor is basic fibroblast growth factor (bFGF).

[0021] In some aspects, the present disclosure provides isolated precursor regulatory cytotrophoblasts (prCTBs), wherein: (i) the prCTBs express beta-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 prCTBs express p53, Ki67, glutamate decarboxylase (GAD65), heat shock protein 70 (HSP70), soluble CD40 ligand (sCD40L), B cell leukemia / lymphoma 2-related protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), or any combination thereof. In some cases, the prCTBs kill cancer cells or pathogens. In some cases, the prCTBs induce apoptosis of cancer cells, for example, by infiltrating cancer cell colonies, optionally wherein the prCTBs themselves do not undergo apoptosis upon contact with cancer cells. In some cases, the cancer cells are solid tumor cells. In some cases, the cancer cells are pancreatic cancer cells, breast cancer cells, liver tumor cells, ovarian tumor cells, lung tumor cells, stomach tumor cells, melanoma cells, or any combination thereof. In some cases, the prCTBs downregulate inflammatory pathways. In some cases, the prCTBs express CD4, CD16, CD56, CD107a, CD8, or any combination thereof. In some cases, the prCTBs lack the expression of syncytin, programmed cell death protein 1 (PD-1), or a combination thereof.In some cases, prCTB also expresses 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), B-cell leukemia / lymphoma 2-related protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), beta-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 8 (IL-8), interleukin 10 (IL-10), CD105, CD146, or any combination thereof. In some cases, prCTB is a human cell. In some cases, prCTB is genetically engineered. In some cases, genetically engineered prCTB contains a polynucleotide encoding a foreign protein, the foreign protein comprising 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 cases, prCTB secretes chemokines, cytokines, growth factors, or any combination thereof, or exosomes carrying any of the foregoing. In some aspects, a pharmaceutical composition comprising the prCTB disclosed herein and a pharmaceutically acceptable excipient is disclosed herein. In some aspects, methods for treating a disease or disorder are disclosed herein, which comprise administering to a subject in need thereof the pharmaceutical composition described herein. In some aspects, a cell population comprising a plurality of the prCTB disclosed herein is disclosed herein. In some cases, at least about 10% of the prCTB population expresses CD16 and CD56. In some cases, at least about 2% of the prCTB population expresses CD4; at least about 2% of the prCTB population expresses CD8; at least about 5% of the prCTB population expresses CD107; or any combination thereof. In some cases, at least about 10% of the prCTB population expresses CD16 and CD56; 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 cases, at least about 2% of the prCTB population expresses CD16, CD56, and CD107. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Various aspects of the present invention are set forth in detail in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description and accompanying drawings which set forth exemplary embodiments in which the principles of the present invention are utilized, wherein:

[0023] Figures 1A-1D Characterization of insulin expression in human trophoblast stem cells (hTS) is shown. Figure 1A Schematic diagram of the molecular mechanism of insulin synthesis and secretion in hTS cells. Figure 1B ChIP-qPCR analysis showed that CREB1 and MAFA levels were reduced, and CREB1 and MAFA antibodies inhibited insulin (INS) gene transcription. Data represent mean ± SD, n = 5; Input: positive control (as 100%); IgG: negative control. Figure 1C Glucose (Gluc; 20 mM) and sulfonylurea (Gli, gliclazide; 10 μM) were shown to promote insulin secretion in hTS cells (1x106 cells); while the VDCC inhibitor nifedipine (Nif; 10 μM) inhibited insulin secretion by RIA in secretome analysis. Data represent mean ± SD, n = 3; Student t-test, *p < 0.05, significant compared to control. Figure 1D Representative imaging of immunoreactive molecules characterizing biological roles in hTS cells is shown. Scale bars are as indicated.

[0024] Figures 2A-2M Gα in insulin-expressing hTS cells is shown q / 11 / PIP2 / IP3 / IP3R / CaMKII / CREB1 and Gβ / PI3K / AKT / GSK3β / MAFA signaling pathways. Figures 2A-2C It was shown that glucose (20 mM) stimulated rapid, transient activation of the sweet taste receptors T1R2 / T1R3 at the cell membrane by qPCR analysis in hTS cells ( Figure 2A ), and thus by Western blot analysis ( Figure 2C ) activates G protein signaling, including Gα, within 15 minutes q / 11 and Gβ( Figure 2B This effect was inhibited by the T1R2 / T1R3 inhibitor 2,4-dichlorophenoxyacetic acid (2,4-D; 100 μg) ( Figure 2B ). Data represent mean ± SD, n = 5. Student t-test: *p < 0.05 statistically significant. Figures 2D-2F Western blot analysis demonstrated the establishment of Gα q / 11 / PIP2 / IP3 / IP3R / CaMKII / CREB1 pathway. Active Gα q / 11Inositol triphosphate (IP3) acts on its receptor IP3R at the endoplasmic reticulum (ER) membrane to elevate intracellular calcium levels, which subsequently activates CaMKII. This effect is inhibited by shGα that links Gα q / 11 and CaMKII molecules. Then CaMKII activates downstream CREB1 through phosphorylation (p), which is further verified by using the CaMKII inhibitor KN93. Figures 2G-2I Depicts the establishment of the Gβ / PI3K / AKT / GSK3α / β pathway. Active Gβ rapidly induces PI3K / AKT signaling through phosphorylation of AKT (pAKT) at the ser473 site and induces its downstream inhibitory GSK3α / β via phosphorylation at the ser21 / 9 site. These effects are inhibited by the PI3K inhibitor LY294002 and the AKT inhibitor MK2206. Then inhibitory pGSKα / β promotes pMAFA for nuclear localization, which is confirmed by its presence in the nucleus, and pMAFA expression is inhibited by the GSK3 inhibitor SB216763. Figure 2J Shows that both pCREB1 and pMAFA enter the nucleus, as demonstrated by cytoplasmic fractionation of the nucleus. β-actin: loading control; α-tubulin and H3 indicate cytoplasmic and nuclear compartments, respectively. Figure 2K Shows that pretreatment with CREB1 shRNA rather than PDX1 shRNA significantly reduces insulin expression in hTS cells in response to glucose stimulation (Glu; 20 mM) by qPCR analysis. C is used as a control and shGFP as a positive control. Student t-test; *: p < 0.05, n = 4. Figure 2L Insulin expression in two hTS cell lines with two different anti-insulin antibodies is shown by flow cytometry. Isotype: control. Figure 2M By immunocytochemistry, native hTS cells do not express stress proteins or the proliferation marker Ki67, but express HSP90. Scale bar: 50 μm.

[0025] Figure 3 Shows representative images of insulin-related immunoreactive molecules (including PDX1, HNF-1β, NGN3, SOX9, NKX6.1, and insulin) in prCTB. Bar scale: 50 μm.

[0026] This article describes Figures 4A to 4F . Figure 4A By immunocytochemistry, hTSC is shown to express TGF-β1. Figure 4B By Western blot assay, bFGF (10 ng / ml) upregulates TGF-β1 and vimentin but downregulates E-cadherin, while Figure 4DThe anti-TGF-β1 antibody was shown to neutralize these effects. Data are represented as mean ± SD, n = 3, Student's t-test: statistical significance: *p < 0.05, **p < 0.001. Figure 4C Immunocytochemistry showed that bFGF (10 ng / ml) upregulated TGF-β1 and vimentin, but downregulated E-cadherin. Figure 4E bFGF (10 ng / ml) was shown to induce morphological changes in hTSCs after 1 day of incubation, with the cell shape changing from long and spindle-shaped to shorter and fatter, while the nuclear shape changed from oval to more rounded. Figure 4F Confirmation of bFGF-induced FOXA2 activation was shown, whereby FOXA2 was attenuated in the presence of shRNA against β-catenin. Cells transfected with shGFP (nonspecific shRNA) were used as controls, and β-actin was used as a loading control.

[0027] Figures 5A-5C Describe the biological characteristics of precursor regulatory cytotrophoblasts (prCTBs). Figure 5A Representative images of immunoreactive molecules related to insulin expression and oxidative stress proteins in chorionic villi of tubal ectopic pregnancies (7 - 8 weeks of gestation) are shown. Scale bar: 50 μm. Figure 5B The distribution of insulin and stress proteins in chorionic villi of normal pregnancies (7 - 8 weeks of gestation) was shown by imaging. Scale bar: 200 μm. Figure 5C Schematic diagram showing the differences between prCTBs and hTS cells in terms of stress protein expression and trophoblast differentiation pathways compared to normal uterine pregnancy cells.

[0028] Figure 6A and 6B Comparison of syncytial knots expressing stress protein p53 and insulin in chorionic villi between normal pregnancy eggs and blighted eggs is shown. Stressed syncytial knots were defined by the expression of insulin and p53 in areas containing more than 15 positive cells counted in a single chorionic villus at 8 weeks of normal pregnancy ( Figure 6A ); while chorionic villi from patients with blighted eggs at 7 - 8 weeks of gestation ( Figure 6B ), showed ( Figure 6B ) a 1.96-fold higher frequency of occurrence of stressed syncytial knots than ( Figure 6A ). The number of syncytial knots was independently counted by two assistants in 80 fields of view ( Figure 6A ) and 50 fields of view ( Figure 6B ). Another 25 fields of view were not shown. Figure 6C Changes in the expression of pluripotent transcription factors during the imaging of cell-converted immunoreactive OCT4 but not CDX2 in the DE stage after 4 hours of induction are shown.

[0029] This article describes Figures 7A to 7Q . Figure 7A Luminex analysis showing exosomes secreted from hTS cells (dark columns on the left) and prCTB cells (light columns on the right) in PLUS-containing medium (upper panel), and exosomes present in native hTS cells (dark columns on the left) and native prCTB (light columns on the right) after excluding the effects of components in the PLUS medium used (lower panel). Figure 7B Showing immunoblot analysis results indicating the presence of many receptor molecules in hTS cells (left) and prCTB (right). Figure 7C Secretome assay results of proteins released from hTS cells and prCTB shown by immunoblot analysis. sHLA-G: soluble HLA-G. n = 3. Figure 7D hTSC expression of angiogenic factors CD105 and CD146 shown by FACS analysis. Figure 7E Showing immunoblot analysis results indicating the presence of many receptor molecules in hTS cells (left) and prCTB (right). Figure 7F Immunoblot analysis showing that bFGF (10 ng / ml) induces upregulation of STAT3, c-JUN, and Fas ligand (FasL), but downregulation of Fas. This effect is inhibited by shFGFR1. shGFP is used as a negative control. Figures 7G-7J Protein blot assay showing that bFGF (10 ng / ml) induces activation of CREB1 signaling. Figure 7G Showing that bFGF activates its receptor FGFR1 and downstream PI3K signaling, which is inhibited by the FGFR inhibitor PD166866. Figure 7H Showing that PI3K phosphorylates (p)AKT to form PI3K / pAKT signaling, which is inhibited by PI3K shRNA. Figure 7I Showing that pAKT activates its downstream CREB1, which is neutralized by using AKT antibodies (including AKT1 shRNA, AKT2 shRNA, and AKT3 shRNA). Figure 7J Immunoprecipitation (IP) assay showing that pAKT activates its downstream CREB1 through direct interaction. Figure 7K Showing that bFGF (10 ng / ml) induces IL-6 (left panel) and IL-8 (right panel) in a time-dependent manner. Data represent mean ± SD, n = 3 independent cell lines, student-t test: statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001. Figure 7LRT-qPCR analysis shows that IL-6 (10 ng / ml) upregulates IL-6R mRNA, CREB1 mRNA, and β-hCG mRNA in hTSCs (left panel), and IL-8 (30 ng / ml) upregulates CREB1 mRNA and CD56 mRNA, but not the conventional receptor IL-8R (right panel). Data are represented as mean + / − SD, n = 4, Student t-test, statistical significance **p < 0.01, ***p < 0.001. Figures 7M-7N Western blot analysis shows that IL-6 generates β-hCG(+)CD56(+)prCTB. Figure 7M IL-6 (10 ng / ml) is shown to induce β-hCG via GnRHR and IL-6R, which is inhibited by the GnRHR inhibitor Elagolix sodium and the CREB1 inhibitor 666-15. Figure 7N IL-8 (30 ng / ml) is shown to induce CD56 via CXCR2 / STAT3 signaling, which is inhibited by the CXCR2 inhibitor SB225002 and the STAT3 inhibitor Stattic. Figure 7O IL-8 (30 ng / ml) is shown to generate CD4(+)prCTB via the CXCR2 / CREB1 and CXCR2 / STAT3 signaling pathways, which is inhibited by the CREB1 inhibitor 666-15, the CRCR2 inhibitor SB225002, and the STAT3 inhibitor Stattic. Figure 7P IL-8 (30 ng / ml) is shown to induce Foxp3 via the CXCR2 / STAT3 signaling pathway, which is inhibited by the CRCR2 inhibitor SB225002 and the STAT3 inhibitor Stattic (E). Data represent mean ± SD, n = 3 independent cell lines, student-t test: statistical significance *p < 0.05, **p < 0.01, ***p < 0.001. Figure 7Q Immunocytochemistry shows that bFGF induces co-expression of CD4 and Foxp3 in prCTB. Scale bar: 50 μm.

[0030] Figure 8 Two tables are shown listing CD molecules in hTS cells and prCTB measured by flow cytometry, and markers of T cell and NK cell subtypes detected in hTS cells and prCTB measured by flow cytometry.

[0031] Figures 9A to 9N Representative flow cytometric analysis of CD biomarker distribution in hTS cells and prCTB is shown. Figures 9A-9KFACS plot analysis of representative samples out of a total of 8 samples, showing undetectable CD3 and CD45 ( Figure 9A ), CD34 ( Figure 9B ), and CD3 and gdTCR ( Figure 9C ) in hTS cells (left panel) and prCTB (right panel), however, detectable CD(16 + 56) and CD107a ( Figure 9D ), CD(16 + 56) ( Figure 9E ), detectable CD(16 + 56) but little CD4 ( Figure 9F ), detectable CD(16 + 56) and CD8 ( Figure 9G ), detectable CD(16 + 56) but no CD19 ( Figure 9H ), detectable CD107a but little CD4 ( Figure 9I ), detectable CD107a and CD8 ( Figure 9J ), and detectable CD107a but no CD19 ( Figure 9K ). Figure 9L and 9M show the distribution of CD molecules ( Figure 9L ) and NK cell and T cell subsets ( Figure 9M ) in hTS cell (black bars) and prCTB (gray bars) populations. Data are represented as mean ± SD, n = 8 independent samples. Figure 9N Expression of CD11b and CD49f in prCTB was shown by immunostaining.

[0032] This article describes Figures 10A to 10K . Figures 10A-10D Results of transwell invasion and migration assays are shown. MCP-1 ( Figure 10A and 10B ) and CXCL2 ( Figure 10C and 10D ) significantly induced the movement of both hTSC and prCTB in a dose-dependent and time-dependent manner. Figure 10E Immunohistochemistry of normal chorionic villi at 7 - 8 weeks of gestation is shown, revealing that invasive EVT expresses immunoreactive p53(+), syncytin, β-hCG, and HLA-G molecules. Figures 10F-10G Shows CD56(+) prCTB ( Figure 10F brown, upper and middle panels) and β-hCG(+) prCTB ( Figure 10G brown, upper panel) moving towards EVT for implantation (red arrows). Abundant CD56(+) (lower panel of F) and β-hCG(+) prCTB ( Figure 10Gin the middle figure). Substituting arterial endothelial cells with β-hCG(+)prCTB in the maternal decidua indicates SA remodeling( Figure 10G in the middle figure) and β-hCG(+)prCTB within the vascular lumen of the decidual vein( Figure 10G in the lower figure), also indicating the invasion of the vein. Figures 10H-10K Photographs showing invasion and migration assays. Figures 10H-10I Showing that MCP-1 drives the movement of hTSC (4×103 cells / ml) in hTSC and prCTB (4×103 cells / ml each) in a dose-dependent manner( Figure 10H ) and in a time-dependent manner( Figure 10I ). Figures 10J-10K Showing that CXCL2 drives the movement of hTSC (4×103 cells / ml) in hTSC and prCTB (4×103 cells / ml each) in a time-dependent manner( Figure 10J ) and in a dose-dependent manner( Figure 10K ). Blue indicates cell staining with 0.2% crystal violet (Sigma-Aldrich; 115940). The number of migrating cells (blue) was counted by using a hemocytometer or flow cytometer. n = 3 independent samples in each experiment.

[0033] This article describes Figures 11A to 11K . Figures 11A-11D Showing co-culture of prCTB (arrow tip) and PANC-1 (arrow) at a ratio of 2:1 (3×104 cells / well) over time, showing apoptotic changes of PANC-1 contained by prCTB by light microscopy( Figure 11A ). Immunocytochemistry shows the interaction of the two live cells: prCTB (blue, CytoCalcein 450 dye) and PANC-1 (red, PKH26 dye) in the co-culture( Figure 11B ). Apoptotic PANC-1 (green, apopxin dye, upper) and intact prCTB (blue, middle) were observed by interaction (merged, arrow, lower)( Figure 11C ), and 3D fluorescence microscopy analysis shows apoptotic PANC-1 (green) and intact prCTB (blue) upon interaction( Figure 11D ). Figure 11E Showing immunocytochemistry, revealing that prCTB expresses PD-L1 (upper, left column) but not PD-1 (lower, left column); while PANC-1 expresses both PD-1 (upper, right column) and PD-L1 (lower, right column). Figure 11FFasL was shown to be present in prCTB (upper, left panel), but not in PANC-1 (lower, right panel), whereas Fas was present in both prCTB (lower, left panel) and PANC-1 (upper, right panel). Scale bar is as indicated. Figures 11G-11K prCTB was shown to induce apoptosis of solid tumor cells upon interaction. Figure 11G prCTB (blue) was shown to induce apoptosis of breast MCF-7 cells (green) upon interaction by 3D cell probing-fluorescence microscopy ( Figure 11G ). Figure 11H prCTB was shown to express PD-L1 but not PD-1 (left panel); whereas PANC-1 expressed both PD-1 and PD-L1 (right panel). Figure 11I prCTB was shown to express both FasL and Fas (left panel); whereas PANC-1 expressed both (right panel). Figure 11J Analysis by RT-qPCR showed that prCTB significantly upregulated anti-apoptotic Mcl-1 mRNA and Bfl-1 mRNA. Data represent mean ± SD, n = 4, student-t test: statistical significance: *p < 0.05, **p < 0.01. Figure 11K prCTB was shown to cause apoptosis (apopxin, green) of Huh7 cells (liver), H1299 cells (lung), MKN45 cells (stomach), PA-1 cells (ovary) and A375 cells (melanoma) at 24 h of co-culture, as analyzed by 3D cell probing-fluorescence microscopy (Nanolive, Swiss). Scale bar is as indicated.

[0034] Figures 12A-12B Cellular processes in the migration of CD56(+)β-hCG(+) cells from EVT to the maternal decidua were shown. In tubal ectopic chorionic villi. Left panel: Immunocytochemistry of CD56-expressing cells. Middle panel: Scattered distribution of CD56(+) cells in the villous stroma, inner layer of villous CTB (black arrow), and concentration in the EVT area. Right panel: Distribution of β-hCG-expressing trophoblasts on the villous surface and in the EVT area. In normal placental villi. Left panel: Histological identification of anchoring villi, EVT and maternal decidual tissues by H&E staining. Middle panel: Appearance of CD56(+) cells in the EVT area and adjacent decidual tissues. Right panel: Significant appearance of CD56(+) cells in the maternal decidua. Similar distribution of β-hCG(+) trophoblasts and CD56(+) cells. Detailed implementation

[0035] In the drawings, details of one or more embodiments of the present invention are set forth in the claims and the specification. Other features, objects and advantages of the embodiments of the present invention disclosed and anticipated herein may be combined with any other embodiment, unless explicitly excluded.

[0036] The present disclosure relates to uniquely generated in vitro precursor regulatory cytotrophoblast cells, compositions thereof, and uses thereof in treating disorders (such as cancer, inflammation, or autoimmune diseases) or improving conditions (such as skin conditions). The precursor regulatory cytotrophoblast cells are different from trophoblast stem cells. The precursor regulatory cytotrophoblast cells are also different from embryonic stem cells. The precursor regulatory cytotrophoblast cells are also different from primitive cytotrophoblasts and cells derived from primitive cytotrophoblasts, including villous cytotrophoblasts (villous CTB), primitive syncytiotrophoblasts (pSTB), syncytiotrophoblasts (STB), and extravillous cytotrophoblasts (EVT), see Figure 5C . The precursor regulatory cytotrophoblast cells are also different from placental cytotrophoblasts. In some cases, the precursor regulatory cytotrophoblast cells are not precursor villous cytotrophoblast cells.

[0037] 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 should be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory and are not restrictive of any claimed subject matter. In this application, unless otherwise stated, the use of the singular includes the plural. It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms such as "comprises", "including", and "contains" is not restrictive.

[0038] As used herein, ranges and amounts may be expressed as "about" a particular value or range, such as ±15% of the recited value. About also includes the exact amount. Thus, "about 5 μL" means "about 5 μL" as well as "5 μL". Generally, the term "about" includes amounts expected to be within experimental error.

[0039] As used herein, the terms "treat", "treatment", etc. refer to obtaining the desired pharmacological and / or physiological effect. In some cases, an individual (e.g., an individual suspected of having and / or genetically predisposed to having a liver-related disease or disorder) is prophylactically treated with the cell preparations described herein, and such prophylactic treatment completely or partially prevents a liver-related disease or disorder or its signs or symptoms. In some cases, an individual is therapeutically treated (e.g., when the individual has a liver-related disease or disorder), and such therapeutic treatment causes partial or complete cure of the disease or disorder and / or reversal of the adverse effects attributable to the disease or disorder and / or stabilization of the disease or disorder and / or delay in the progression of the disease or disorder and / or causing regression of the disease or disorder.

[0040] Administering (e.g., transplanting) the cells disclosed herein to an area in need of treatment is accomplished by, for example but not limited to, local infusion during surgery, by injection, by catheter, or by an implant, which is a porous, non-porous, or gel-like material, including membranes such as sialastic membranes, or fibers.

[0041] "Transplanting" a composition into a mammalian body refers to introducing the composition into the mammalian body by any method established in the art. The introduced composition is a "graft", and the mammalian is a "recipient". The graft and the recipient can be syngeneic, allogeneic, or xenogeneic. Additionally, the transplant can be an autograft.

[0042] When referring to the use of cells or cell populations, the term "isolated" refers to the state in which the cells or cell populations are separated from the host organism from which the cells or cell populations are derived. In some cases, the isolated cells are in contact with other cells isolated from the same host organism. In some cases, the isolated cells are separated from any other cells. In some cases, the isolated prCTB is derived in vitro from progenitor cells. In some cases, the isolated prCTB is obtained from and separated from the host organism.

[0043] "Effective amount" is the amount of a therapeutic agent sufficient to achieve the intended purpose. The effective amount of a composition for treating or ameliorating a disorder is the amount of the composition sufficient to alleviate or eliminate the symptoms of the disorder.

[0044] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0045] Cells and Compositions

[0046] In some cases, the present disclosure provides in vitro isolated precursor regulatory cytotrophoblasts (prCTBs), wherein the isolated prCTBs express one or more of the following proteins: 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 signal receptor (Fas), Fas ligand (FasL), CD335 (NKp46), CD11b, CD49f, CD3, CD19, CD34, B cell leukemia / lymphoma 2-related protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), or any combination thereof; or the prCTBs secrete chemokines, cytokines, growth factors, or any combination thereof, or secrete exosomes carrying chemokines, cytokines, growth factors, or any combination thereof; and / or wherein the prCTBs express p53, Ki67, glutamate decarboxylase (GAD65), heat shock protein 70 (HSP70), soluble CD40 ligand (sCD40L), or any combination thereof. In some cases, the isolated prCTBs express CD4, CD16, CD56, CD107a, CD8, or any combination thereof. In some cases, the prCTBs induce apoptosis of cancer cells, for example, by infiltrating cancer cell colonies, optionally wherein the prCTBs themselves do not undergo apoptosis upon contact with cancer cells. In some cases, the cancer cells are solid tumor cells. In some cases, 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 cases, the cancer cells express PD-1 and PD-L1, while the prCTBs express PD-L1 but not PD-1. In some cases, the cancer cells express Fas but not FasL, while the prCTBs express Fas and FasL. In some cases, the prCTBs express Bfl-1 and Mcl-1.

[0047] In some cases, the present disclosure provides isolated cell populations comprising precursor regulatory cytotrophoblasts (prCTBs), wherein the prCTB population expresses one or more of the following proteins: 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 signal receptor (Fas), Fas ligand (FasL), CD335 (NKp46), B-cell leukemia / lymphoma 2-related protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), CD11b, CD49f, CD3, CD19, CD34, or any combination thereof; or the prCTBs secrete chemokines, cytokines, growth factors, or any combination thereof, or secrete exosomes carrying chemokines, cytokines, growth factors, or any combination thereof; and / or wherein the prCTB population expresses p53, Ki67, glutamate decarboxylase (GAD65), heat shock protein 70 (HSP70), soluble CD40 ligand (sCD40L), or any combination thereof. In some cases, at least about 10% of the population is prCTBs expressing CD16 and CD56. In some cases, at least about 2% of the population is prCTBs expressing CD4. In some cases, at least about 2% of the population is prCTBs expressing CD8. In some cases, at least about 5% of the population is prCTBs expressing CD107.

[0048] In some cases, the present disclosure provides isolated cell populations comprising precursor regulatory cytotrophoblasts (prCTBs), wherein: (i) at least about 10% of the population is prCTBs expressing CD16 and CD56; (ii) at least about 2% of the population is prCTBs expressing CD4; (iii) at least about 2% of the population is prCTBs expressing CD8; or (iv) at least about 5% of the population is prCTBs expressing CD107, or any combination thereof. In some cases, the present disclosure provides isolated cell populations comprising precursor regulatory cytotrophoblasts (prCTBs), wherein: (i) at least about 10% of the population is prCTBs expressing CD16 and CD56; (ii) at least about 2% of the population is prCTBs expressing CD4; (iii) at least about 2% of the population is prCTBs expressing CD8; and (iv) at least about 5% of the population is prCTBs expressing CD107, or any combination thereof. In some cases, the cell population comprises at least about 2% of the population as prCTBs expressing CD16, CD56, and CD107.

[0049] In some cases, the isolated prCTB expresses interleukin 15 (IL-15). In some cases, 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-related protein A1 (BCL2A1 or Bfl-1), myeloid cell leukemia sequence 1 (Mcl-1), CD11b, CD49f, CD3, CD19, CD34, or any combination thereof. In some cases, the isolated prCTB also expresses beta-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 cases, the isolated prCTB lacks the expression of syncytin, programmed cell death protein 1 (PD-1), or a combination thereof. In some cases, the isolated prCTB secretes chemokines, cytokines, growth factors, or any combination thereof, or carries exosomes containing chemokines, cytokines, growth factors, or any combination thereof. In some cases, the cytokines include 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 (CXCL1), chemokine (C-X-C motif) ligand 2 (CXCL2), 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 (CXCL10), fractalkine, and chemokine (C-C motif) ligand 4 (CCL4), or any combination thereof. In some cases, the cytokines include interleukin 1α (IL-1α), interleukin 1β (IL-1β), 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 cases, the cytokines include interferon α (IFN-α) or interferon γ (IFN-γ).In some cases, the growth factors include 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 α (TNFα), interleukin 1β (IL-1β), or any combination thereof. In some cases, the isolated prCTB has a higher level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN than the progenitor cells from which it is differentiated in vitro, as measured by immunoblotting. In some cases, the level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN in the isolated prCTB is 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 than that of the progenitor cells from which the isolated prCTB is differentiated in vitro, as measured by immunoblotting. In some cases, the level of SOX2 protein expressed by the isolated prCTB is 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 than that of the progenitor cells from which the isolated prCTB is differentiated in vitro. In some cases, the chorionic villus-derived progenitor cells lack the expression of glutamate decarboxylase (GAD65), Ki67, heat shock protein 70 (HSP70), p53, soluble CD40 ligand (sCD40L), or any combination thereof. In some cases, both the chorionic villus-derived progenitor cells and the isolated prCTB express heat shock protein 90 (HSP90). In some cases, the isolated prCTB is a human cell. In some cases, the isolated prCTB is derived from a rodent, rabbit, bovine, sheep, pig, dog, cat, monkey, or ape. In some cases, the isolated prCTB is present in a pharmaceutical composition that also contains a pharmaceutically acceptable excipient.

[0050] In some cases, the cells provided herein, such as prCTB, are genetically modified. In some cases, 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 transferred into an organism. For example, a transgene can be an extension or segment of DNA containing a gene introduced into an organism. When a transgene is transferred into an organism, that organism is then referred to as a transgenic organism. A transgene can retain its ability to produce RNA or a polypeptide (such as a protein) in the transgenic organism. A transgene can consist of different nucleic acids, such as RNA or DNA. A transgene can encode an engineered T cell receptor, such as a TCR transgene. A transgene can contain a TCR sequence. A transgene can contain an oncogene. A transgene can contain an immuno-oncogene. A transgene can contain a recombination arm. A transgene can contain an engineered site. In some cases, the transgene is an oncogene. In some cases, the transgene is an immuno-oncogene. In some cases, the transgene is a tumor suppressor gene. In some cases, the transgene encodes a protein that directly or indirectly promotes proteolysis. In some cases, the transgene is an oncolytic gene. In some cases, the transgene can help lymphocytes target tumor cells. In some cases, the transgene is a T cell enhancing gene. In some cases, the transgene is an oncolytic virus gene. In some cases, the transgene inhibits tumor cell growth. In some cases, the transgene is an anti-cancer receptor. In some cases, the transgene is an anti-angiogenic factor. In some cases, the transgene is a cytotoxic gene. Exemplary transgenes include, but are not limited to, CD28, inducible costimulator (ICOS), CD27, 4-1BB (CD137), ICOS-L, CD70, 4-1BBL, Signal 3, cytokines 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, truncated CD19, VEGF, caspase, chemokine, or one or more genes encoding an antibody (such as a monoclonal antibody) to any of the foregoing, or any combination thereof. In some cases, the transgene encodes a protein involved in cell or tissue repair (such as proteins related to DNA repair, immune responses (such as interferons and interleukins), and structural proteins). In some cases, the transgene encodes a growth factor receptor. In some cases, prCTB as described herein contains a transgene encoding a TCR, B cell receptor (BCR), chimeric antigen receptor (CAR), or any combination thereof. In some cases, the CAR can contain an antigen recognition domain, a hinge region, a transmembrane domain, and an intracellular signaling domain.In some cases, the antigen recognition domain can be exposed to the exterior of the cell and can interact with potential target molecules. The antigen recognition domain can comprise the variable regions of monoclonal antibodies that can be linked as single-chain variable fragments. In some cases, the single-chain variable fragment can comprise the variable light and variable heavy chains of immunoglobulins that can be linked to a linker peptide. In some cases, a ligand or receptor system can be used as an alternative to the antibody-based antigen recognition domain. The hinge domain can be designed to combine the transmembrane domain with the antigen recognition domain. In some cases, the hinge region can be a peptide that can be designed to increase the flexibility of the antigen recognition domain. The transmembrane domain can comprise a hydrophobic α-helix that can span the cell membrane. For example, the CD28 transmembrane domain can be used as the transmembrane domain in a CAR. In some cases, 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 intracellular. In some cases, when the target protein binds to the antigen recognition domain, the intracellular signaling domain can transmit an activation signal. In some cases, activation of the signaling domain can include phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs). The intracellular signaling domain can comprise a modified ITAM. In some cases, the modified ITAM can comprise a signaling domain such as the CD3-ζ domain. In some cases, the modified ITAM signaling domain can comprise: CD3-ζ, CD3-ε, CD3-γ, CD3-δ, derivatives thereof, or any combination thereof. In some cases, the intracellular signaling domain can comprise one or more co-stimulatory domains. In some cases, the intracellular signaling domain can comprise one or more signaling domains and one or more co-stimulatory signaling domains. In some cases, the co-stimulatory domain can comprise a signaling domain from CD28, CD27, CD40, CD134, CD137, inducible co-stimulator (ICOS), DAP10, derivatives thereof, or any combination thereof. In some cases, prCTB as described herein comprises a transgene encoding a cancer gene receptor.

[0051] In some cases, a composition comprising the cells disclosed herein is formulated as a pharmaceutical composition for intravenous administration to a mammal, including a human. In some cases, the composition for intravenous administration is a solution in a sterile, buffered aqueous vehicle. If necessary, the composition also includes a local anesthetic to ameliorate any pain at the injection site. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

[0052] In one aspect, the present disclosure provides compositions (e.g., pharmaceutical compositions) comprising the cells disclosed herein. In some cases, the compositions further comprise a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, and combinations thereof. In other instances, colloidal dispersion systems are used. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0053] Secretome and compositions

[0054] In some aspects, the present disclosure provides compositions comprising the secretome of the trophoblast stem cells or prCTBs described herein. In some cases, the secretome comprises exosomes secreted by the trophoblast stem cells or prCTBs and other soluble molecules (e.g., proteins, nucleic acids, and lipids) secreted by the trophoblast stem cells or prCTBs.

[0055] In some cases, the present disclosure provides compositions comprising chemokines, cytokines, growth factors, or any combination thereof. In some cases, the composition comprises exosomes and a pharmaceutically or cosmetically acceptable excipient, wherein the exosomes carry chemokines, interleukins, growth factors, or any combination thereof. In some cases, the composition is cell-free. In some cases, the composition comprises or the exosomes carry: (i) chemokines, which include CXCL2, MCP-1, fractalkine, IP-10, MCP-3, eotaxin, MIP-1β, or any combination thereof; (ii) interleukins, which include 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, which comprise PDGF-AA, VEGF, bFGF, G-CSF, Flt-3L, GM-CSF, or any combination thereof.

[0056] In some cases, the composition comprises MCP-1 and one, two, three or all of CXCL2, IL-6, IL-8 and VEGF proteins. In some cases, the weight ratio of MCP-1 to CXCL2 in the composition is from about 1:1 to about 2.5:1. For example, the weight ratio of MCP-1 to CXCL2 in the composition is 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, 2.1:1, 2.2:1, 2.3:1, 2.4:1 or 2.5:1. In some cases, the weight ratio of MCP-1 to CXCL2 in the composition is about 2.0. In some cases, the weight ratio of MCP-1 to CXCL2 in the composition is from about 3:1 to about 4:1 or from about 3:1 to about 5:1. For example, the weight ratio of MCP-1 to CXCL2 in the composition is 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 cases, the weight ratio of MCP-1 to CXCL2 in the composition is about 3.2.

[0057] In some cases, the composition comprises MCP-1 and one, two, three or all of CXCL2, IL-6, IL-8 and VEGF proteins. In some cases, the weight ratio of MCP-1 to CXCL2 in the composition is from about 1:7 to about 1:4. For example, the weight ratio of MCP-1 to CXCL2 in the composition is about 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 cases, the weight ratio of MCP-1 to CXCL2 in the composition is about 1:5.0. In some cases, the weight ratio of MCP-1 to CXCL2 in the composition is from about 1:4 to about 1:1.5. For example, the weight ratio of MCP-1 to CXCL2 in the composition is 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 cases, the weight ratio of MCP-1 to CXCL2 in the composition is from about 1:4 to about 1:2.5.

[0058] In some cases, the weight ratio of MCP-1 to IL-6 in the composition is from about 2:1 to about 3:1. For example, the weight ratio of MCP-1 to IL-6 in the composition is 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 cases, the weight ratio of MCP-1 to IL-6 in the composition is about 2.3. In some cases, the weight ratio of MCP-1 to IL-6 in the composition is about 2.5. In some cases, the weight ratio of MCP-1 to IL-6 in the composition is from about 3:1 to about 4:1. For example, the weight ratio of MCP-1 to IL-6 in the composition is 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 cases, the weight ratio of MCP-1 to IL-6 in the composition is about 3.6. In some cases, the weight ratio of MCP-1 to IL-6 in the composition is about 3.8.

[0059] In some cases, the weight ratio of MCP-1 to IL-8 in the composition is from about 4:1 to about 6:1. For example, the weight ratio of MCP-1 to IL-8 in the composition is 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 cases, the weight ratio of MCP-1 to IL-8 in the composition is about 4.6. In some cases, the weight ratio of MCP-1 to IL-8 in the composition is about 4.4. In some cases, the weight ratio of MCP-1 to IL-8 in the composition is about 4.9. In some cases, the weight ratio of MCP-1 to IL-8 in the composition is about 4.5.

[0060] In some cases, the weight ratio of MCP-1 to VEGF in the composition is from about 5:1 to about 7:1. For example, the weight ratio of MCP-1 to VEGF in the composition is 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 cases, the weight ratio of MCP-1 to VEGF in the composition is about 5.6. In some cases, the weight ratio of MCP-1 to VEGF in the composition is about 6.0. In some cases, the weight ratio of MCP-1 to VEGF in the composition is from about 7:1 to about 9:1. For example, about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0. In some cases, the weight ratio of MCP-1 to VEGF in the composition is about 7.6:1. In some cases, the weight ratio of MCP-1 to VEGF in the composition is about 7.3:1.

[0061] In some cases, the composition further comprises PDGF-AA. In some cases, MCP-1 and PDGF-AA are present in a weight ratio of from about 3:1 to about 5:1, such as 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 cases, MCP-1 and PDGF-AA are present in a weight ratio of about 3.5. In some cases, MCP-1 and PDGF-AA are present in a weight ratio of from about 6:1 to about 9:1, such as 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 cases, MCP-1 and PDGF-AA are present in a weight ratio of about 7.8.

[0062] In some cases, the composition further comprises PDGF-AA. In some cases, MCP-1 and PDGF-AA are present in a weight ratio of from 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 cases, MCP-1 and PDGF-AA are present in a weight ratio of about 0.6. In some cases, MCP-1 and PDGF-AA are present in a weight ratio of from 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 cases, MCP-1 and PDGF-AA are present in a weight ratio of about 1.2.

[0063] In some cases, the composition further comprises PDGF-AA. In some cases, MCP-1 and PDGF-AA are present in a weight ratio of from about 3:1 to about 5:1, such as 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 cases, MCP-1 and PDGF-AA are present in a weight ratio of about 3.5. In some cases, MCP-1 and PDGF-AA are present in a weight ratio of from about 6:1 to about 9:1, such as 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 cases, MCP-1 and PDGF-AA are present in a weight ratio of about 7.8.

[0064] In some cases, the composition further comprises PDGF-AA and G-CSF. In some cases, the composition further comprises PDGF-AA and FGF-2 (bFGF). In some cases, the composition further comprises one or more proteins selected from 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, IFNα, IFNγ, PDGF-AB / BB or EGF.

[0065] In some cases, the composition further comprises nucleic acids, such as mRNA, siRNA, shRNA, or DNA. In some cases, the composition further comprises lipid molecules secreted from prCTB or trophoblast stem cells.

[0066] In some cases, the compositions disclosed herein may be sterile. In some cases, the composition may contain resident microorganisms. The microorganisms can be viruses, bacteria, eukaryotic cells, or any combination thereof. In some cases, the microorganisms may not be pathogenic. In some cases, the composition may contain one or more bacteria at a concentration 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 cases, the composition may contain 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 cases, the composition may be substantially free (e.g., at least 95% free) or free of: Staphylococcus aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, Pseudomonas spp., Klebsiella pneumoniae, or any combination thereof.

[0067] In some cases, the compositions disclosed herein may be free of heavy metals such as lead, bithionol, chlorofluorocarbon propellants, nitrosamines, chloroform, halosalicylanilides, hexachlorophene, mercury compounds, 1,4-dioxane, dichloromethane, prohibited bovine materials, sunscreen compounds, vinyl chloride, zirconium-containing complexes, or any combination thereof. In some cases, prohibited bovine materials may include brain, skull, eye, trigeminal ganglion, spinal cord, spine, dorsal root ganglion, tonsil, distal ileum of the small intestine, or any combination thereof. In some cases, the composition may contain lead at a level of 10 parts per million (ppm) or less.

[0068] In some cases, the compositions herein do not contain coloring additives, fragrances, parabens, phthalates, alcohols, or any combination thereof. In some cases, coloring additives, fragrances, parabens, phthalates, or alcohols are present in the composition at negligible levels, such as less than: 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%. In some cases, excipients may not have an effect on the technology / structure, function, or any combination thereof in the composition, such as not having active ingredients. In some cases, the composition does not contain.

[0069] In some cases, the excipients disclosed herein may include water, glycerin, saline, vegetable oils (such as seed oils), fruit oils, flower extracts, mineral oils, synthetic oils, sugar compounds, silicates, calcium salts, magnesium salts, sodium chloride, potassium chloride, lactic acid, starch, sugar alcohols, cellulose, activated carbon, glycerin, butter, amino acids, paraffin, honey, wax, beeswax, agar, calcium carbonate, citric acid, tartaric acid, stearic acid, xanthan gum, benzoic acid, polyethylene glycol, silicon, its derivatives, its salts, or any combination thereof.

[0070] Method of Use

[0071] In some cases, the isolated prCTB is administered to a subject intravenously, subcutaneously, percutaneously, by inhalation, orally, intramuscularly, or intratumorally. In some cases, the subject is a mammal. In some cases, the subject is a primate. In some cases, the subject is a human. In some cases, the isolated prCTB expresses interleukin 15 (IL-15). In some cases, the isolated prCTB expresses CD4, CD16, CD56, CD107a, CD8, or any combination thereof. In some cases, 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), CD11b, CD49f, CD3, CD19, CD34, or any combination thereof. In some cases, the isolated prCTB further expresses beta-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 10 (IL-10), CD105, CD146, or any combination thereof. In some cases, the isolated prCTB lacks the expression of syncytin, programmed cell death protein 1 (PD-1), or a combination thereof. In some cases, the isolated prCTB secretes chemokines, cytokines, growth factors, or any combination thereof, or carries exosomes containing chemokines, cytokines, growth factors, or any combination thereof. In some cases, the cytokines include chemokine (C-C motif) ligand 5 (CCL5), monocyte chemoattractant protein-1 (MCP-1), monocyte chemoattractant protein-3 (MCP-3), chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-X-C motif) ligand 2 (CXCL2), 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 (CXCL10), fractalkine, chemokine (C-C motif) ligand 4 (CCL4), or any combination thereof.In some cases, cytokines include interleukin 1α (IL-1α), interleukin 1β (IL-1β), 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 cases, cytokines include interferon α (IFN-α) or interferon γ (IFN-γ). In some cases, growth factors include 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 α (TNFα), interleukin 1β (IL-1β), or any combination thereof. In some cases, isolated prCTB has a higher level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN than the progenitor cells from which it differentiates in vitro, as measured by immunoblotting. In some cases, the level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN in isolated prCTB is 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 than that of the progenitor cells from which it differentiates in vitro, as measured by immunoblotting. In some cases, the level of SOX2 protein expressed by isolated prCTB is 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 than that of the progenitor cells from which it differentiates in vitro. In some cases, the progenitor cells lack the expression of p53, glutamate decarboxylase (GAD65), Ki67, heat shock protein 70 (HSP70), soluble CD40 ligand (sCD40L), or any combination thereof. In some cases, isolated prCTB is a human cell. In some cases, isolated prCTB is derived from a rodent, rabbit, bovine, sheep, pig, dog, cat, monkey, or ape.

[0072] In some cases, the present disclosure provides methods for killing antigen-bearing target cells, which include administering precursor regulatory cytotrophoblasts (prCTBs) to a subject in need, wherein the isolated prCTBs express one or more proteins comprising: 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 signal receptor (Fas), Fas ligand (FasL), CD335 (NKp46), B cell leukemia / lymphoma 2-related 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 cases, the antigen-bearing cells are not antigen-presenting cells, such as not dendritic cells, macrophages, or B cells. In some cases, the antigen-bearing target cells are cancer cells. In some cases, the cancer cells are solid tumor cells. In some cases, the cancer cells are blood cancer cells. In some cases, the cancer cells include bladder cancer cells, bone cancer cells, brain cancer cells, breast cancer cells, cervical cancer, 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 cases, the antigen-bearing target cells are pathogens. In some cases, the pathogens include viruses, bacteria, protozoa, prions, fungi, or any combination thereof. In some cases, 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 the population of antigen-bearing target cells.

[0073] In some cases, methods for downregulating inflammatory pathways are disclosed herein, which include administering precursor regulatory cytotrophoblasts (prCTBs) to a subject in need, (i) wherein the isolated prCTBs express one or more proteins, which include: (a) 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 signal receptor (Fas), Fas ligand (FasL), CD335 (NKp46), B cell leukemia / lymphoma 2-related 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) wherein the isolated prCTBs secrete chemokines, cytokines, growth factors, or any combination thereof, or secrete exosomes carrying chemokines, cytokines, growth factors, or any combination thereof.

[0074] In some cases, the method treats the following diseases or conditions: transplant rejection, infection, endotoxin shock associated with infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic 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, gallbladder disease, Pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, Lyme arthritis, meningoencephalitis, immune-mediated inflammatory diseases of the central and peripheral nervous systems, pancreatitis, surgical trauma, graft-versus-host disease, heart disease, bone resorption, burn patients, myocardial infarction, Paget's disease, osteoporosis, sepsis, liver or lung fibrosis, periodontitis or achlorhydria. In some cases, the method treats autoimmune diseases, the 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, cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's disease, bullous pemphigoid, discoid lupus, ulcerative colitis, dense deposit disease, inflammatory bowel disease or psoriasis. In some instances, the method treats type 1 diabetes. In some cases, the method improves transplant rejection.

[0075] In some cases, methods for modulating skin disorders are disclosed herein, including administering a composition (e.g., a pharmaceutical composition) to a subject in need, the composition comprising a chemokine, a cytokine such as interleukin, a growth factor, or any combination thereof, or exosomes carrying a chemokine, a cytokine such as interleukin, a growth factor, or any combination thereof. In some cases, the method improves the skin disorder such that the skin disorder has one or more better characteristics after application of the method compared to before application of the method. In some cases, the chemokine includes GRO, MCP-1, fractalkine, IP-10, MCP-3, Eotaxin, MIP-1β, or any combination thereof. In some cases, the composition comprises an interleukin, the 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 cases, the growth factor includes PDGF-AA, VEGF, bFGF, G-CSF, Flt-3L, GM-CSF, or any combination thereof. In some cases, the method provides a cosmetic application. In some cases, the method tightens the skin. In some cases, the method hydrates the skin. In some cases, the method rejuvenates the skin. In some cases, the composition is the culture medium after passage of stem cells. In some cases, the stem cells are the isolated precursor regulatory cell trophoblast (prCTB) described herein. In some cases, the number of passages is at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, the stem cells are cultured in the culture medium for at least about 1 - 3 days, such as 1 - 2 days, for example, before collecting the culture medium. In some cases, the stem cells are cultured in the culture medium for at least about 12 - 24 hours, for example, before collecting the culture medium. In some cases, the number of passages is 5 to 10. In some cases, passage occurs approximately every 1, 2, or 3 days. In some cases, passage occurs approximately every 2, 4, 6, 8, 12, 16, 20, or 24 hours. In some cases, the culture medium is free of stem cells. In some cases, the composition is in the form of a cream, liquid, gel, lotion, liquid spray, capsule, or facial mask. In some cases, the method can be used to treat skin diseases. In some cases, the skin disease can be eczema, psoriasis, acne, rosacea, ichthyosis, vitiligo, urticaria, seborrheic dermatitis, herpes zoster, sunburn, burn, contact dermatitis, rash, or any combination thereof. In some cases, the method can reduce the appearance of skin aging, photoaging, or any combination thereof. In some cases, the method can reduce the appearance of scars. In some cases, the method can improve wound healing. In some cases, the method can prevent, reduce, or eliminate bruising, benign growths, age spots, cancerous growths, ulcers, infections, or any combination thereof. In some cases, the method can prevent, reduce, or eliminate skin striae, wrinkles, or any combination thereof.In some cases, the dermal or epidermal lines can be crow's feet, smile lines, frown lines, forehead lines, tear troughs, bunny lines, nasolabial folds, commissure lines, chin lines, necklines, age-related wrinkles, wrinkle lines, elastic wrinkles, expression lines, gravitational folds, dynamic wrinkles, static wrinkles, atrophic wrinkles, atrophic rhytides or any combination thereof. In some cases, the method can prevent, reduce or eliminate loss of skin volume, elasticity or any combination thereof. In some cases, the method can prevent, reduce or eliminate skin sagging, dull skin color, mottled discoloration, skin roughness, dry skin, itchy skin, thinning of the skin or any combination thereof. In some cases, the method can enhance or improve skin condition, skin disease or any combination thereof. In some cases, the method can moisturize, tighten, lift or rejuvenate the skin. In some cases, the method can restore or maintain healthy, smooth, flawless, translucent, elastic or any combination thereof skin. In some cases, the method can restore, treat, remedy or otherwise address glycosaminoglycans, dermis, collagen and elastin of the skin. In some cases, 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 , histological examination or any combination thereof. In some cases, improved skin health can be measured by diagnostic images such as magnetic resonance imaging (MRI). In some cases, the measurements can be compared before and after administration of the composition. In some cases, the measurements can be compared to a standard. In another aspect, a method of treating a disorder in a subject is disclosed herein, which comprises administering to the subject a pharmaceutical composition comprising an amount of the cells herein effective to engraft the cells into the subject (e.g., into the liver of the subject). In some cases, the cells are administered in a pharmaceutically acceptable carrier. In some cases, the pharmaceutically acceptable carrier comprises saline, such as phosphate buffered saline, or fetal bovine serum. In some cases, the cells are administered at a concentration of about 1×10

[0076] to about 100×10 6 cells / ml, about 1×10 6 to about 250×10 6 cells / ml, about 1×10 6 to about 500×10 6 cells / ml or about 10×10 6 to about 40×10 6 to about 40×10 6Administration of the suspension at a cell density of cells / ml. In some cases, 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 cases, the subject is a human. In some cases, the administration includes injection, such as intravenous injection. In some cases, the injection is performed at the hepatic vein. In some cases, the injection is performed at the hepatic artery. In some cases, the disorder is a liver - related disease or disorder. In some cases, the disorder is liver failure. In some cases, the liver - related diseases or disorders include Alagille syndrome, α1 - antitrypsin deficiency, autoimmune hepatitis, benign liver tumors, biliary atresia, cirrhosis, hepatic cystic diseases, fatty liver diseases (including alcohol - related liver diseases and non - alcoholic fatty liver disease (NAFLD)), galactosemia, cholelithiasis, Gilbert syndrome, hemochromatosis, liver cysts, liver cancer, liver diseases in pregnancy (optionally, acute fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, pre - eclampsia or HELLP syndrome (hemolysis, elevated liver tests, low platelets)), neonatal hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, porphyria, Reye's syndrome, sarcoidosis, toxic hepatitis, type 1 glycogen storage disease, tyrosinemia, viral hepatitis, Wilson's disease, or any combination thereof.

[0077] The administration modes of the cells disclosed herein include, but are not limited to, systemic intravenous injection and direct injection into the intended active site (e.g., endoscopic retrograde injection). The formulation can be administered by any convenient route, such as by infusion or bolus injection, and can be co - administered with other bioactive agents. In some cases, the administration is systemic local administration.

[0078] In some aspects, the present disclosure provides compositions and methods for transplanting the cells disclosed herein into a subject. In some cases, the subject is by cell injection (e.g., intravenous, intramuscular, percutaneous, endoscopic retrograde injection, or intraperitoneal). In some cases, the subject is not treated with an immunosuppressant before transplantation. In some cases, the method further includes treating the patient with an immunosuppressant, such as FK - 506, cyclosporine, or GAD65 antibody.

[0079] In some cases, the cells described herein are delivered to the target site (e.g., the defective part of the liver) through a delivery system adapted to target the cells to a specific tissue. For example, the cells are encapsulated in a delivery vehicle that allows for slow release of the cells at the target site. The delivery vehicle is modified to specifically target a particular tissue. The surface of the targeted delivery system is modified in various ways. In the case of a liposome - based targeted delivery system, lipid groups are incorporated into the lipid bilayer of the liposome to maintain stable binding of the targeting ligand to the liposome bilayer.

[0080] The administration of the cells described herein is optionally customized for an individual by: (1) increasing or decreasing the amount of cells injected; (2) varying the number of injections; or (3) altering the method of delivering the cells.

[0081] Detection method

[0082] Methods for determining the expression or presence of the biomarkers described above are well known in the art and can be measured, for example, by flow cytometry, immunohistochemistry, western blotting, immunoprecipitation, magnetic bead selection, and quantification of cells expressing any of these cell surface markers. Biomarker RNA expression levels can be measured by RT-PCR, Qt-PCR, microarray, RNA blotting, or other similar techniques.

[0083] "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. Thus, "detecting expression" includes situations in which the biomarker is determined to be not expressed, not detectably expressed, expressed at a low level, expressed at a normal level, or overexpressed.

[0084] In some cases, the expression or presence of the biomarkers described herein is determined at the nucleic acid level using, for example, immunohistochemical techniques or nucleic acid-based techniques such as in situ hybridization and RT-PCR. In some cases, the expression or presence of one or more biomarkers is carried out by means of nucleic acid amplification, nucleic acid sequencing, using nucleic acid microarrays (DNA and RNA), or in situ hybridization using specifically labeled probes.

[0085] In some cases, the expression or presence of a biomarker is determined by gel electrophoresis. In some cases, the determination is carried out by transferring to a membrane and hybridizing with a specific probe. In some cases, the expression or presence of a biomarker is determined by diagnostic imaging techniques. In some cases, the expression or presence of a biomarker is determined by a detectable solid matrix. In some cases, the detectable solid matrix is a paramagnetic nanoparticle functionalized with an antibody.

[0086] In some cases, the expression or presence of a biomarker is at the RNA (e.g., mRNA) level. In some cases, techniques for detecting RNA (e.g., mRNA) levels include, but are not limited to, DNA or RNA blot analysis, polymerase chain reaction analysis, and probe arrays.

[0087] A method for detecting mRNA levels includes contacting isolated mRNA with a nucleic acid molecule (probe) that hybridizes to the mRNA encoded by the gene being detected. The nucleic acid probe includes, for example, full-length cDNA or a portion thereof, such as an oligonucleotide having a length of at least 7, 15, 30, 50, 100, 250, or 500 nucleotides and sufficient to specifically hybridize to the mRNA or genomic DNA encoding the biomarker described herein under stringent conditions. Hybridization of the mRNA to the probe indicates that the biomarker or other target protein is being expressed.

[0088] In some cases, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by electrophoresing the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane such as a nitrocellulose membrane. In some cases, 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 readily adapt known mRNA detection methods to detect the levels of mRNA encoding a biomarker or other target protein.

[0089] Alternative methods for determining the level of target mRNA in a sample include processes of nucleic acid amplification, such as by RT-PCR, ligase chain reaction, self-sustained sequence replication, transcription-based amplification systems, Q-β replicase, rolling circle replication, or any other nucleic acid amplification method, followed by detecting the amplified molecules using techniques well known to those skilled in the art. These detection schemes are particularly useful for detecting nucleic acid molecules if they are present in very low amounts. In some cases, biomarker expression is evaluated by quantitative fluorescence RT-PCR (e.g., the TAQMAN system).

[0090] The expression level of the target RNA is monitored using a membrane blot (such as for hybridization assays such as RNA, dot blot, etc.) or a micro-well, sample tube, gel, bead, or fiber (or any solid support containing bound nucleic acid). Detection of expression also includes using nucleic acid probes in solution.

[0091] In some cases, microarrays are used to determine the expression or presence of one or more biomarkers. Nucleic acid microarrays provide a method for simultaneously measuring 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 hybridizes to the complementary probes on the array and is then detected by laser scanning. The hybridization intensity of each probe on the array is measured and converted into a quantitative value representing the relative gene expression level. High-density oligonucleotide arrays are particularly suitable for determining the gene expression profiles of large amounts of RNA in a sample.

[0092] In some cases, arrays are fabricated on surfaces of almost any shape or even on multiple surfaces. In some cases, the array is a planar array surface. In some cases, the array includes beads, gels, polymer surfaces, peptides or nucleic acids on fibers such as optical fibers, glass, or any other suitable substrate. In some cases, the array is encapsulated in a manner that permits diagnostic or other operations of a fully functional device.

[0093] In some cases, the expression or presence of a biomarker as described herein is assayed at the protein level using, for example, an antibody specific for a particular biomarker protein. These antibodies are used in a variety of methods such as Western blotting, ELISA, multiplexing techniques, immunoprecipitation, or immunohistochemical techniques. In some cases, the detection of the biomarker is accomplished by ELISA. In some cases, the detection of the biomarker is accomplished by electrochemiluminescence (ECL).

[0094] Any means is contemplated for specifically identifying and quantifying a biomarker in a biological sample. Thus, in some cases, the expression level of a target biomarker protein in a biological sample is detected by way of a binding protein that is capable of specifically interacting with the biomarker protein or a bioactive variant thereof. In some cases, a labeled antibody, a binding portion thereof, or other binding partner is used. As used herein, the term "label" refers to a detectable compound or composition that is conjugated, either directly or indirectly, to an antibody to produce a "labeled" antibody. In some cases, the label itself is detectable (e.g., a radioisotope label or a fluorescent label), or in the case of an enzyme label, catalyzes a chemical change in a detectable substrate compound or composition.

[0095] Antibodies used to detect biomarker proteins are of monoclonal or polyclonal origin, or are synthetic or recombinantly produced. Standard protein detection methods known to those of skill in the art are used to determine the amount of the complexed protein, e.g., the amount of biomarker protein that binds to a binding protein (e.g., an antibody that specifically binds the biomarker protein). Detailed reviews of immunological assay design, theory, and protocols can be found in many textbooks in the art.

[0096] The choice of marker for labeling antibodies will vary according to the application. However, those skilled in the art can readily determine the choice of marker. These labeled antibodies are used in immunoassays as well as histological applications to detect the presence of any biomarker or target protein. The labeled antibodies are polyclonal or monoclonal. In addition, the antibodies used to detect target proteins are labeled with radioactive atoms, enzymes, chromogenic or fluorescent moieties, or colorimetric tags as described elsewhere herein. The choice of label also depends on the desired detection limit. Enzyme assays (e.g., ELISA) generally allow the detection of colored products formed by the interaction of an enzyme-labeled complex with an enzyme substrate. Radionuclides used 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 used as detectable labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and glucose-6-phosphate dehydrogenase. Chromophore moieties include, but are not limited to, fluorescein and rhodamine. The antibodies are conjugated to these labels by methods known in the art. For example, enzymes and chromogenic molecules are conjugated to antibodies by coupling agents such as dialdehydes, carbodiimides, dimaleimides, etc. Alternatively, conjugation is carried out via ligand-receptor pairs. Examples of suitable ligand-receptor pairs are biotin-avidin or biotin-streptavidin and antibody-antigen.

[0097] In some cases, the expression or presence of one or more biomarkers or other target proteins in a biological sample is determined by radioimmunoassay or enzyme-linked immunosorbent assay (ELISA), competitive binding ELISA, dot blot, western blot, chromatography such as high performance liquid chromatography (HPLC), or other assays known in the art. Thus, the detection assay includes steps such as, but not limited to, immunoblotting, immunodiffusion, immunoelectrophoresis, or immunoprecipitation.

[0098] Methods for obtaining precursor regulatory cytotrophoblasts

[0099] In some embodiments, the prCTB herein (e.g., human prCTB) is derived in vitro from pluripotent stem cells, such as progenitor cells from chorionic villi. In some cases, the prCTB differentiates from pluripotent stem cells in a medium supplemented with one or more differentiation factors. In some cases, the stem cells are progenitor cells from chorionic villi. In some cases, the progenitor cells from chorionic villi include mammalian trophoblast stem cells, such as human trophoblast stem cells.

[0100] In some cases, methods for obtaining precursor regulatory cytotrophoblasts (prCTB) are disclosed herein, which include: in vitro differentiating pluripotent stem cells by contacting the pluripotent stem cells with fibroblast growth factor in a culture medium. In some cases, the culture medium contains nucleosides, L-glutamine, a dipeptide containing L-glutamine, platelet lysate, or a combination thereof. In some cases, the culture medium contains nucleosides, a dipeptide, and platelet lysate. In some cases, the culture medium contains from about 2 mM to about 200 mM of L-glutamine.

[0101] In some cases, pluripotent stem cells, such as human trophoblast stem cells, are contacted with fibroblast growth factor for about 24 hours to 48 hours to produce prCTB. In some cases, 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 cases, 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.

[0102] In some cases, pluripotent stem cells, such as human trophoblast stem cells, are contacted with fibroblast growth factor when the pluripotent stem cells are at passage 5 to 10. In some cases, pluripotent stem cells are contacted with fibroblast growth factor when the pluripotent stem cells are at passage 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0103] In some cases, the method includes culturing the stem cells in a culture medium before contacting the stem cells with fibroblast growth factor to differentiate into prCTB.

[0104] In some cases, the culture medium for obtaining and / or maintaining prCTB does not contain antibiotics, such as penicillin, streptomycin, or any combination thereof. In some cases, the culture medium for obtaining and / or maintaining prCTB does not contain retinoic acid. In some cases, the culture medium for obtaining and / or maintaining prCTB does not contain 2-mercaptoethanol, nicotinamide, or a combination thereof. In some cases, 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 cases, the culture medium for obtaining and / or maintaining prCTB is xeno-free, such as free of animal components. In some cases, the culture medium for obtaining and / or maintaining prCTB does not contain components of human origin and components of animal origin, such as being a chemically defined medium. In some cases, the culture medium for obtaining and / or maintaining prCTB does not contain serum. In some cases, the culture medium for obtaining and / or maintaining prCTB does not contain fetal bovine serum.

[0105] In some cases, the fibroblast growth factor is basic fibroblast growth factor (bFGF). In some cases, the stem cells are contacted with from about 1 ng / ml to about 100 ng / ml bFGF. In some cases, the stem cells are contacted with from about 2 ng / ml to about 50 ng / ml, from about 4 ng / ml to about 30 ng / ml, from about 6 ng / ml to about 15 ng / ml, or from about 8 ng / ml to about 12 ng / ml bFGF. In some cases, the stem cells are contacted with about 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, or 15 ng / ml bFGF. In some cases, the stem cells are contacted with about 10 ng / ml bFGF.

[0106] Method for obtaining mammalian trophoblast stem cells

[0107] In some embodiments, the mammalian trophoblast stem cells herein (e.g., human trophoblast stem cells, hTS cells) can be isolated from umbilical cord, amniotic fluid, amnion, Wharton's jelly, chorionic villi, placenta, or ectopic pregnancy in a manner that does not interfere with or destroy the embryo.

[0108] In some cases, the mammalian trophoblast stem cells described herein are cultured in a medium without antibiotics such as penicillin, streptomycin, or any combination thereof. In some cases, the medium used to obtain the mammalian trophoblast stem cells does not contain retinoic acid. In some cases, the medium for obtaining and / or passaging mammalian trophoblast stem cells does not contain mercaptoethanol, nicotinamide, or any combination thereof. In some cases, the 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 cases, the medium for obtaining and / or passaging mammalian trophoblast stem cells is xenogeneic-free, e.g., does not contain animal components. In some cases, the medium for obtaining and / or passaging mammalian trophoblast stem cells does not contain components of human origin and components of animal origin, e.g., is a chemically defined medium. In some cases, the medium for obtaining and / or passaging mammalian trophoblast stem cells does not contain serum. In some cases, the medium for obtaining and / or passaging mammalian trophoblast stem cells does not contain fetal bovine serum.

[0109] In one case, the mammalian trophoblast stem cells (e.g., hTS cells) of the present disclosure can be isolated from an amniocentesis biopsy sample or from amniotic fluid. In one instance, amniocentesis can be a procedure for obtaining a small sample of the amniotic fluid surrounding the fetus during pregnancy. In one case, amniocentesis can be offered to women between the 15th and 20th weeks of pregnancy who have an increased risk of chromosomal abnormalities, e.g., women over 35 years of age at the time of delivery, or women with abnormal maternal serum (blood) screening tests indicating an increased risk of chromosomal abnormalities or neural tube defects. In one instance, a needle, e.g., a long, thin, hollow needle, can be used with ultrasound guidance to enter the uterus and amniotic sac through the abdomen. A predetermined amount of amniotic fluid, e.g., one ounce, can be aspirated into a syringe.

[0110] In another case, the mammalian trophoblast stem cells (e.g., hTS cells) of the present disclosure can be obtained from a blastomere biopsy during preimplantation genetic diagnosis (PGD), e.g., in combination with reproductive therapies such as in vitro fertilization (IVF). In one case, the cells of the present disclosure can be generated by a method for blastocyst biopsy, where the remaining portion of the blastocyst is implanted and results in pregnancy and subsequent live birth, e.g., the zona pellucida is removed from the blastocyst and then the blastocyst is biopsied.

[0111] In another case, the mammalian trophoblast stem cells (e.g., hTS cells) of the present disclosure can be obtained from prenatal chorionic villus sampling (CVS). In one instance, CVS can be a prenatal test that involves removing a tissue sample from the placenta to test for chromosomal abnormalities and certain other genetic problems. In one case, CVS can be performed between the 10th and 12th weeks of pregnancy. In one instance, the CVS procedure is transcervical, e.g., a catheter is inserted through the cervix into the placenta to obtain a tissue sample. In one case, the CVS procedure is transabdominal, e.g., a needle is inserted through the abdomen and uterus into the placenta to obtain a tissue sample.

[0112] In one case, the mammalian trophoblast stem cells (e.g., hTS cells) in the present disclosure can be obtained from a placental biopsy after full-term pregnancy. In one case, the mammalian trophoblast stem cells (e.g., hTS cells) of the present disclosure can be isolated from the placenta after vaginal delivery or cesarean section.

[0113] In some embodiments, the mammalian trophoblast stem cells (e.g., hTS cells) of the present disclosure can be obtained from chorionic villus sampling during the first trimester of pregnancy (e.g., 8 +3 to 12 +0Separation of full-term placenta delivered by vaginal delivery at term (e.g., 38-40 weeks of gestation) or by cesarean section. The chorionic tissue can be separated from the amnion, minced, and / or enzymatically digested (e.g., with 0.05% trypsin-EDTA, e.g., for 20 minutes). Subsequently, the cells are centrifuged (e.g., at 1500 rpm, e.g., for 5 minutes), counted, and / or re-plated in a medium (e.g., Dulbecco's modified Eagle's medium + 10% fetal bovine serum) (e.g., 104 cells / cm 2 ). In one case, the isolated cells can be adherent. In one case, the cells can be used at passages 4-8.

[0114] In one case, the mammalian trophoblast stem cells (e.g., hTS cells) herein can be isolated from full-term (e.g., 38-40 weeks of gestation) placenta according to the following procedure. The umbilical cord blood is emptied from the placenta, and the placenta is then carefully dissected. The harvested tissue pieces are washed several times (e.g., in 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 resuspended in a complete medium (e.g., Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and / or 100 μg / ml streptomycin). The cell culture is maintained under suitable conditions, e.g., 37 °C and a water-saturated atmosphere and 5% CO2. The medium is changed regularly, e.g., once to twice a week. When the cells reach the desired confluence level, e.g., more than 80% confluence, they are harvested, e.g., with 0.25% trypsin / EDTA, and re-plated at a dilution of, e.g., 1:3.

[0115] In another scenario, the mammalian trophoblast stem cells of the present disclosure (e.g., hTS cells) can be isolated from a post-partum human placenta according to the following procedure. The chorion is separated from the amnion by peeling them off. The decidual tissue is scraped off (e.g., mechanically) and washed (e.g., in Dulbecco's phosphate buffered saline), and then cut into small pieces (e.g., ~2×2 cm). The chorion is cut into small pieces and subjected to enzymatic treatment (e.g., 0.5% trypsin-EDTA, e.g., for 5 minutes), followed by digestion with collagenase I (e.g., at 0.3% in a 37°C incubator for 20 to 30 minutes). Then the mobilized cells are collected and passed through a cell strainer (e.g., 100 μm). The filtered cells are collected by centrifugation (e.g., at 2,500 rpm, e.g., for 5 minutes). The cells are resuspended in a medium (e.g., α-modified minimal essential medium supplemented with 10% fetal bovine serum and / or 1% penicillin-streptomycin) and cultured in a container (e.g., a T25 flask) under suitable conditions (e.g., at 37°C and / or 5% CO2). Periodically, for example, the medium is changed every 3 days until the chorionic MSCs reach the desired confluence level, e.g., 70% confluence.

[0116] In another case, with approval from the Institutional Review Board and the Ethics Committee of KMUH Human Subjects Research, by performing laparoscopic surgery on the fallopian tubes of women, chorionic villi can be obtained from the non-viable, unruptured pre-implantation embryos in the fallopian tubes of women with ectopic pregnancies (e.g., gestational age: 5 - 7 weeks). Small villous tissues can be minced thoroughly in a suitable medium (e.g., serum-free α-MEM), identified under a microscope, then trypsinized (e.g., with 0.025% trypsin / EDTA) for a period of time (e.g., 15 minutes), and medium (e.g., α-MEM containing 10% FBS) is added to stop the reaction. Adherent cells can be obtained and cultured under suitable conditions (e.g., in conditioned α-MEM, 10% FBS, and 1% penicillin-streptomycin, at 5% CO2 in a 37°C incubator). After two passages, the hCG level may become undetectable as measured by a commercial kit (e.g., Dako, Carpinteria, CA).

[0117] Kit / Product

[0118] The present disclosure provides kits and products for use with one or more of the methods and compositions described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers, such as vials, tubes, etc., each container including one of the individual elements used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In some cases, the containers are formed from a variety of materials such as glass or plastic.

[0119] The articles provided herein include packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, vials, and any packaging material suitable for the selected formulation and intended mode of use.

[0120] For example, the container includes hTS cells, optionally in the compositions disclosed herein. Such a kit optionally includes explicit instructions or labels or a manual regarding its use in the methods described herein.

[0121] The kit generally includes a label listing the contents and / or a manual of use, as well as a package insert with instructions for use. A set of instructions is also generally included.

[0122] In some cases, the label is on the container or associated with the container. In some cases, the label is on the container when the letters, numbers, or other characters forming the label are attached, molded, or etched into the container itself; the label is associated with the container when the label is present in a receptacle or carrier that also holds the container, such as a package insert. In some cases, the label is used to indicate that the contents are for a specific therapeutic application. The label also indicates the usage of the contents, e.g., for use in the methods described herein.

[0123] Examples

[0124] The following examples are non-limiting and only represent various aspects and features of the invention.

[0125] Example 1

[0126] Transformation characteristics of human trophoblast stem (hTS) cells upon stress.

[0127] To examine how blastocyst trophoblasts trigger maternal hyperglycemia threat during embryo transport in the oviduct, the effect of high glucose (20 mM) on hTS cells was tested. It was found that glucose rapidly induced transient activation of the sweet taste receptor T1R2 / T1R3 on the cell membrane to subsequently activate G protein signaling in the cells, thereby generating the Gαq / 11 / CaMKII / CREB1 and Gβ / GSK3β / MAFA signaling pathways ( Figure 1A ). The regulatory molecular mechanisms are described in the Supplementary Information ( Figures 2A to 2J ). In the nucleus, the two G-protein pathways synergistically targeted the promoter of the insulin gene for transcription to produce insulin ( Figure 1A ). Knockdown of the transcription factors CREB1 and MAFA by specific shRNA reduced insulin expression by ChIP-qPCR analysis ( Figure 1B)。Meanwhile, activated T1R2 / T1R3 signaling induces glucose sensors and the transporter GLUT2 to facilitate glucose entry into the cell. Glucose increases ATP production to open L-type voltage-gated Ca2+ channels (VGCCs), resulting in extracellular calcium entry into the cell, which promotes insulin secretion into the culture medium detected by radioimmunoassay (RIA). This effect was confirmed by insulin secretion promoted by sulfonylureas, while the VGCC inhibitor nifedipine blocked insulin secretion measured by RIA( Figure 1C ). These results indicate that glucose can control insulin synthesis and secretion in hTS cells. This insulin expression can be attributed to CREB1 signaling rather than the conventional pancreatic and duodenal homeobox 1 (PDX1) measured by qPCR analysis( Figure 2K ).

[0128] In addition, immunofluorescence imaging studies showed that not only insulin, CREB1, CaMKII, GLUT2, and MAFA, but also β-hCG, the histocompatibility antigen HLA-G, and the pluripotency transcription factor CDX2, rather than OCT4 (octamer-binding transcription factor 4), were expressed in hTS cells( Figure 1D ). The stress protein heat shock protein HSP90 was also expressed. However, hTSCs also did not express the proliferation marker Ki-67, the protein folding activator HSP70, the tumor suppressor p53, the autoantigen GAD65, and the cell-cell fusion protein syncytin( Figure 2M ), thus supporting the concept that hTSCs are in the first position of the TE-differentiated trophoblast.

[0129] Further examination of the stress effect of glucose on the morphology of hTS cells showed that high glucose rapidly induced cells to change from adherent fibroblast-like characteristics to 3D aggregated cell clusters, which were positive for zinc chelator dithizone (DTZ) staining, a specific staining for β-cells in in vitro culture. Withdrawal of high glucose led to the rapid release of cells from the cell clusters to re-adhere to the culture dish again, thus reverting to the original fibroblast-like characteristics. The cell process was recorded by a computerized microscopic video system (Olympus, IX-81, DP30, MIU-IBC-IF-2) as shown in the supplementary online video (VideoS1). In addition, ultrastructural studies of the cell clusters revealed a large cytoplasmic-to-nuclear ratio with desmosomal junctions between two cells. Various empty vesicles, abundant mitochondria, and immature granules in vesicles were observed in the cytoplasmic compartment, similar to those in pancreatic β-cells. Therefore, it was demonstrated that hTS cells may have the ability to express insulin and the stress protein HSP90 in response to external threats, where hTS cells may be highly sensitive to glucose stimulation, which can cause reversible cell transformation of hTS cells.

[0130] Example 2

[0131] In vitro induction of precursor regulatory cytotrophoblasts (prCTBs).

[0132] In this example, the effect of bFGF on hTS cells derived from the trophectoderm was examined. In one experiment, prCTBs were induced from hTS cells in vitro.

[0133] bFGF induces epithelial-mesenchymal transition (EMT) through TGF-β1.

[0134] Through Western blot analysis ( Figure 4B ) and immunocytochemistry ( Figure 4C ), it was found that bFGF induced TGF-β1 expression in hTSCs ( Figure 4A ), upregulated the mesenchymal cell marker vimentin, but downregulated the cell-cell adhesion protein E-cadherin. These effects were neutralized by pretreatment with a TGF-β1 antibody ( Figure 4D ). Optical microscopy showed a change in cell shape from elongated spindles to a plump and shortened morphology with a more rounded nucleus ( Figure 4E ). These phenomena suggest that bFGF induces EMT to acquire the ability to migrate and invade.

[0135] bFGF promotes the imprinting of definitive endoderm (DE) through mRNA-124a.

[0136] It was found that bFGF targets the receptor FGFR1 on the cell membrane to induce the activation of the PI3K / AKT / CREB1 signaling pathway. Through in silico investigation with DIANA-mirGen 2.0, a consensus CREB binding sequence (TGACGTCA) at the promoter of bFGF-induced CREB1-targeted microRNA-124a (miR-124a) was confirmed by qPCR analysis, and knockdown of CREB1 decreased miR-124a expression. A positive correlation between CREB1 and miR-124a was observed.

[0137] To gain insight into the downstream effectors of miR-124a, plasmids for SMAD4, GSK3β, and CDX2 were constructed using the pGL4.51 vector (Promega, Madison, WI) for luciferase reporter assays. First, miR-124a was shown to target the SMAD4 gene, resulting in inhibitory SMAD4. Since SMAD4 can interact with SMAD2 / 3 to bind to sequences in the proximal promoter of the MIXL1 gene for transcription, inhibitory SMAD4 causes the inhibitory homeodomain protein MIXL1. This was verified by using SMAD4 shRNA and supporting imaging studies (revealing that bFGF (10 ng / ml) induced a marked expression of MIXL1 at 15 minutes compared to the control. The intensity of MIXL1 expression decreased at 4-hour induction, indicating transient expression). Second, miR-124a was shown to inhibit glycogen synthase kinase 3β (GSK3β), which was confirmed by pretreatment with miR-124a and anti-miR-124a antibodies. As a result, inhibitory GSK3β caused the accumulation of downstream β-catenin, leading to nuclear translocation to target the FOXA2 gene for transcription, which was demonstrated by using shRNA against β-catenin assay( Figure 4F ). In turn, forkhead box protein A2 (FOXA2) controls PDX1 expression, thus contributing to the pro-endocrine transcription factor neurogenin 3 (NGN3) to participate in the specification of endocrine cell differentiation. Third, miR-124a targeted the CDX2 gene to inhibit CDX2 synthesis, which was verified by pretreatment with miR-124a and anti-miR-124a antibodies. Downregulated CDX2 led to the upregulation of OCT4. In turn, OCT4 targeted the SOX17 gene for transcription to produce SOX17 (SRY-box containing protein 17), which was supported by imaging studies. In summary, it was demonstrated that bFGF could promote miR-124a in an 8-hour induction to obtain the imprint of definitive endoderm (DE) by upregulating SOX17, FOXA2, and OCT4 and downregulating MIXL1, which could contribute to further insulin expression.

[0138] bFGF induced the generation of prCTB from hTS cells.

[0139] At 8 hours of bFGF induction, a significant upregulation of pancreatic progenitor cell biomarkers was noted, including PDX1, pancreatic transcription factor 1 protein (PTF1a), SOX9, and homeobox 1 protein NKX (NKX6.1). The appearance of these molecular signals triggered the expression of NGN3, insulinogen C peptide, and insulin at 20 hours of induction. In addition to insulin, immunofluorescence imaging confirmed the presence of multiple pancreatic progenitor and endocrine cell markers in bFGF-treated hTS cells, 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)( Figure 3 ).

[0140] Next, it was found that initially bFGF-induced hTS cells expressed CDX2 but not OCT4( Figure 5C ). As differentiation progressed, OCT4 was upregulated while CDX2 was downregulated, thus regulating pluripotency at the DE stage at 8 hours of induction. When OCT4 was gradually downregulated and NANOG was upregulated to a peak at 12 hours of induction, it was similar to the stage of mesendoderm. Among them, HSP90 maintained the levels of OCT4 and NANOG in the differentiated trophoblast. When differentiation entered the progenitor cell stage, NANOG was downregulated, but SOX2 remained upregulated until the end of the day after 12 hours of induction, where insulin-expressing prCTB was formed. Notably, these molecular processes were similar to the development of β-cells in the pancreas but differed from glucose-induced insulin expression in hTS cells as previously described. The spatio-temporal switching of pluripotency transcription factors was mainly attributed to a reciprocal negative autoregulatory mechanism, and SOX2 played a major role in maintaining the stemness of prCTB. For this reason, our experiments demonstrated that bFGF could effectively induce isolated hTS cells to differentiate into a new pancreatic ectopic tissue-specific phenotype expressing insulin, which is herein referred to as precursor regulatory cytotrophoblast (prCTB). As demonstrated herein, prCTB can be maintained mainly by SOX2 during the in vitro trophoblast differentiation process.

[0141] Example 3

[0142] Differences between prCTB and hTS cells and primary prCTB

[0143] Immunohistochemistry showed that most prCTB cells were stained by insulin, GLUT2, CaMKII, Ki67 (a proliferation factor), β-hCG, and HLA-G, indicating that prCTB had proliferative characteristics( Figure 5A ). In addition to syncytin, stress proteins including GAD65, HSP70, HSP90, and p53 were also expressed in prCTB, but were different from the findings in hTS cells( Figure 2M)。These results suggest that the action of extracellular stressors can drive the conversion of hTS cells into prCTB in vitro.

[0144] In normal uterine pregnancy, primitive cytotrophoblasts (pCTB) can give rise to: 1) villous cytotrophoblasts; 2) primitive syncytiotrophoblasts (pSTB) and late syncytiotrophoblasts (STB); and 3) extravillous cytotrophoblasts (EVT). It was found that villous cytotrophoblasts express HSP90 and insulin, but not p53 and syncytin ( Figure 5B ). STB expresses insulin, p53, and syncytin, but not HSP90. EVT expresses significant amounts of insulin and p53, but not HSP90 and syncytin, suggesting an invasive rather than a proliferative phenotype. Figure 5C Such a developmental process has been described.

[0145] Example 4

[0146] Secretomes or exosomes from hTS cells and prCTB.

[0147] The levels of chemokines, cytokines, and growth factors in the culture media of hTS cells and prCTB were measured by Milliplex assays based on Luminex technology. The results showed that both hTS cells and prCTB were able to release significant amounts of various secretomes or exosomes ( Figure 7A , upper panel), including:

[0148] 1) Chemokines

[0149] This group includes RANTES (also known as CCL5), MCP-1 (also known as CCL2, monocyte chemoattractant protein-1), GROα (also known as CXCL1, CXCL2, macrophage inflammatory protein 2-α or MIP2-α), MCP-3 (also known as CCL7), IL-8, Eotaxin (also known as CCL11, CCL24, and CCL26), MDC (also known as CCL22), IP-10 (also known as IFNγ-induced CXCL10), fractalkine (also known as CX3CL1), MIP-1β (also known as CCL4), and soluble CD40 ligand (also known as sCD40L, CD154).

[0150] 2) Cytokines and growth factors

[0151] This group includes IL-6, IL-10, IL-4, IL-7, IL-15, IL-13, IL-1α, IL-1β, IL-12p40, IL-3 and IL-2. IFN-γ and IFN-α are also secreted. And the growth factors include: 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.

[0152] 3) Other proteins

[0153] In addition, hTS cells and prCTB can release soluble human leukocyte antigen G (sHLA-G), transforming growth factor β1 (TGF-β1), plasminogen activator inhibitor-1 (PAI-1) and IL-10 through secretome analysis ( Figure 7B ).

[0154] However, the commercial human platelet lysate used (i.e., PLUS, Compass) also secretes exosomes in the medium. By excluding the basal effect of PLUS, it was found that the original hTS cells and prCTB can actually secrete exosomes, including chemokines such as major GRO, MCP-1, fractalkine, IP-10, MCP-3, Eotaxin and a smaller amount of MIP-1β; and cytokines including IL-6 and IL-8 along with a smaller amount of IL-4, IL-1RA, IL-10, IL-12P40, IL-15, IL-1α and IL-17A ( Figure 7A , as shown in the figure below). And the growth factors include PDGF-AA, VEGF, bFGF and G-CSF as well as a smaller amount of Flt-3L and GM-CSF ( Figure 7A , as shown in the figure below). These data indicate that the original hTS cells and prCTB may have the ability to release multiple secretomes or exosomes to perform functions.

[0155] Figure 7C Another summary showing the production of cytokines IL-6 and IL-8; chemokines MCP-1 and CXCL2; and angiogenic factors PDGF-AA and VEGF in hTSC and prCTB. prCTB expresses the angiogenic molecules CD105 (endoglin, a receptor through which TGF-β acts in angiogenesis) and CD146 (vascular endothelial cadherin) ( Figure 7D ). These results indicate that prCTB has the ability to function in the decidual tissue at the fetal-maternal interface.

[0156] Example 5

[0157] prCTB expression of immune cell-related biomarkers

[0158] Expression of immune cell-related biomarkers was examined in prCTB using 8 independent cell lines by FACS analysis, showing a similar pattern for NK and T cell biomarkers. The results showed that both expressed CD biomarkers, including CD4+, CD8+, CD107a+ and (CD16+CD56)+, with a similar CD biomarker profile to T cells and NK cells. Representative cytometric analysis is shown in the table of Figure 8 and Figures 9A-9M . (CD16+CD56) + cells and CD107(+) cells showed the highest expression in both hTSC and prCTB ( Figure 8 and 9L ). Their combinatory panel also occupied the largest part of the cell population in the distribution ( Figure 8 and 9M ). These results indicate that prCTB has the ability to perform immune cell-like functions at the fetal-maternal interface. prCTB was also found to express CD11b and CD49f by immunostaining ( Figure 9N ).

[0159] In addition, immunoblot analysis determined that hTS cells and prCTB express ILT-2 (leukocyte Ig-like receptor 1, also known as LILRB1), ILT-4 (also known as LILRB2), TCR (T cell receptor), and especially KIR2DL4 (killer cell Ig-like receptor), which is expressed by NK cells and CD8+ T cell subsets to inhibit the cytolytic NK cell function ( Figure 7E ). They also express PD-L1 (programmed death-ligand 1), Fas (apoptosis signal receptor, also known as APO-1), FasL (Fas ligand) that induces apoptosis of infiltrating lymphocytes, and NKp46 (major NK cell activation receptor) ( Figure 7E ), which are involved in the elimination of target cells. Interestingly, bFGF activates signal transducer and activator of transcription 3 (STAT3) and transcription factor c-JUN, thereby promoting the expression of FasL in the differentiation of prCTB ( Figure 7F ). This effect was confirmed by shRNA knockdown of FGFR1. As a result, the (CD16+CD56) molecules of prCTB produce IFN-γ to stimulate the production of PD-L1, thereby recognizing the homologous receptor PD-1 in tumor cells and downregulating the immune response against malignancies.

[0160] bFGF induces IL-6 and IL-8 in prCTB through the FGFR1 / CREB1 signaling pathway.

[0161] To investigate how prCTB generates cytokines IL-6 and IL-8, hTSC was incubated with bFGF for 1 day to mimic a short stay in the fallopian tube. Mechanistically, bFGF activates its receptor FGFR1 on the cell membrane to induce PI3K / phosphorylated (p)AKT signaling. Subsequently, pAKT interacts with and phosphorylates downstream pCREB1 (cAMP response element-binding protein 1) to activate pCREB1 signaling. These molecular processes were verified by using the FGFR1 inhibitor PD166866 and specific shRNAs for PI3K and pAKT ( Figures 7G-7J ). In the nucleus, CREB1 targets 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), as confirmed by ELISA assays ( Figure 7C ). These molecular processes occur spatiotemporally in concert with EMT. In summary, bFGF induces the transformation of hTSC into prCTB and the production of IL-6 and IL-8 in prCTB via autocrine / paracrine manners.

[0162] In prCTB, IL-6 induces the trophoblast marker β-hCG and IL-8 induces the NK cell marker CD56

[0163] In prCTB, IL-6 binds to its receptor IL-6R on the cell membrane to activate CREB1 signaling, leading to the production of β-hCG as determined by RT-qPCR ( Figure 7L , left panel). Interestingly, by Western blot analysis, IL-6 is able to bind another receptor GnRHR to consistently activate CREB1 signaling, resulting in the production of β-hCG ( Figure 7M ). Meanwhile, by RT-qPCR assay, IL-8 induces the production of CD56 (also known as NCAM), but not through the IL-8R ( Figure 7L , right panel). However, we found that IL-8 can alternatively bind another receptor CXCR2 to activate STAT3 (signal transducer and activator of transcription 3) signaling and then target genes for transcription to produce CD56 by Western blot analysis ( Figure 7N ). These molecular processes were verified by using the GnRHR inhibitor elagolix and the CREB1 inhibitor 666-15 for β-hCG expression ( Figure 7L ); while the CXCR2 inhibitor SB225002 and the STAT3 inhibitor Stattic were used for CD56 expression ( Figure 7N) These results indicate that IL-6 induces the trophoblast biomarker β-hCG; while IL-8 consistently induces the NK cell biomarker CD56 in prCTB in an autocrine / paracrine manner. For this reason, we demonstrated that bFGF induces the transformation of hTSC into prCTB, generating unique β-hCG(+)CD56(+) prCTB.

[0164] IL-8 induces the T cell marker CD4 in prCTB via CXCR2 / CREB1 signaling

[0165] Meanwhile, IL-8 binds to and activates the receptor CXCR2 on the cell membrane of prCTB in an autocrine / paracrine manner to induce CREB1 signaling, thereby allowing its nuclear translocation. In the nucleus, by Western blot analysis, CREB1 targets the CD4 gene for transcription to produce CD4 molecules ( Figure 7O ) However, we found that IL-8 is also capable of binding to the receptor CXCR2, thereby activating STAT3 signaling, leading to the nuclear translocation of STAT3. In the nucleus, STAT3 targets different sites of the CD4 gene for transcription to produce CD4 ( Figure 7P ) These molecular processes were verified by using the CXCR2 inhibitor SB225002, the CREB1 inhibitor 666-15, and the STAT3 inhibitor Stattic ( Figure 7O and 7P )

[0166] IL-8 induces Foxp3 to form CD4(+)Foxp3(+) Treg cell-like prCTB

[0167] It was found that IL-8-induced CXCR2 signaling can bind to and thereby activate STAT3 signaling for the nuclear translocation of STAT3. Subsequently, by Western blot analysis, STAT3 targets the Foxp3 gene for transcription to produce the Foxp3 protein (also known as scurfin involved in immune system responses) ( Figure 7P ) These molecular processes were verified by using the CXCR2 inhibitor SB225002 and the STAT3 inhibitor Stattic ( Figure 7P ) These results indicate that IL-8 produces CD4(+) and Foxp3(+) molecules in prCTB, mimicking CD4(+)Foxp3(+) Treg cells. Immunocytochemical co-staining of CD4(+)Foxp3(+) biomarkers was achieved in prCTB ( Figure 7Q )

[0168] Example 6

[0169] The prCTB barrier at the fetal-maternal interface.

[0170] The prCTB expression factor promotes angiogenesis in decidual tissue.

[0171] By Milliplex assay, PrCTB was able to secrete VEGF and PDGF-AA( Figure 7C ), and by ELISA assay, secrete plasminogen activator inhibitor-1 (PAI-1) and IL-10( Figure 7B ), and by FACS analysis express CD105(+) and CD146(+) markers( Figure 7D ). The expression of all these molecules indicates that prCTB has the ability to promote angiogenesis and vasculogenesis, for example, in the SA remodeling of decidual tissue at the feto-maternal interface.

[0172] MCP-1 and CXCL2 synergistically drive the motility of prCTB.

[0173] Transwell invasion and migration assays revealed that MCP-1 significantly induced the invasion and migration of prCTB in a dose-dependent manner( Figure 10A ) and in a time-dependent manner( Figure 10B ). However, CXCL2 also promoted cell migration in a dose-dependent manner in prCTB( Figure 10C ), and promoted cell migration in a time-dependent manner in both hTSC and prCTB( Figure 10D ). These results indicate that MCP-1 and CXCL2 have the ability to synergistically drive the motility of prCTB in a time- and dose-dependent manner.

[0174] Formation of a new prCTB barrier at the feto-maternal interface.

[0175] Immunohistochemical imaging showed that prCTB migrated towards EVT, which expressed syncytin, p53, β-hCG, and HLA-G, while HSP90 appeared in the inner CTB layer( Figure 10E ). At the feto-maternal interface, CD56(+) prCTB was sporadically distributed in the villous stromal tissue by immunohistochemistry, which had the tendency to move and aggregate at the invading EVT( Figure 10F , upper and middle panels). These results indicate that prCTB differentiates into EVT expressing syncytin, p53, β-hCG, and HLA-G to synergistically invade the decidual epithelial cell layer of the maternal decidua for implantation.

[0176] It was found that both CD56(+) and β-hCG(+) prCTB could anchor and invade the maternal decidua( Figure 10F , middle panel; Figure 10G, as shown in the upper figure). Through invasion and migration, CD56(+) prCTB occupies a large number of cellular components, similar to conventional decidual natural killer (dNK) cells in decidual tissue ( Figure 10F , as shown in the lower figure), while β-hCG(+) prCTB behaves in a similar manner ( Figure 10G , as shown in the middle figure). It was found that β-hCG(+) prCTB replaced endothelial cells at arterial vessels ( Figure 10G , as shown in the middle figure), and also appeared in the vascular lumen of veins ( Figure 10G , as shown in the lower figure), suggesting SA remodeling and the phenomenon of prCTB invading decidual veins. Finally, a large number of CD56(+)β-hCG(+) prCTB can accumulate on the decidual side to form a cellular barrier, which is called the "prCTB barrier" at the feto-maternal interface.

[0177] Example 7

[0178] prCTB induces apoptosis of solid tumor cells upon interaction.

[0179] Pancreatic cancer cells (PANC-1, CRL-1469)

[0180] How pancreatic cancer cells (PANC-1) interact with prCTB was studied. Co-culture of prCTB and PANC-1 revealed by light microscopy that prCTB was able to migrate PANC-1 to surround and infiltrate into the cell colonies of PANC-1, resulting in apoptosis of PANC-1 ( Figure 11A ). The apoptosis / necrosis detection kit (blue, green, red) was used according to the manufacturer's instructions (ab176749, Abcam) to further demonstrate this apoptosis phenomenon. Figure 11B Shows the interaction of two live cells, while Figure 11C Shows the apoptosis of PANC-1 upon interaction. This interaction was further demonstrated by 3D fluorescence microscopy, which showed apoptosis of PANC-1 ( Figure 11D ).

[0181] Mechanistically, it was found that prCTB expresses the protein PD-L1 (programmed cell death-ligand-1), but does not express PD-1 (programmed cell death protein 1) ( Figure 11E , left column), while PANC-1 expresses PD-L1 and PD-1 ( Figure 11E, right panel). This means that prCTB can transmit the PD-L1 / PD-1 cell death signal to the target PANC-1 cells, thereby inducing apoptosis of PANC-1 cells. However, the PD-L-1 of PANC-1 cells cannot send a death signal to prCTB because PD-1 is lacking in prCTB, which explains why apoptosis does not occur in prCTB. When we addressed the Fas / FasL cell death signaling pathway, we found that prCTB expresses Fas ligand (FasL) and Fas (as the receptor for FasL), while PANC-1 expresses Fas but not FasL( Figure 11F ), thus, prCTB may deliver the apoptotic FasL / Fas signal to the target PANC-1, thereby inducing apoptosis of PANC-1. Conversely, prCTB expresses Fas, but FasL is lacking in PANC-1, indicating that FasL / Fas cell death signaling does not occur in prCTB upon interaction.

[0182] Breast cancer cells (MCF-7, HTB22)

[0183] Co-culture of prCTB with the breast cancer cell line (MCF-7) revealed attraction and interaction, leading to apoptosis of MCF-7 observed by 3D fluorescence microscopy( Figure 11G ). The PD-L1 expressed by prCTB will send a death signal to its receptor PD-1 on MCF-7 cells, resulting in an apoptotic response( Figure 11H , upper panel). MCF-7 also expresses PD-L1, but the lack of the PD-1 receptor in prCTB prevents any apoptosis( Figure 11H , lower panel). However, when we examined the other FasL / Fas cell death axis, we found that prCTB expresses FasL while MCF-7 expresses Fas, thus allowing FasL / Fas death signaling to occur in MCF-7( Figure 11I , upper panel).

[0184] prCTB contains the anti-apoptotic proteins Bfl-1 and Mcl-1

[0185] Interestingly, prCTB expresses Fas, while MCF-7 expresses FasL( Figure 11I , lower panel). This fact implies that prCTB may undergo apoptosis, but this is not the case. To explain this, we found by RT-qPCR analysis that prCTB expresses significantly higher levels of the anti-apoptotic Bfl-1 and Mcl-1 mRNAs in prCTB( Figure 11J ). Both Bfl-1 and Mcl-1, members of the Bcl-2 family of proteins, contain anti-apoptotic capabilities to avoid death signaling in cancer cells, thereby promoting cell survival.

[0186] Other solid tumor cells

[0187] Therefore, co - cultures of prCTB with a series of solid tumor cells were performed and detected by using a combination of immunocytochemistry and 3D fluorescence microscopy. All solid tumor cells, including liver Huh7 cells, ovarian PA - 1 (CRL - 1572) cells, lung H1299 (CRL - 5803) cells, gastric MNK45 (TCP - 1008) cells, and melanoma A375 (CRL - 1619) cells, showed apoptosis during co - culture( Figure 11K ). These results indicate that prCTB has the ability to eradicate multiple solid tumor cells, depending on which cell death signaling pathway is involved.

[0188] Example 8

[0189] Similarities between dNK cells and prCTB.

[0190] dNK cells in chorionic tissue were obtained from women who had miscarriages for medical reasons and women with ectopic pregnancies at 8 - week gestation with consent. First, the presence of the CD56 biomarker in dNK cells was confirmed by immunocytochemistry (CD56 is also expressed in prCTB)( Figure 12A , upper figure). Subsequently, immunohistochemistry in chorionic villi showed scattered CD56(+) dNK cells visible in the inner chorionic CTB and chorionic stroma, while accumulating in the EVT region( Figure 12A , middle figure). β - hCG(+) dNK cells were observed in the chorionic trophoblast and accumulated in the EVT region( Figure 12A , right figure). In normal implantation, accumulated CD56(+) dNK cells and β - hCG(+) CTB were found in the EVT region and were scattered in the nearby decidual tissue( Figure 12B , left sides of the upper and lower figures respectively). Interestingly, a large number of CD56(+) dNK cells and β - hCG(+) CTB were expressed in the maternal decidual tissue( Figure 12B , left sides of the upper and lower figures respectively). Through secreted exosomes, prCTB, like dNK cells, will have the ability to migrate into the decidual tissue, communicate with decidual stromal cells, regulate with the maternal immune system, and ultimately complete implantation.

[0191] Example 9

[0192] Materials and methods for the experiments described in Examples 1 - 8.

[0193] Experimental model and subject details.

[0194] Human trophoblast stem cells (hTS) are derived from trophoblast tissues. Trophoblast tissues were obtained from women with non-viable tubal ectopic pregnancies at 7-8 weeks of gestation under informed consent. This study was approved by the Institutional Review Board of KMUH. The original hTS cells were cultured and passaged at 37 °C in a humidified atmosphere containing 5% CO2 in α-MEM supplemented with 10% (v / v) fetal bovine serum (FBS; SAFC Biosciences). Cultures were manually passaged every 2-3 days at a split ratio of 1:3-1:6. Low seeding density and new media were tested for the growth of hTS cells, including 1) MesenCult TM -ACF Plus medium, with MesenCult TM -ACF PLUS 500x supplement and L-glutamine, with or without a substrate such as Cell Attachment Substrate, and 2) α-MEM containing nucleosides, GlutaMAX TM supplement and 10% Stemulate TM human platelet lysate cell culture supplement, with or without a substrate. For flow cytometric analysis of CD molecules, FBS was replaced with CMP grade PLUS (Compass Biochemical). Characteristic biomarkers including HLA-G, β-hCG, CDX2 and undetectable CD34 and CD45 were stably expressed. Induction of hTSC cells into prCTB was performed by treating hTS cells at passages 5-10 with 10 ng / ml bFGF for 24 hours. Several media for induction were tested, including 1) MesenCult TM -ACF Plus medium, with MesenCult TM -ACF PLUS 500x supplement and L-glutamine, with or without a substrate such as Cell Attachment Substrate, and 2) α-MEM containing nucleosides, GlutaMAX TM supplement and 10% Stemulate TM human platelet lysate cell culture supplement, with or without a substrate. The seeding density was approximately 10,000 cells / cm 2 . Cultures were free of penicillin, streptomycin, mercaptoethanol and / or nicotinamide. Cultures could also be free of animal components, serum such as fetal bovine serum, antibiotics, retinoic acid, dexamethasone, recombinant human oncostatin M, BMP4 and / or HGF. The differentiation protocol was determined empirically (data not shown). Stage-specific differentiation of lineages refers to multiple cell biomarkers described previously. Cells were harvested at designated times for different analyses.

[0195] Transfection experiments.

[0196] hTS cells were transfected with siRNA or shRNA or 3'UTR reporter plasmids using TransIT-LT1 transfection reagent (Mirus Bio LLC). Transfection was carried out with 2 μg siRNA or shRNA plus 4 μl transfection reagent in 100 μl OPTI-MEM (Gibco). After incubation at room temperature for 10 minutes, the transfection mixture was gently added to the cells overnight. Then the transfected cells were further incubated with α-MEM supplemented with 10% FBS for further processing.

[0197] Plasmid construction and dual-luciferase reporter assay.

[0198] To construct the luciferase-3'UTR reporter plasmids, we amplified the 3'UTR fragments from genomic DNA extracts of hTS cells. The 3'UTR regions were PCR amplified using a forward primer with PsiI site and a reverse primer with MfeI site for the 3'UTR reporter constructs, which are listed as follows: for the Cdx2 3'UTR region: 5'-aaattataagctgtttgggttgttggtct-3' and 5'-aaacaattgcccccataatttctgactgc-3'; for the Smad4 3'UTR region 1: 5'-aaattataactcccaaagtgctgggatta-3' and 5'-aaacaattgctgcactgttcacaggagga-3'; for the Smad4 3'UTR region 2: 5'-aaattataacagttgtcccagtgctgcta-3' and 5'-aaacaattgatgacttgcccaaaggtcac-3'; for the GSK3β 3'UTR region: 5'-aaattataacccacaactggggtaaaaga-3' and 5'-aaacaattgctgtggaaggggcaaagata-3'. After digestion with a combination of PsiI and MfeI (NEB), the 3'UTR insert fragments were subcloned into the pGL4.51 plasmid (Promega) using T4 DNA ligase (Takara).

[0199] For dual-luciferase assays, the firefly luciferase reporter (500 ng) or an empty vector without any 3'UTR was co-transfected with the pGL4.74 vector, the Renilla luciferase plasmid (500 ng, Promega), and a non-specific control miRNA (30 pmol) or the miR-124a precursor (30 pmol; System Bioscience) into hTS cells (1.5 × 104 cells per well). Twenty-four hours after changing the transfection medium, luciferase activity was analyzed by the Dual-Luciferase Reporter Assay System (Promega) and the Centro LB960 Microplate Luminometer (Berthold Technologies). For evaluation, first, the Renilla luciferase values were normalized to the firefly luciferase activity, and the calculated activity of each 3'UTR reporter was further normalized relative to the activity of the control vector. Data are represented as mean ± SD, n = 8, with p < 0.05 considered statistically significant. Whole cell extracts prepared in cell lysis buffer were immunoblotted with CDX2, SMAD4, GSK3β, and β-actin antibodies.

[0200] Secretomics analysis.

[0201] Harvest the hTS cell culture medium (10 ml) at 80 - 90% confluence, and then centrifuge (3,000 rpm, 30 minutes, 4°C). Further concentrate the supernatant to 1 ml using a 3 kDa Vivaspin concentrator (Sigma). Further detect TGF-β1, HLA-G, and PAI-1 in the concentrated supernatant by immunoblot analysis. Measure the IL-10 level using an OptEIA ELISA assay kit according to the supplier's instructions (BD Pharmingen, San Diego). The detectable IL-10 concentration range is 2 - 2000 pg / ml. Measure aliquots of 100 μl samples in triplicate. Measure the total protein in the supernatant using a Pierce BCA protein assay kit (Thermo Scientific). To measure C-peptide and insulin levels in the glucose stimulation test, add high glucose (20 mM) to α-MEM medium (5 ml) when the cells are more than 80% confluent after bFGF treatment. Collect the medium at different times (5, 10, 20, 30, 60, and 120 minutes), freeze-dry it using a freeze dryer (VirTis; Warminster), and rehydrate it with sterile water (400 μl) for radioimmunoassay (RIA). Measure C-peptide and insulin levels in 5 assays respectively by C-PEP II-RIA-CT (DIAsource ImmunoAssays S.A.) and Coat-A-Count insulin (Siemens Healthcare Diagnostics).

[0202] Exosome analysis.

[0203] Cell culture supernatants were harvested from: 1) hTS cell cultures (1×1.86 cells / 10 ml) for 24 hours 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 20 instrument (R&D system, USA) at the National Experimental Reaserch Laboratories, (Taiwan, China), and the data were analyzed using Milliplex analysis software (5.1.0.0.).

[0204] Transmission electron microscopy analysis.

[0205] After high glucose stimulation, cell clusters formed by hTS cells on the culture dish were dissected with tungsten needles. For transmission electron microscopy analysis, 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) sucrose (Merck) at room temperature for 1 hour and overnight at 4 °C. Before and after osmication in Palade's fixative containing 1% (v / v) OsO4 (Sigma) for 2 hours at 4 °C, the samples were washed with PBS, treated with uranyl acetate dihydrate (Merck), dehydrated through a graded series of ethanol solutions, and embedded using an 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-2000EXII (JEOL, Tokyo).

[0206] Western blotting.

[0207] In cell culture, hTS cells were treated with bFGF and harvested at the indicated times and placed in RIPA lysis buffer (Millipore) supplemented with protease (Thermo Scientific) and phosphatase inhibitors (Cell Signaling Technology). After electrophoresis of 30 μg of lysate on a polyacrylamide gel, electroblotting was performed onto a PVDF membrane (Millipore). The target protein was incubated with the primary antibody by blocking with 5% non-fat milk in PBS at room temperature (1 hour). All membranes were incubated with a chemiluminescent agent (Millipore) and the images were captured using a ChemiDoc XRS system (Bio-RAD). The antibodies used are listed in the Key Resources Table. The data were analyzed using AlphaEaseFC (version 4.0.0).

[0208] Immunofluorescence imaging.

[0209] For immunocytochemistry: Briefly, glass slides with cultured cells were fixed in 95% (v / v) ethanol for 30 minutes at room temperature, washed three times in PBS, and incubated for 60 minutes in blocking buffer PBS containing 0.05% (v / v) Tween 20 (PBST; Sigma) and 5% (v / v) normal donkey serum (Millipore). Primary and secondary antibodies were diluted in the blocking buffer as indicated. The primary antibody was incubated overnight at 4 °C or for 2 hours at room temperature. After incubation with the specific primary antibody in the blocking buffer, a secondary antibody conjugated with appropriate fluorescein isothiocyanate (FITC, Invitrogen) or Alexa Fluor 488, 594, 647 (Invitrogen) or Dylight 488, 594 (BioLegend) was added for 1 hour at room temperature. The slides were mounted with 50% glycerol by nuclear counterstaining with DAPI. Images were captured by confocal laser scanning microscopy (LSM700; Zeiss Z1 or Olympus FluoView 1000 confocal laser scanning microscope) or Countess II FL (Invitrogen), or 3D detection-fluorescence microscopy (Nanolive, Swiss), or TissueFAXS system (TissueQnostics GmbH).

[0210] For immunohistochemistry: All procedures were performed on a Leica Bond-III automated system (Leica microsystems, Bannockburn). Staining was performed using diaminobenzidine and hematoxylin from the Bond Polymer Refine Detection Kit (catalog number DS9800, Leica). When the run was complete and the slide tray was removed, the cover was carefully lifted up by the neck to remove it. Before coverslipping, the slides were dehydrated by two changes each of 95% and 100% ethanol and two changes of xylene.

[0211] TaqMan miRNA and quantitative real-time PCR analysis.

[0212] RNA was isolated from hTS cells in triplicate or quintuplicate samples using TRIzol reagent (Invitrogen) and on-column digestion with DNAase I (Qiagen) according to the manufacturer's protocol. Total RNA (500 ng) was used for reverse transcription with the iScript cDNA Synthesis Kit (Bio-Rad). PCR was performed in duplicate using 1 / 40 of the cDNA per reaction and 400 nM forward and reverse primers. For miRNA stem-loop qPCR, we used the single-tube TaqMan miRNA Assay according to the manufacturer's instructions (Applied Biosystems). All RT reactions, including no-template controls and RT-controls, were performed in a GeneAmp PCR 9700 thermal cycler (Applied Biosystems). Comparative real-time PCR was performed in triplicate or quintuplicate, including no-template controls, using primers specific for miR-124 or RNU6B (Applied Biosystems). U6 snRNA (RNU6B; Applied Biosystems) was used as an endogenous control. Relative expression was calculated using SDS 2.2.2 software (Applied Biosystems) and used for comparative ΔCt analysis.

[0213] Immunoprecipitation (IP) assay.

[0214] Cell lysates of bFGF-treated hTS cells were collected. Total protein (100 μg) was treated overnight with the listed specific primary antibodies by incubation with Protein G-agarose (Millipore) for 30 minutes. After treatment with Protein G-agarose beads for 2 hours, the samples were washed three times with RIPA lysis buffer (Millipore), followed by the addition of protein loading dye and boiling for 5 minutes. Samples were resolved by 8% SDS-PAGE and subjected to immunoblot analysis.

[0215] Chromatin immunoprecipitation (ChIP) assay.

[0216] The ChIP assay was performed using the ChIP-IT Express Chromatin Immunoprecipitation kit (ActiveMotif) according to the manufacturer's instructions. Briefly, immunoprecipitated DNA fragments were extracted from hTS cells (1×106). Anti-CREB1 or anti-OCT4 or anti-β-catenin antibodies were used. The conserved binding sites at the promoter regions of miR-124a or SOX17 or FOXA2 were amplified using specific primers, which are listed as follows: For the promoter of miR124-2: forward, 5'-tctgcggctctttggtttca-3', and reverse, 5'-tctgccttcagcacaagagg-3'; and forward, 5'-gcggctctttggtttcaagg-3'; 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'.

[0217] Exosome analysis.

[0218] Cell culture supernatants were harvested from: 1) hTS cell cultures (1×1.8 6 cells / 10 ml) for 24 hours and 2) hTS cells treated with bFGF (10 ng / ml) for 24 hours (prCTB). These supernatants were analyzed by Milliplex using a Luminex LX 200 instrument (R&D system, USA) at the National Experimental Research Laboratories, (Taiwan, China), and the data were analyzed by Milliplex analysis software (5.1.0.0.).

[0219] Flow cytometry

[0220] For insulin analysis, hTS cells were collected by scraping or trypsinizing with 1X TrypLE (Thermo Fisher Scientific) and washed with PBS. Cells (5×106 cells / ml) were incubated in blocking buffer (PBST plus 5% donkey serum) on ice for 1 hour, followed by resuspension in blocking buffer with Alexa 647-conjugated anti-insulin antibody (9008s, Cell signaling) or unconjugated anti-insulin antibody (sc-7839, Santa Cruz) for 30 minutes. Cells were washed twice in blocking buffer and stained with the unconjugated antibody, followed by incubation with Alexa 647-conjugated secondary antibody in blocking buffer in the dark on ice for 30 minutes. After washing twice, cells were passed through a polystyrene round-bottom tube (BD Falcon) with a cell strainer cap prior to flow cytometry analysis (LSR-II flow cytometer; BD Biosciences). Results were analyzed with FlowJo software.

[0221] For pluripotent transcription factor analysis, hTS cells were transfected with one or more non-specific shRNAs against CDX2 or OCT4 or SOX2 or NANOG. Cells (5×106 cells / ml) were then incubated with specific primary antibodies for 30 minutes. Primary antibodies conjugated to appropriate fluorescent dyes were incubated at 4°C for 1 hour at a regulated dilution, the samples were washed and resuspended in PBS, followed by passing them through a polystyrene round-bottom tube (BD Falcon) with a cell strainer cap prior to flow cytometry analysis (FACScan, BD Biosciences, San Jose, CA). Data were analyzed with Cell-Quest software (BD Biosciences).

[0222] For CD biomarker analysis, hTS cells or prCTB (1×10 5 ~1×10 6)Suspended in 240 μl of 1X FCM buffer (Leinco, F1175). The cells (30 μl) were stained with 7-AAD (BD, 5599257), fluorescently labeled antibodies (BD multitest 6-color TBNK (BD, 337166) + BV421-labeled anti-CD107a (BioLegend, 328625) or only BV421-labeled anti-CD34 (BD, 562577) 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 TM 5.5-labeled IgG1κ (BD, 552834) + PE-Cy TM 7-labeled IgG1κ (BD, 557872) + APC-labeled IgG1κ (BD, 550854) + APC-Cy7-labeled IgG1κ (BD, 557873) + BV421-labeled IgG1κ (BD, 562438) or only BV421-labeled IgG1κ (BD, 562438). After incubation for 15 minutes at room temperature, the cells were washed with 1 ml of 1X FCM buffer and resuspended in 200 μl of 1X FCM buffer. Finally, the cell samples were analyzed by using a FACSVerse flow cytometer (BD, 651155) and FACSuite software.

[0223] Cell apoptosis analysis

[0224] Various cancer cells (2,000 cells), including PANC-1 cells (pancreas), MCF-7 cells (breast), H1299 cells (lung), MKN45 cells (stomach), HepG2 cells (liver), PA-1 cells (ovary), A375 cells (melanoma), and PC-3 cells (prostate) were seeded and cultured in a medium in a 35 mm glass-bottom culture dish (ibidi; cat#81158), and incubated at 5% CO2, 37 °C. After the cells adhered overnight, bFGF-induced hTS cells (2 × 104 cells) were added and co-cultured for 24 hours. For apoptosis assay, the co-cultured cells were stained using an apoptosis / necrosis kit (ab176749, Abcam, Cambridge, England) according to the manufacturer's instructions. Briefly, after removing the medium, the cells were washed twice with assay buffer. Staining was performed by adding Apopxin green indicator (apoptotic cells / green) and CytoCalcein 450 (healthy cells / blue), and the cells were incubated at room temperature for approximately 40 minutes. After washing twice with buffer, the cells were observed under a 3D cell explorer-fluo (Nanolive, Swiss).

[0225] The co-culture of prCTB and PANC-1 cells at a ratio of 2:1 (3 × 104 cells / well) was performed in a 12-well plate at 37 °C for 6 days and observed by light microscopy. For apoptosis assay, the co-cultured cells (24 hours) were analyzed using an apoptosis / necrosis kit (ab176749, Abcam, Cambridge, England) according to the manufacturer's instructions. After removing the medium, the cells were washed twice with assay buffer. Then, Apopxin green indicator (apoptotic cells) and CytoCalcein 450 (healthy cells) were added to each well for incubation at room temperature for 60 minutes. The cells were washed with assay buffer and analyzed by fluorescence microscopy.

[0226] Transwell assay

[0227] prCTB (1x10 6Cells / ml) and incubated at 37 °C and 5% CO2 for 10 minutes to allow the cells to settle. For this, an extracellular matrix (ECM) material is added on top of the transwell membrane, and then cells are added on top of the ECM. For example, Matrigel is melted and liquefied on ice, then 30 - 50 μl of Matrigel is added to a 24-well transwell insert and cured in a 37 °C incubator for 15 - 30 minutes to form a thin gel layer. The cell solution is added on top of the Matrigel coating to simulate invasion through the extracellular matrix. Transwell cell migration assays measure the chemotactic ability of cells towards a chemoattractant. However, transwell cell invasion assays measure both cell chemotaxis and the invasion of cells through the extracellular matrix, which is a process commonly found in cancer metastasis or embryonic development.

[0228] Using a pipette, very carefully add 600 μl of the desired chemoattractant to the bottom of the lower chamber in a 24-well plate. Add the chemoattractant without moving the transwell insert and avoid creating bubbles. Ensure that the chemoattractant liquid in the bottom well contacts the membrane in the upper well to form a chemotactic gradient. The incubation time depends on the cell type and the chemoattractant used. Note: Further testing may be required to determine the incubation period. Note: For adherent cells, the migrating cells will attach to the other side of the membrane 1,8 . Quantification of the migrating cells can be performed after steps 2.4 to 2.8 (steps 2.4 - 2.8 do not need to be performed in a sterile environment). For non-adherent cells, the migrating cells will fall into the medium in the lower chamber. The number of migrating cells can be counted by using a hemocytometer or a flow cytometer 5 .

[0229] Remove the transwell insert from the plate. Use a cotton swab applicator as many times as needed to carefully remove the medium and the remaining cells that did not migrate from the top of the membrane without damaging the membrane.

[0230] Add 600 - 1,000 μl of 70% ethanol to the wells of the 24-well plate. Place the transwell insert in 70% ethanol for 10 minutes to fix the cells. Remove the transwell insert from the 24-well plate and use a cotton swab applicator to remove the remaining ethanol from the top of the membrane. Allow the transwell membrane to dry (usually 10 - 15 minutes).

[0231] Add 600 - 1,000 μl of 0.2% crystal violet to the wells of the 24-well plate and place the membrane in it for staining. Incubate at room temperature for 5 - 10 minutes.

[0232] Gently remove the crystal violet from the top of the membrane using a pipette tip or cotton swab applicator. Very carefully, to avoid washing off the fixed cells, dip the membrane into distilled water multiple times as needed to remove the excess crystal violet. Allow the transwell membrane to dry.

[0233] Observe and count the number of cells in different fields of view under an inverted microscope to obtain the average total number of cells that migrated through the membrane towards the chemoattractant and attached to the underside of the membrane.

[0234] Statistical analysis.

[0235] The experiments in immunoblot analysis, qPCR analysis, reporter gene analysis, and insulin and IL-10 analysis were performed in triplicate or quadruplicate as indicated and repeated twice. The p-values were calculated by Student's t-test with a two-sided distribution, and p < 0.05 was considered statistically significant.

[0236] When appropriate, one or more embodiments, situations, or aspects disclosed herein can be combined with any other embodiments, situations, or aspects disclosed herein.

[0237] Although some embodiments have been shown and described herein, these embodiments are provided by way of example only. Many variations, changes, and substitutions can be made without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention.

Claims

1. An isolated human precursor regulatory cytotrophoblast (prCTB) derived from chorionic villus precursor cells, wherein the isolated human prCTB expresses β-human chorionic gonadotropin (β-hCG), human leukocyte antigen G (HLA-G), CD56, insulin, heat shock protein 90 (HSP90), CD4, CD16, CD107a, CD8, 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), p53, Ki67, glutamate decarboxylase 65 (GAD65), heat shock protein 70 (HSP70), B-cell leukemia / lymphoma 2-related protein A1 (BCL2A1), and myeloid cell leukemia sequence 1 (Mcl-1), and wherein the isolated human prCTB lacks the expression of syncytin and programmed cell death protein 1 (PD-1).

2. The isolated human prCTB according to claim 1, wherein the isolated human prCTB further expresses interleukin 15 (IL-15).

3. The isolated human prCTB according to claim 1, wherein the isolated human prCTB further expresses CD335 (NKp46).

4. The isolated human prCTB according to claim 1, wherein the isolated human prCTB further expresses 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.

5. The isolated human prCTB according to claim 1, wherein the isolated human prCTB secretes chemokines, cytokines, growth factors, or any combination thereof, or exosomes carrying any of the above.

6. The isolated human prCTB according to claim 5, wherein the prCTB secretes chemokines, and the chemokines include chemokine (C-C motif) ligand 5 (CCL5), monocyte chemoattractant protein-1 (MCP-1), monocyte chemoattractant protein-3 (MCP-3), chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-X-C motif) ligand 2 (CXCL2), 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 (CXCL10), fractalkine, and chemokine (C-C motif) ligand 4 (CCL4), or any combination thereof.

7. The isolated human prCTB according to claim 5, wherein the prCTB secretes cytokines, and the cytokines include interleukin 1α (IL-1α), interleukin 1β (IL-1β), 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.

8. The isolated human prCTB according to claim 5, wherein the prCTB secretes cytokines, and the cytokines include interferon α (IFN-α) or interferon γ (IFN-γ).

9. The isolated human prCTB according to claim 5, wherein the prCTB secretes growth factors, and the growth factors include 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 (FLT3LG), soluble CD40 ligand (sCD40L), tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), or any combination thereof.

10. The isolated human prCTB according to claim 1, wherein, as determined by immunoblotting, the isolated human prCTB has a higher level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN compared to the progenitor cells from which the isolated human prCTB differentiates in vitro.

11. The isolated human prCTB according to claim 1, wherein, as determined by immunoblotting, the isolated human prCTB has a level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN that is at least 1.1, 1.2, 1.5, 1.5, 2, 2.2, 2.5, 2.8, 3, 3.5, 4, 5, 8, or 10 times higher than that of the progenitor cells from which the isolated human prCTB differentiates in vitro.

12. The isolated human prCTB according to claim 1, wherein, as determined by immunoblotting, the isolated human prCTB expresses a level of SOX2 protein that is at least 1.1, 1.2, 1.5, 1.5, 2, 2.2, 2.5, 2.8, 3, 3.5, 4, 5, 8, or 10 times higher than that of the progenitor cells from which the isolated human prCTB differentiates in vitro.

13. The isolated human prCTB according to claim 1, wherein the isolated human prCTB is differentiated in vitro from chorionic villus-derived progenitor cells lacking the expression of glutamate decarboxylase 65 (GAD65), Ki67, heat shock protein 70 (HSP70), p53, soluble CD40 ligand (sCD40L), or any combination thereof.

14. The isolated human prCTB according to claim 1, wherein the isolated human prCTB is differentiated in vitro from chorionic villus-derived progenitor cells, and wherein both the chorionic villus-derived progenitor cells and the isolated human prCTB express heat shock protein 90 (HSP90).

15. The isolated human prCTB according to claim 1, wherein the isolated human prCTB is genetically engineered.

16. The isolated human prCTB according to claim 15, wherein the isolated human prCTB comprises an exogenous polynucleotide encoding a cell receptor, an immune checkpoint protein, a cytokine, or any combination thereof.

17. The isolated human prCTB according to claim 16, wherein the isolated human prCTB comprises an exogenous polynucleotide encoding the cell receptor, and the cell receptor comprises a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof.

18. A pharmaceutical composition comprising: a pharmaceutically acceptable excipient; and the isolated human prCTB according to claim 1.

19. Use of the isolated human prCTB according to any one of claims 1-17 in the preparation of a medicament for treating cancer in a subject in need thereof, wherein the isolated human prCTB kills cancer cells; and wherein the cancer cells include pancreatic cancer cells, breast cancer cells, liver cancer cells, lung cancer cells, ovarian cancer cells, gastric cancer cells, melanoma, or any combination thereof.

20. An isolated cell population comprising a plurality of the isolated human precursor regulatory trophoblast (prCTB) according to claim 1.

21. The cell population according to claim 20, wherein at least 10% of the population is isolated human prCTB expressing CD16 and CD56.

22. The cell population according to claim 20, wherein at least 2% of the population is isolated human prCTB expressing CD4.

23. The cell population according to claim 20, wherein at least 2% of the population is isolated human prCTB expressing CD8.

24. The cell population according to claim 20, wherein at least 5% of the population is isolated human prCTB expressing CD107.

25. The cell population according to claim 20, wherein: (i) at least 10% of the population is isolated human prCTB expressing CD16 and CD56; (ii) at least 2% of the population is isolated human prCTB expressing CD4; (iii) at least 2% of the population is isolated human prCTB expressing CD8; and (iv) at least 5% of the population is isolated human prCTB expressing CD107.

26. The cell population according to claim 20, wherein at least 2% of the population is isolated human prCTBs that express CD16, CD56, and CD107.

27. The cell population according to claim 20, wherein the plurality of isolated human prCTBs further express interleukin 15 (IL-15).

28. The cell population according to claim 20, wherein the plurality of isolated human prCTBs further express CD335 (NKp46).

29. The cell population according to claim 20, wherein the plurality of isolated human prCTBs further express 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.

30. The cell population according to claim 20, wherein the plurality of isolated human prCTBs secrete chemokines, cytokines, growth factors, or any combination thereof, or carry exosomes carrying any of the foregoing.

31. The cell population according to claim 30, wherein the prCTBs secrete the chemokines, and the chemokines include chemokine (C-C motif) ligand 5 (CCL5), monocyte chemoattractant protein-1 (MCP-1), monocyte chemoattractant protein-3 (MCP-3), chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-X-C motif) ligand 2 (CXCL2), 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 (CXCL10), fractalkine, and chemokine (C-C motif) ligand 4 (CCL4), or any combination thereof.

32. The cell population according to claim 30, wherein the prCTBs secrete the cytokines, and the cytokines include interleukin 1α (IL-1α), interleukin 1β (IL-1β), 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.

33. The cell population according to claim 30, wherein the prCTBs secrete the cytokines, and the cytokines include interferon α (IFN-α) or interferon γ (IFN-γ).

34. The cell population according to claim 30, wherein the prCTB secretes the growth factor, and 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 (FLT3LG), soluble CD40 ligand (sCD40L), tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), or any combination thereof.

35. The cell population according to claim 20, wherein, as determined by immunoblotting, the plurality of isolated human prCTBs have a higher level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN compared to the progenitor cells from which the plurality of isolated human prCTBs are differentiated in vitro.

36. The cell population according to claim 20, wherein, as determined by immunoblotting, the plurality of isolated human prCTBs have a level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN that is at least 1.1, 1.2, 1.5, 1.5, 2, 2.2, 2.5, 2.8, 3, 3.5, 4, 5, 8, or 10 times higher than that of the progenitor cells from which the plurality of isolated human prCTBs are differentiated in vitro.

37. The cell population according to claim 20, wherein, as determined by immunoblotting, the plurality of isolated human prCTBs express a level of SOX2 protein that is at least 1.1, 1.2, 1.5, 1.5, 2, 2.2, 2.5, 2.8, 3, 3.5, 4, 5, 8, or 10 times higher than that of the progenitor cells from which the plurality of isolated human prCTBs are differentiated in vitro.

38. The cell population according to claim 20, wherein the plurality of isolated human prCTBs are differentiated in vitro from chorionic villus-derived progenitor cells lacking the expression of glutamate decarboxylase 65 (GAD65), Ki67, heat shock protein 70 (HSP70), p53, soluble CD40 ligand (sCD40L), or any combination thereof.

39. The cell population according to claim 20, wherein the plurality of isolated human prCTBs are differentiated in vitro from chorionic villus-derived progenitor cells, and wherein both the chorionic villus-derived progenitor cells and the plurality of isolated human prCTBs express heat shock protein 90 (HSP90).

40. The cell population according to claim 20, wherein the plurality of isolated human prCTBs are genetically engineered.

41. The cell population according to claim 40, wherein the plurality of isolated human prCTBs comprise an exogenous polynucleotide encoding a cell receptor, an immune checkpoint protein, a cytokine, or any combination thereof.

42. The cell population according to claim 40, wherein the plurality of isolated human prCTBs comprise exogenous gene polynucleotides encoding a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof.

43. A pharmaceutical composition comprising: a pharmaceutically acceptable excipient; and the cell population according to claim 20.

44. Use of the cell population according to any one of claims 20-42 in the preparation of a medicament for treating cancer, wherein the cell population kills antigen-carrying cancer cells; and wherein the cancer cells include pancreatic cancer cells, breast cancer cells, liver cancer cells, lung cancer cells, ovarian cancer cells, gastric cancer cells, or any combination thereof.

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