WO2020227314A1 - Precursory regulatory cytotrophoblast cells and uses thereof - Google Patents
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Definitions
- inventive embodiments provided in this Brief Summary of the Invention are meant to be illustrative only and to provide an overview of selective embodiments disclosed herein.
- the Brief Summary of the Invention, being illustrative and selective, does not limit the scope of any claim, does not provide the entire scope of inventive embodiments disclosed or
- prCTB cytotrophoblast
- the prCTB expresses beta-hormone human chorionic gonadotropin (b- hCG), human leukocyte antigen G (HLA-G), CD56, insulin, heat shock protein 90 (HSP90), CD4, CD 16, CD56, CD 107a, 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), CDl lb, CD49f, CD3, CD19, CD34, or any combination thereof; and (ii) the prCTB expresses beta-hormone human chorionic gonadotropin (b- h
- prCTBs precursory regulatory cytotrophoblast
- the population of cells express beta-hormone human chorionic gonadotropin (b-hCG), human leukocyte antigen G (HLA-G), CD56, insulin, heat shock protein 90 (HSP90), CD4, CD 16, CD56, CD 107a, 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), CDl lb, CD49f, CD3, CD19, CD34, or any combination thereof; and (i) the population of cells express beta-hormone human chorionic gonadotropin (b-hCG), human
- At least about 10% of the population are prCTBs expressing CD 16 and CD56. In some cases, at least about 2% of the population are prCTBs expressing CD4. In some cases, at least about 2% of the population are prCTBs expressing CD8. In some cases, at least about 5% of the population are prCTBs expressing CD 107.
- an isolated population of cells comprising precursory regulatory cytotrophoblast (prCTBs), wherein: (i) at least about 10% of the population are prCTBs expressing CD16 and CD56; (ii) at least about 2% of the population are prCTBs expressing CD4; (iii) at least about 2% of the population are prCTBs expressing CD8; or (iv) at least about 5% of the population are prCTBs expressing CD 107, or any combination thereof.
- precursory regulatory cytotrophoblast prCTBs
- the population of cells comprise at least about 2% of the population are prCTBs expressing CD 16, CD56, and CD 107.
- the prCTB or a plurality of the prCTBs express interleukin 15 (IL-15).
- the prCTB or a plurality of the prCTBs express leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), leukocyte immunoglobulin-like receptor subfamily B member 2
- LILRB2 T cell receptor
- TCR T cell receptor
- KIR2DL4 killer cell immunoglobulin-like receptor 2DL4
- PD-L1 programmed death-ligand 1
- Fas Fas Ligand
- NKp46 B cell leukemia/lymphoma 2 related protein A1
- BCL2A1 or Bfl-1 myeloid cell leukemia sequence 1 (Mcl-1), or any combination thereof.
- the prCTB or a plurality of the prCTBs further express beta-hormone human chorionic gonadotropin (b- hCG), soluble human leukocyte antigen G (sHLA-G), transformation growth factor b ⁇ (TGF- b ⁇ ), Plasminogen activator inhibitor- 1 (PAI-1), interleukin 10 (IL-10), CD105, CD146, or any combination thereof.
- the prCTB or a plurality of the prCTBs lack expression of syncytin, programmed cell death protein 1 (PD-1), or a combination thereof.
- the prCTB or a plurality of the prCTBs secrete a chemokine, a cytokine, a growth factor, or any combination thereof or an exosome carrying a chemokine, a cytokine, a growth factor, or any combination thereof.
- the cytokine comprises chemokine (C-C motif) ligand 5 (CCL5), monocyte chemoattractant protein- 1 (MCP-1), monocyte chemoattractant protein- 1 (MCP-3), chemokine (C-X-C motif) ligand 1 (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.
- CCL5 monocyte chemoattractant protein- 1
- MCP-3 monocyte chem
- the cytokine comprises interleukin la (IL-la), interleukin 1b (IL-Ib), 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.
- the cytokine comprises interferon a (IFN-a) or interferon g (IFN-g).
- 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), a fibroblast growth factor (FGF) family protein, FMS-like tyrosine kinase 3 ligand (Flt3L), soluble CD40 ligand (sCD40L), tumor necrosis factor a (TNFa), interleukin 1b (IL-Ib), or any combination thereof.
- PDGF- AA platelet-derived growth factor homodimer AA
- PDGF-BB PDGF homodimer BB
- PDGF-AB PDGF heterodimer
- VEGF vascular endothelial growth factor
- GM-CSF granulocyte-m
- the prCTB or a plurality of the prCTB s have a higher level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN than a progenitor cell from which the isolated prCTB is differentiated in vitro , as measured by immunoblotting.
- the prCTB has a level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN 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 a progenitor cell from which the isolated prCTB is differentiated in vitro , as measured by immunoblotting.
- the prCTB expresses a higher level of SOX2 protein 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 a progenitor cell from which the isolated prCTB is differentiated in vitro , as measured by immunoblotting.
- the isolated prCTB is differentiated in vitro from a chorionic villi-derived progenitor cell that lacks expression of glutamate decarboxylase (GAD65), Ki67, heat shock protein 70 (HSP70), p53, soluble CD40-ligand (sCD40L), or any combination thereof.
- the isolated prCTB is differentiated in vitro from a chorionic villi- derived progenitor cell, and wherein both the chorionic villi-derived progenitor cell and the isolated prCTB express heat shock protein 90 (HSP90).
- HSP90 heat shock protein 90
- the isolated prCTB is a human cell.
- the isolated prCTB is originated from a rodent, rabbit, cow, sheep, pig, dog, cat, monkey, or ape.
- the isolated prCTBs are genetically engineered.
- the isolated prCTBs comprise an exogenous polynucleotide encoding a cellular receptor, an immunological checkpoint protein, a cytokine, or any combination thereof. In some cases, the isolated prCTBs comprise an exogenous polynucleotide encoding a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof.
- TCR T cell receptor
- BCR B cell receptor
- CAR chimeric antigen receptor
- composition comprising: a pharmaceutically acceptable excipient or carrier; and the prCTB or the population of cells as described herein.
- Disclosed herein is a method for treating a disease or condition, comprising administering to a subject in need thereof the prCTB or the population of cells as described herein.
- the method kills an antigen-bearing target cell.
- the antigen-bearing cell is not an antigen-presenting cell, for example not being a dendritic cell, macrophage, or B cell.
- the antigen-bearing target cell is a cancer cell.
- the cancer cell comprises bladder cancer cell, bone cancer cell, brain cancer cell, breast cancer cell, carcinoma of cervix, colorectal cancer cell, esophageal cancer cell, gastrointestinal cancer cell, hematopoietic malignancy, head and neck squamous cell carcinoma, leukemia, liver cancer cell, lung cancer cell, lymphoma, myeloma, nasal cancer cell, nasopharyngeal cancer cell, oral cancer cell, oropharyngeal cancer cell, ovarian cancer cell, prostate cancer cell, sarcoma, stomach cancer cell, melanoma, thyroid cancer cell, or any combination thereof.
- the antigen-bearing target cell is a pathogen.
- the pathogen comprises virus, bacterium, protozoa, prion, fungus, or any combination thereof.
- the method kills at least about 5%, at least about 10%, at least about 20%, at least about 50%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100% of a population of antigen-bearing target cells.
- the method downregulates an inflammatory pathway.
- the method treats a disease or condition that comprises transplant rejection, infection, endotoxic shock associated with infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic onset juvenile idiopathic arthritis (JIA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), asthma, pelvic inflammatory disease, Alzheimer's Disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, ankylosing spondylitis, dermatomyositis, uveitis, Peyronie's Disease, coeliac 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 system, pancreatitis, trauma from surgery, graft-versus-host disease, heart disease, bone
- the method treats an autoimmune disease.
- 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 thrombocytopenia 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.
- the method treats Type 1 diabetes.
- the method treats Type 1 diabetes.
- compositions comprising secretomes including an exosome, wherein the secretome comprise or exosome carries a chemokine, a cytokine for example an interleukin, a growth factor, or any combination thereof, and a pharmaceutically or cosmetically acceptable excipient, and wherein the composition is free from a cell.
- the secretome comprise or exosome carries: (i) a chemokine that comprises CXCL2, MCP-1, Fractalkine, IP- 10, MCP-3, Eotaxin, MIR-Ib, or any combination thereof; (ii) an interleukin that comprises IL-6, IL-8, IL-4, IL-1RA, IL-10, IL-12P40, IL-15, IL- la, IL-17A, or any combination thereof; and (iii) a growth factor that comprises PDGF-AA, VEGF, bFGF, G-CSF, Flt-3L, GM-CSF, or any combination thereof.
- a chemokine that comprises CXCL2, MCP-1, Fractalkine, IP- 10, MCP-3, Eotaxin, MIR-Ib, or any combination thereof
- an interleukin that comprises IL-6, IL-8, IL-4, IL-1RA, IL-10, IL-12P40, IL-15, IL- la,
- the composition comprises MCP-1 and one, two, three, or all of CXCL2, IL-6, IL-8, and VEGF proteins.
- MCP-1 and CXCL2 in the composition have a weight ratio of about 1 : 1 to about 2: 1.
- MCP-1 and CXCL2 in the composition have a weight ratio of about 3: 1 to about 4: 1.
- MCP-1 and IL-6 in the composition have a weight ratio of from about 2: 1 to about 3: 1.
- MCP-1 and IL-6 in the composition have a weight ratio of from about 3: 1 to about 4: 1.
- compositions have a weight ratio of from about 4: 1 to about 6: 1. In some cases, MCP-1 and VEGF in the composition have a weight ratio of from about 4: 1 to about 6: 1. In some cases, MCP-1 and VEGF in the composition have a weight ratio of from about 7: 1 to about 9: 1. In some cases, the composition further comprises 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 comprises 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 comprises PDGF-AA and G-CSF.
- the composition further comprises PDGF-AA and FGF-2 (bFGF).
- the composition further comprises one or more proteins of IP- 10, Eotaxin, Flt-3L, GM-CSF, MIP-la, MIP-lb, IL-la, IL-1RA, IL-4, IL-7, IL-10, IL-12P40, IL-13, IL-15, IL-17A, CCL5 (RANTES), MDC, MCP-3, IL-12P70, IFNa, IFNr, PDGF-AB/BB, or EGF.
- a method for modulating a skin condition comprising administering to a subject in need thereof the composition as described herein.
- the method treats a disease or provides a cosmetic application.
- the method tightens skin.
- the method hydrates skin.
- the method rejuvenates skin.
- the composition is free from a stem cell.
- the composition is administered to the subject topically, subcutaneously, percutaneously, intramuscularly, or intratumorally.
- the composition is a dosage 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.
- the subject is a mammal.
- the subject is a primate.
- the subject is a human.
- a method of obtaining precursory regulatory cytotrophoblasts comprising: differentiating pluripotent stem cells in vitro by contacting the stem cells with a fibroblast growth factor and a culture medium comprising nucleosides, L-glutamine, a dipeptide comprising L-glutamine, platelet lysate, or a combination thereof.
- the culture medium comprises nucleosides, the dipeptide, and platelet lysate.
- the culture medium comprises from about 2mM to about 200 mM of L- glutamine.
- the contacting lasts for about 24 hours to 48 hours.
- the stem cells are chorionic villi-derived progenitor cells.
- the method comprises culturing the stem cells with the culture medium before contacting them with the fibroblast growth factor to differentiate into the prCTBs.
- the culture medium is free from an antibiotic.
- the antibiotic is penicillin, streptomycin, or any combination thereof.
- the culture medium is free from retinoic acid.
- the culture medium is free from mercaptoethanol, nicotinamide, or a combination thereof. In some cases, the culture medium is free from dexamethasone, recombinant human oncostatin M, BMP4, HGF, or any combination thereof. In some cases, the culture medium is free from an animal component. In some cases, the culture medium is free from a human derived component. In some cases, the culture medium is free from a serum. In some cases, the culture medium is free from fetal bovine serum. In some cases, the fibroblast growth factor is basic fibroblast growth factor (bFGF).
- bFGF basic fibroblast growth factor
- an isolated precursory regulatory cytotrophoblast wherein: (i) the prCTB expresses beta-hormone human chorionic gonadotropin (b- hCG), human leukocyte antigen G (HLA-G), CD56, insulin, heat shock protein 90 (HSP90), CD4, CD 16, CD 107a, 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), CDl lb, CD49f, CD3, CD19, CD34, or any combination thereof; and (ii) the prCTB expresses beta-hormone human chorionic gonadotropin (b-
- the prCTB kills a cancer cell or a pathogen. In some instances, the prCTB induces apoptosis of a cancer cell, e.g., by infiltrating a colony of cancer cells, optionally wherein the prCTB itself does not undergo apoptosis from contacting the cancer cell.
- the cancer cell is a solid tumor cell. In some instances, the cancer cell is a pancreatic cancer cell, a breast cancer cell, liver tumor cell, ovarian tumor cell, lung tumor cell, stomach tumor cell, melanoma cell, or any combination thereof. In some instances, the prCTB downregulates an inflammatory pathway.
- the prCTB expresses CD4, CD 16, CD56, CD 107a, CD8, or any combination thereof. In some instances, the prCTB lacks expression of syncytin, programmed cell death protein 1 (PD-1), or a combination thereof.
- PD-1 programmed cell death protein 1
- the prCTB further 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-hormone human chorionic gonadotropin (b-hCG), soluble human leukocyte antigen G (sHLA-G), transformation growth factor b ⁇ (TGF-bI), Plasminogen activator inhibitor-1 (PAI-1), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 15
- the prCTB is a human cell.
- the prCTB is genetically engineered.
- the genetically engineered prCTB comprises a polynucleotide encoding an exogenous protein comprising a cellular receptor, an immunological checkpoint protein, a cytokine, a T cell receptor (TCR), a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof.
- the prCTB secretes a chemokine, a cytokine, a growth factor, or any combination thereof, or an exosome carrying any of the foregoing.
- a pharmaceutical composition that comprises the prCTB disclosed herein and a pharmaceutically acceptable excipient.
- a method for treating a disease or condition comprising administering to a subject in need thereof the pharmaceutical composition described herein.
- a population of cells that comprise a plurality of prCTBs disclosed herein. In some instances, at least about 10% of the population of the prCTBs express expressing CD 16 and CD56. In some instances, at least about 2% of the population of the prCTBs express CD4; at least about 2% of the population of the prCTBs express CD8; at least about 5% of the population of the prCTBs express CD107; or any combination thereof.
- At least about 10% of the population of the prCTBs express expressing CD 16 and CD56; at least about 2% of the population of the prCTBs express CD4; at least about 2% of the population of the prCTBs express CD8; and at least about 5% of the population of the prCTBs express CD 107. In some instances, at least about 2% of the population of the prCTBs express CD 16, CD56, and CD 107.
- Figures 1A-1D show characteristics of insulin expression in human trophoblast stem (hTS) cells.
- Figure 1A is a schematic illustration of molecular mechanisms of insulin synthesis and secretion in hTS cells.
- Figures 2A-2M illustrate Ga q/ n/PIP2/IP3/IP3R/CaMKII/CREBl and
- Figures 2D-2F illustrate establishment of the
- Ga q/i i /PIP2/IP3/IP3R/CaMKII/CREBl pathway by blotting analysis.
- Active Ga q/ n induced inositol trisphosphate (IP3) to act on its receptor IP3R at the membrane of endoplasmic reticulum (ER) to elevate intracellular calcium levels which consequently activate CaMKII.
- This action was inhibited by shGa that links Goq / n and CaMKII molecules.
- CaMKII then activates downstream CREB1 via phosphorylation (p), further verified by using CaMKII inhibitor KN93.
- Figures 2G-2I depict establishment of the Ob/R 13 K/ A KT/GS K3 a/b pathway.
- FIG. 2K shows that pre-treatment with CREB1 shRNA, but not PDX1 shRNA, significantly reduced insulin expression in response to glucose stimulation (Glu; 20 mM) in hTS cells by qPCR analysis.
- C control
- shGFP positive control.
- Student /-test; * : p ⁇ 0.05, n
- Figure 2L shows insulin expression in two hTS cell lines with two different anti-insulin antibodies by flow cytometry. Isotype: control.
- Figure 2M shows that naive hTS cells do not express stress proteins or proliferation marker Ki67, but express HSP90 by
- Figure 3 shows representative images of insulin-related immunoreactive molecules, including PDX1, HNF-Ib, NGN3, SOX9, NKX6.1, and insulin in prCTBs. Bar Scale bar: 50 pm.
- Figures 4 A to 4F are described herein.
- Figure 4A shows hTSCs express TGF-bI immunocytochemically.
- Figure 4B shows that bFGF (10 ng/ml) up-regulated TGF-bI and vimentin but down-regulated E-cadherin by Western blot assay, while Figure 4D shows that anti-TGF-bI antibody neutralized these actions.
- Data representing Mean ⁇ SD, n 3, Student t-test: statistical significance: *p ⁇ 0.05, **p ⁇ 0.001.
- Figure 4C shows that bFGF (10 ng/ml) up- regulated TGF-bI and vimentin but down-regulated E-cadherin by immunocytochemistry.
- Figure 4E shows that bFGF (10 ng/ml) induced morphological changes of hTSCs after one-day incubation, switching from long and spindle towards shortened and fat in cell shape, while from oval to more round in nucleal shape.
- Figure 4F shows confirmation of bFGF-induced FOXA2 activation by which FOXA2 was attenuated at the presence of shRNAs against b-catenin. Cells transfected with shGFP (non-specific shRNA) were used as control and b-actin as loading control.
- Figures 5 A-5C illustrate biological characteristics of precursory regulatory
- FIG. 5 A shows representative images of immunoreactive molecules associated with insulin expression and oxidative stress proteins in the chorionic villi of tubal ectopic pregnancy (7-8 weeks’ gestation). Scale bar: 50 pm.
- Figure 5B shows distribution of insulin and stress proteins in the chorionic villi of normal pregnancy (7-8 weeks’ gestation) by imaging. Scale bar: 200 pm.
- Figure 5C is a schematic illustration of differences of prCTBs and hTS cells in expression of stress proteins and also compared to cells in normal uterine pregnancy in the pathways of trophoblast differentiation.
- Figures 6A and 6B show comparison of stress protein p53- and insulin-expressing syncytial knots in chorionic villi between normal pregnancy and blighted ovum.
- the stressed syncytial knots are defined by expressing insulin and p53 at an area containing over 15 positive cells counted in a single chorionic villi at 8 weeks of normal gestation ( Figure 6A); while chorionic villi of patient with blighted ovum at 7-8 weeks of gestation ( Figure 6B), showing that the frequency of stress syncytial knot occurs in (Figure 6B) is 1.96-fold higher than that in (Figure 6A).
- Figures 6A and 6B show change in expression of pluripotent transcription factors during cell transformation immunoreactive OCT4 but not CDX2 imaging at DE stage by 4 hr induction.
- Figures 7A to 7Q are described herein.
- Figure 7A shows Luminex analysis of exosomes secreted from hTS cells (dark column on the left) and prCTB cells (light column on the right) in culture media with PLUS (upper panel), and exosomes presented in naive hTS cells (dark column on the left) and naive prCTB s (light column on the right) after excluding the effect of components in PLUS media used (lower panel).
- Figure 7B shows immunoblotting assay results demonstrating the presence of numerous receptor molecules in hTS cells (the left) and prCTBs (the right).
- Figure 7C shows secretomic assay results of proteins released from hTS cells and prCTBs by immunoblotting analysis.
- sHLA-G soluble HLA-G.
- n 3.
- Figure 7D shows that hTSCs express angiogenetic factors CD105 and CD146 by FACS analysis.
- Figure 7E shows immunoblotting assay results demonstrating the presence of numerous receptor molecules in hTS cells (the left) and prCTBs (the right).
- Figure 7F shows that bFGF (10 ng/ml) induced upregulation of STAT3, c-JUN, and Fas ligand (FasL), but downregulation of Fas by
- FIGS. 7G-7J show that bFGF (10 ng/ml) induced activation of CREB1 signaling by Western blot assay.
- Figure 7G shows that bFGF activated its receptor FGFR1 and downstream PI3K signal, which was inhibited by FGFR inhibitor PD 166866.
- Figure 7H shows that PI3K phosphorylated (p) AKT to form PI3K/pAKT signaling, which was inhibited by PI3K shRNA.
- Figure 71 shows that pAKT activated its downstream CREB1, which was neutralized by using AKT antibodies, including AKT1 shRNA, AKT2 shRNA, and AKT3 shRNA.
- Figure 7J shows that pAKT activated its downstream CREB 1 through direct interaction by immunoprecipitation (IP) assay.
- Figure 7K shows that bFGF (10 ng/ml) induced IL-6 (left panel) and IL-8 (right panel) in a time-dependent manner.
- Data representing Mean ⁇ SD, n 3 independent cell lines, Student-t test: statistical significance: *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
- Figure 7L shows RT- qPCR analysis showing that IL-6 (10 ng/ml) upregulated IL-6R mRNA, CREBl mRNA, and b- hCG mRNA in hTSCs (left panel) and IL-8 (30 ng/ml) upregulates CREBl mRNA and CD56 mRNA but not conventional receptor IL-8R (right panel).
- Data representing mean+/-SD, n 4, Student t-test, statistical significance **p ⁇ 0.01, ***p ⁇ 0.001.
- Figures 7M-7N show that IL-6 generated P-hCG(+)CD56(+) prCTBs by Western blot assay.
- Figure 7M shows that IL-6 (10 ng/ml) induced b-hCG via GnRHR and IL-6R, inhibited by GnRHR inhibitor Elagolix sodium and CREB1 inhibitor 666-15.
- Figure 7N shows that IL-8 (30 ng/ml) induced CD56 via GnRHR and IL-6R.
- Figure 70 shows that IL-8 (30 ng/ml) generated CD4(+) prCTBs via both
- FIG. 7P shows that IL-8 (30 ng/ml) induced Foxp3 via CXCR2/STAT3 signaling pathway, inhibited by CRCR2 inhibitor SB225002 and STAT3 inhibitor Stattic (E).
- Data representing Mean ⁇ SD, n 3 independent cell lines, Student-t test: statistical significance *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
- Figure 7Q shows that bFGF induced co-expression of CD4 and Foxp3 in prCTBs immunocytochemically. Bar scale: 50 pm.
- Figure 8 shows two tables listing CD molecules in hTS cells and prCTBs as measured by flow cytometry, and markers of subtypes of T cells and NK cells detected in hTS cells and prCTBs as measured by flow cytometry.
- Figures 9Ato 9N show representative flow cytometric analysis on distribution of CD biomarkers in hTS Cells and prCTBs.
- Figures 9A-9K show FACS plots analysis in a
- Figure 9N shows expression of CD1 lb and CD49f in prCTBs by immunostaining.
- Figures 10A to 10K are described herein.
- Figures 10A-10D show results of transwell invasion and migration assay.
- MCP-1 Figures 10A and 10B
- CXCL2 Figures IOC and 10D
- Figure 10E shows immunohistochemistry in normal chorionic villi of 7-8 gestational weeks, revealing that the invading EVTs expressed immunoreactive p53(+), syncytin, b-hCG, and HLA-G molecules.
- Figures 10F-10G show that CD56(+) prCTBs (brown, upper and middle panels of Figure 10F) and P-hCG(+) prCTBs (brown, upper pane of Figure 10G) move towards EVTs for implantation (red arrow).
- CD56(+) lower panel of F
- b- hCG(+) prCTBs middle panel of Figure 10G
- Figures 10H-10K show photographs of Invasion and Migration assay.
- Figures 1 OH- 101 show that MCP-1 drove movement of hTSCs (4 x 103 cells/ml) in a dose-dependent manner ( Figure 10H) and in a time- dependent manner ( Figure 101) in both hTSCs and prCTBs (each 4 x 103 cells/ml).
- Figures 11 A to 1 IK are described herein.
- Figures 11 A-l ID show co-culture of prCTBs (arrowhead) and PANC-1 (arrow) at ratio of 2: 1 (3 c 104 cells/well) in time course showing apoptotic changes of PANC-1 encompassed by prCTBs by light microscope ( Figure 11 A).
- Immunocytochemistry showing interaction of two live cells: prCTBs (blue, CytoCalcein 450 stain) and PANC-1 (red, PKH26 stain) in co-culture (Figure 11B).
- Figure 1 IF shows that FasL appeared in prCTBs (upper, left column) but not PANC-1 (lower, right column), while Fas was in both prCTBs (lower, left column) and PANC-1 (upper, right column). Bar scale as indicated.
- Figures 1 lG-1 IK show that prCTBs induced apoptosis of solid tumor cells upon interaction.
- Figure 11G shows by 3D cell explorer-fluo microscopy that prCTBs (blue) induced apoptosis of breast MCF-7 cells (green) upon interaction (Figure 11G).
- Figure 11H shows that prCTBs expressed PD-L1 but no PD-1 (left column); while PANC-1 expressed both PD-1 and PD-L1 (right column).
- Figures 111 shows that prCTBs expressed both FasL and Fas (left column); while PANC-1 expressed both of them (right column).
- Figure 1 IK shows that prCTBs caused apoptosis (apopxin, green) of Huh7 cells (liver), H1299 cells (lung), MKN45 cells (stomach), PA-1 cells (ovary), and A375 cells
- Figures 12A-12B show cellular processes of CD56(+) b-hCG (+) cells in migration from EVT to maternal decidua. In tubal ectopic chorionic villi.
- Left panel CD56-expressing cells immunocytochemistry.
- Middle panel Sporadic distribution of CD56(+) cells among villous stroma, inner layer of villous CTBs (black arrow), and concentrated at the EVT areas.
- Right panel Distribution of b-hCG-expressing trophoblasts at the villous surface and EVT areas. In normal placental villi.
- Left panel Histological identification of anchoring villi, EVTs, and maternal decidual tissues by H&E stain.
- Middle panel Appearance of CD56(+) cells at the EVT areas and nearby decidual tissues.
- Right panel Prominent CD56(+) cells appear at maternal decidua. Similar distribution of b-hCG (+) trophoblasts as CD56(+) cells.
- precursory regulatory cytotrophoblast cells produced in vitro , compositions thereof, and uses thereof in treating disorders (e.g., cancers, inflammations, or autoimmune diseases) or improving conditions (e.g., skin conditions).
- the precursory regulatory cytotrophoblast cells are distinct from trophoblast stem cells.
- the precursory regulatory cytotrophoblast cells are also distinct from embryonic stem cells.
- the precursory regulatory cytotrophoblast cells are also distinct from primitive cytotrophoblasts and primitive cytotrophoblasts derived cells including villous cytotrophoblasts (villous CTBs), primitive syncytiotrophoblasts (pSTBs), syncytiotrophoblasts (STBs), and extravillous cytotrophoblasts (EVTs), see FIG. 5C.
- the precursory regulatory cytotrophoblast cells are also distinct from placental cytotrophoblasts. In some instances, precursory regulatory cytotrophoblast cells are not precursor villous cytotrophoblast cells.
- ranges and amounts can be expressed as“about” a particular value or range, e.g., ⁇ 15% of a referenced numeral value. About also includes the exact amount. Hence “about 5 pL” means“about 5 pL” and also“5 pL.” Generally, the term“about” includes an amount that would be expected to be within experimental error.
- the terms“treating,”“treatment,” and the like are used herein to mean obtaining a desired pharmacologic and/or physiologic effect.
- an individual e.g., an individual suspected to be suffering from and/or genetically pre-disposed to a liver-associated disease or disorder is treated prophylactically with a preparation of cells described herein and such prophylactic treatment completely or partially prevents a liver-associated disease or disorder or sign or symptom thereof.
- an individual is treated therapeutically (e.g., when an individual is suffering from a liver-associated disease or disorder), such therapeutic treatment causes a partial or complete cure for the disease or disorder and/or reverses an adverse effect attributable to the disease or disorder and/or stabilizes the disease or disorder and/or delays progression of the disease or disorder and/or causes regression of the disease or disorder.
- Administration e.g., transplantation
- “Transplanting” a composition into a mammal refers to introducing the composition into the body of the mammal by any method established in the art.
- the composition being introduced is the“transplant”, and the mammal is the“recipient”.
- the transplant and the recipient can be syngeneic, allogeneic or xenogeneic. Further, the transplantation can be an autologous transplantation.
- isolated when used in relation to a cell or a population of cells, refers to the state of the cell or population of cells being separate from a host organism, from which the cell or the population of cells are derived.
- an isolated cell is in contact with other cells that are isolated from the same host organism.
- an isolated cell is separate from any other cells.
- an isolated prCTB is derived in vitro from a progenitor cell.
- an isolated prCTB is obtained from a host organism and separated from the host organism.
- An“effective amount” is an amount of a therapeutic agent sufficient to achieve the intended purpose.
- An effective amount of a composition to treat or ameliorate a disorder is an amount of the composition sufficient to reduce or remove the symptoms of the disorder.
- cytotrophoblast wherein the isolated prCTB expresses one or more proteins of:
- HSP90 interleukin 15
- IL-15 interleukin 15
- LILRB1 leukocyte immunoglobulin-like receptor subfamily B member 1
- LILRB2 leukocyte immunoglobulin like receptor subfamily B member 2
- TCR T cell receptor
- the prCTB secretes a chemokine, a cytokine, a growth factor, or any combination thereof, or secretes an exosome carrying a chemokine, a cytokine, a growth factor, or any combination thereof; and/or wherein the prCTB expresses p53, Ki67, glutamate decarboxylase (GAD65), heat shock protein 70 (HSP70), soluble CD40-ligand (sCD40L), or any combination thereof .
- the isolated prCTB expresses CD4, CD 16, CD56, CD 107a, CD8, or any combination thereof.
- the prCTB induces apoptosis of a cancer cell, e.g., by infiltrating a colony of cancer cells, optionally wherein the prCTB itself does not undergo apoptosis from contacting the cancer cell.
- the cancer cell is a solid tumor cell.
- the cancer cell is a pancreatic cancer cell, a breast cancer cell, liver tumor cell, ovarian tumor cell, lung tumor cell, stomach tumor cell, melanoma cell, or any combination thereof.
- the cancer cell expresses PD-1 and PD-L1, while the prCTB expresses PD-L1 but not PD-1. In some instances, the cancer cell expresses Fas but not FasL, while the prCTB expresses Fas and FasL. In some instances, the prCTB expresses Bfl-1 and Mcl-1.
- an isolated population of cells comprising precursory regulatory cytotrophoblast (prCTBs), wherein the population of prCTBs express one or more proteins of: HSP90, insulin, CD4, CD 16, CD56, CD 107a, 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), CDl lb, CD49f, CD3, CD 19, CD34, or any combination thereof;
- prCTBs precursory regulatory
- At least about 10% of the population are prCTBs expressing CD 16 and CD56. In some cases, at least about 2% of the population are prCTBs expressing CD4. In some cases, at least about 2% of the population are prCTBs expressing CD8. In some cases, at least about 5% of the population are prCTBs expressing CD 107.
- prCTBs precursory regulatory cytotrophoblast
- prCTBs precursory regulatory cytotrophoblast
- the population of cells comprises at least about 2% of the population are prCTBs expressing CD 16, CD56, and CD 107.
- the isolated prCTB expresses interleukin 15 (IL-15). In some instances, the isolated prCTB expresses leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), Leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB1), Leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB1), Leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB1), Leukocyte immunoglobulin-like receptor subfamily B member 2
- LILRB2 T cell receptor
- TCR T cell receptor
- KIR2DL4 killer cell immunoglobulin-like receptor 2DL4
- PD-L1 programmed death-ligand 1
- Fas apoptosis signal receptor
- Fas Ligand FasL
- CD335 NKp46
- BCL2A1 or Bfl-1 B cell leukemia/lymphoma 2 related protein A1
- Mcl-1 myeloid cell leukemia sequence 1
- CD1 lb CD49f
- CD3, CD 19, CD34 or any combination thereof.
- the isolated prCTB further expresses beta-hormone human chorionic gonadotropin (b-hCG or hCG-b), soluble human leukocyte antigen G (sHLA-G), transformation growth factor b ⁇ (TGF-bI), Plasminogen activator inhibitor-1 (PAI-1), interleukin 10 (IL-10), CD105, CD146, or any combination thereof.
- the isolated prCTB lacks expression of syncytin, programmed cell death protein 1 (PD-1), or a combination thereof.
- the isolated prCTB secretes a chemokine, a cytokine, a growth factor, or any combination thereof, or an exosome carrying a chemokine, a cytokine, a growth factor, or any combination thereof.
- the cytokine comprises chemokine (C-C motif) ligand 5 (CCL5), monocyte chemoattractant protein- 1 (MCP-1), monocyte chemoattractant protein- 1 (MCP-3), chemokine (C-X-C motif) ligand 1 (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
- the cytokine comprises interleukin la (IL-la), interleukin 1b (IL-Ib), 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.
- the cytokine comprises interferon a (IFN-a) or interferon g (IFN-g).
- 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), a fibroblast growth factor (FGF) family protein, FMS-like tyrosine kinase 3 ligand (Flt3L), soluble CD40 ligand (sCD40L), tumor necrosis factor a (TNFa), interleukin 1b (IL-Ib), or any combination thereof.
- PDGF-AA platelet-derived growth factor homodimer AA
- PDGF-BB PDGF homodimer BB
- PDGF-AB PDGF heterodimer
- VEGF vascular endothelial growth factor
- GM- CSF granulocyte-macrophage
- the isolated prCTB has a higher level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN than a progenitor cell from which the isolated prCTB is differentiated in vitro , as measured by immunoblotting. In some instances, the isolated prCTB has a level of activated signal transducer and activator of transcription 3 (STAT3) or
- transcription factor c-JUN 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 a progenitor cell from which the isolated prCTB is differentiated in vitro , as measured by immunoblotting.
- the isolated prCTB expresses a higher level of SOX2 protein 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 a progenitor cell from which the isolated prCTB is differentiated in vitro , as measured by immunoblotting.
- the chorionic villi-derived progenitor cell lacks expression of glutamate decarboxylase (GAD65), Ki67, heat shock protein 70 (HSP70), p53, soluble CD40- ligand (sCD40L), or any combination thereof.
- both the chorionic villi-derived progenitor cells and the isolated prCTB express heat shock protein 90 (HSP90).
- the isolated prCTB is a human cell.
- the isolated prCTB is originated from a rodent, rabbit, cow, sheep, pig, dog, cat, monkey, or ape.
- the isolated prCTB is present in a pharmaceutical composition that further comprises a pharmaceutically acceptable excipient.
- the cells provided herein are genetically modified.
- the cell is genetically modified to express an exogenous gene, e.g. , transgene.
- transgene can refer to a gene or genetic material that is transferred into an organism.
- a transgene can be a stretch or segment of DNA containing a gene that is introduced into an organism.
- a transgene can retain its ability to produce RNA or polypeptides (e.g, proteins) in a transgenic organism.
- a transgene can be composed of different nucleic acids, for example RNA or DNA.
- a transgene may encode for an engineered T cell receptor, for example a TCR transgene.
- a transgene may comprise a TCR sequence.
- a transgene can comprise an oncogene.
- a transgene can comprise an immune oncogene.
- a transgene can comprise recombination arms.
- a transgene can comprise engineered sites.
- a transgene is an oncogene.
- a transgene is an immune oncogene.
- a transgene is a tumor suppressor gene.
- a transgene encodes a protein that directly or indirectly promotes proteolysis.
- a transgene is an oncolytic gene. In some cases, a transgene can aid a lymphocyte in targeting a tumor cell. In some cases, a transgene is a T cell enhancer gene. In some cases, a transgene is an oncolytic virus gene. In some cases, a transgene inhibits tumor cell growth. In some cases, a transgene is an anti-cancer receptor. In some cases, a transgene is an anti- angiogenic factor. In some cases, a transgene is a cytotoxic gene.
- transgenes include, but are not limited to, CD28, inducible co-stimulator (ICOS), CD27, 4-1BB (CD137), ICOS-L, CD70, 4-1BBL, Signal 3, a cytokine such as IL-2, IL-7, IL-12, IL-15, IL-21, ICAM-1 (CD54), LFA-3 (CD58), HLA class I genes, B7, CD80, CD83, CD86, CD32, CD64, 4-1BBL, CD3, CDld, CD2, membrane-bound IL-15, membrane-bound IL-17, membrane-bound IL-21, membrane-bound IL-2, truncated CD 19, VEGF, Caspase, a chemokine, or one or more genes encoding an antibody (e.g., a monoclonal antibody) to any of the above, or any combination thereof.
- a cytokine such as IL-2, IL-7, IL-12, IL-15, IL-21, ICAM-1 (CD54
- a transgene encodes a protein involved in cell or tissue repair (e.g., proteins associated with DNA repair, the immune response (e.g., interferons and interleukins), and structural proteins).
- a transgene encodes a growth factor receptor.
- a prCTB as described herein comprises a transgene coding for a TCR, a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof.
- the CAR can comprise an antigen recognition domain, a hinge region, a transmembrane domain, and an intracellular signaling domain.
- the antigen recognition domain can be exposed to the outside of the cell and may interact with a potential target molecule.
- the antigen recognition domain can comprise variable regions of a monoclonal antibody that may be linked as a single chain variable fragment.
- the single chain variable fragment can comprise a variable light chain and a variable heavy chain of an immunoglobin that may be connected with a linker peptide.
- a ligand or receptor system may be used as an alternative to the antibody-based antigen recognition domain.
- the hinge domain may be designed to combine the transmembrane domain to the antigen recognition domain.
- the hinge region can be a peptide that may be designed to increase the flexibility of the antigen recognition domain.
- the transmembrane domain can comprise a hydrophobic alpha helix that may span the cell membrane.
- a CD28 transmembrane domain can be used as the transmembrane domain in the CAR.
- the transmembrane domain can anchor the CAR to the plasma membrane.
- the intracellular T-cell signaling domain can be connected to the transmembrane domain and may be inside the cell.
- activation of a signaling domain can comprise phosphorylation of immunoreceptor tyrosine-based activation motif (IT AM).
- An intracellular signaling domain can comprise a modified IT AM.
- a modified IT AM can comprise a signaling domain, such as a CD3-zeta domain.
- a modified IT AM signaling domain can comprise: CD3-zeta, CD3-epsilon, CD3-gamma, CD3-delta, a derivative thereof, or any combination thereof.
- a intracellular signaling domain may comprise one or more co-stimulatory domains.
- an intracellular signaling domain can comprise one or more signaling domains and one or more co-stimulatory signaling domains.
- a costimulatory domain can comprise a signaling domain from CD28, CD27, CD40, CD134, CD137, inducible costimulatory (ICOS), DAPIO, a derivative thereof, or any combination thereof.
- a prCTB as described herein comprises a transgene coding for an oncogene receptor.
- compositions for intravenous administration are solutions in sterile tonic aqueous buffer. Where necessary, the composition also includes a local anesthetic to ameliorate any pain at the site of the injection. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients are mixed prior to administration.
- compositions comprising a cell disclosed herein.
- the compositions further comprise a pharmaceutically acceptable carrier or excipient.
- a carrier includes, but is not limited to, saline, buffered saline, dextrose, water, and combinations thereof.
- a colloidal dispersion system is used. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
- composition comprising a secretome of a trophoblast stem cell or a prCTB as described herein.
- the secretome comprises exosomes secreted by the trophoblast stem cell or the prCTB and other soluble molecules (e.g ., proteins, nucleic acids, and lipids) secreted by the trophoblast stem cell or the prCTB.
- compositions comprising a chemokine, a cytokine, a growth factor, or any combination thereof.
- the composition comprises an exosome, wherein the exosome carries a chemokine, an interleukin, a growth factor, or any combination thereof, and a pharmaceutically or cosmetically acceptable excipient.
- the composition is free from a cell.
- the composition comprises or exosome carries: (i) a chemokine that comprises CXCL2, MCP-1, Fractalkine, IP-10, MCP-3, Eotaxin, MIR-Ib, or any combination thereof; (ii) an interleukin that comprises IL-6, IL-8, IL-4, IL-1RA, IL-10, IL-12P40, IL-15, IL-la, IL-17A, or any combination thereof; and (iii) a growth factor that comprises PDGF-AA, VEGF, bFGF, G-CSF, Flt-3L, GM-CSF, or any combination thereof.
- a chemokine that comprises CXCL2, MCP-1, Fractalkine, IP-10, MCP-3, Eotaxin, MIR-Ib, or any combination thereof
- an interleukin that comprises IL-6, IL-8, IL-4, IL-1RA, IL-10, IL-12P40, IL-15, IL-la, IL
- the composition comprises MCP-1 and one, two, three, or all of CXCL2, IL-6, IL-8, and VEGF proteins.
- MCP-1 and CXCL2 in the composition have a weight ratio of about 1 : 1 to about 2.5: 1.
- MCP- 1 and CXCL2 in the composition have a weight ratio of about: 1.0: 1, 1.1 : 1, 1.2: 1, 1.3: 1, 1.4: 1, 1.5: 1, 1.6: 1, 1.7: 1, 1.8: 1, 1.9: 1, 2.0: 1, 2.1 :, 2.2: 1, 2.3: 1, 2.4: 1, or 2.5: 1.
- MCP-1 and CXCL2 in the composition have a weight ratio of about 2.0.
- MCP-1 and CXCL2 in the composition have a weight ratio of about 3:1 to about 4:1 or about 3:1 to about 5:1.
- MCP-1 and CXCL2 in the composition have a weight ratio of about 3:1, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.5, 5, or 4.0.
- MCP-1 and CXCL2 in the composition have a weight ratio of about 3.2.
- the composition comprises MCP-1 and one, two, three, or all of CXCL2, IL-6, IL-8, and VEGF proteins.
- MCP-1 and CXCL2 in the composition have a weight ratio of about 1 :7 to about 1 :4.
- MCP-1 and CXCL2 in the composition have a weight ratio of 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.
- MCP-1 and CXCL2 in the composition have a weight ratio of about 1:5.0.
- MCP-1 and CXCL2 in the composition have a weight ratio of about 1:4 to about 1:1.5.
- MCP-1 and CXCL2 in the composition have a weight ratio of about 1:4.0, 1:3.8, 1:3.6, 1:3.4, 1:3.2, 1:3.0, 1:2.8, 1:2.6, 1:2.4, 1:2.2,
- MCP-1 and CXCL2 in the composition have a weight ratio of about 1 :4 to about 1:2.5.
- MCP-1 and IL-6 in the composition have a weight ratio of from about 2:
- MCP-1 and IL-6 in the composition have a weight ratio of from 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, MCP-1 and IL-6 in the composition have a weight ratio of about 2.3. In some cases, MCP-1 and IL-6 in the composition have a weight ratio of about 2.5. In some cases, MCP-1 and IL-6 in the
- composition have a weight ratio of from about 3 : 1 to about 4: 1.
- MCP-1 and IL-6 in the composition have a weight ratio of from about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8.,
- MCP-1 and IL-6 in the composition have a weight ratio of about 3.6. In some cases, MCP-1 and IL-6 in the composition have a weight ratio of about 3.8.
- MCP-1 and IL-8 in the composition have a weight ratio of from about 4:
- MCP-1 and IL-8 in the composition have a weight ratio of from 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.
- MCP-1 and IL-8 in the composition have a weight ratio of about 4.6.
- MCP-1 and IL-8 in the composition have a weight ratio of about 4.4.
- MCP-1 and IL-8 in the composition have a weight ratio of about 4.9.
- MCP-1 and IL-8 in the composition have a weight ratio of about 4.5.
- MCP-1 and VEGF in the composition have a weight ratio of from about 5: 1 to about 7: 1.
- MCP-1 and VEGF in the composition have a weight ratio of from 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.
- MCP-1 and VEGF in the composition have a weight ratio of about 5.6.
- MCP-1 and VEGF in the composition have a weight ratio of about 6.0.
- MCP-1 and VEGF in the composition have a weight ratio of from about 7: 1 to about 9: 1. for instance, 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.
- MCP-1 and VEGF in the composition have a weight ratio of about 7.6: 1.
- MCP-1 and VEGF in the composition have a weight ratio of about 7.3 : 1.
- the composition further comprises PDGF-AA.
- MCP-1 and PDGF-AA are present in a weight ratio of from about 3 : 1 to about 5: 1, for instance, 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.
- MCP-1 and PDGF-AA are present in a weight ratio of about 3.5.
- MCP-1 and PDGF-AA are present in a weight ratio of from about 6: 1 to about 9: 1, for instance 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.
- the composition further comprises PDGF-AA.
- MCP-1 and PDGF-AA are present in a weight ratio of from about 1 :2.5 to about 1 : 1.5, for instance, 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.
- MCP-1 and PDGF-AA are present in a weight ratio of about 0.6.
- MCP-1 and PDGF-AA are present in a weight ratio of from about 1 : 1.5 to about 1.5: 1, for instance 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.
- the composition further comprises PDGF-AA.
- MCP-1 and PDGF-AA are present in a weight ratio of from about 3: 1 to about 5: 1, for instance, 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.
- MCP-1 and PDGF-AA are present in a weight ratio of from about 3.5.
- MCP-1 and PDGF-AA are present in a weight ratio of from about 6: 1 to about 9: 1, for instance 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 from about 7.8.
- 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 of IP- 10, Eotaxin, Flt-3L, GM-CSF, MTP- la, MIP-lb, IL-la, IL-1RA, IL-4, IL-7, IL-10, IL-12P40, IL-13, IL-15, IL-17A, CCL5
- RANTES RANTES
- MDC MCP-3
- IL-12P70 IFNa, IFNr, PDGF-AB/BB, or EGF.
- the composition further comprises nucleic acids, such as, mRNA, siRNA, shRNA, or DNA.
- the composition further comprises lipid molecules that are secreted from the prCTB or trophoblast stem cells.
- a composition disclosed herein can be aseptic.
- the composition can comprise resident microbes.
- the microbes can be viruses, bacteria, eukaryotic cells or any combination thereof. In some instances, the microbes may not be pathogenic.
- the composition can comprise a bacterium or bacteria at a concentration of less than about: 10 colony forming units (CFU)/gram (g), 50 CFU/g 100 CFU/g, 150 CFU/g, 200 CFU/g, 300 CFU/g, 400 CFU/g, 500 CFU/g, 600 CFU/g, 700 CFU/g, 800 CFU/g, 900 CFU/g, or 1000 CFU/g.
- CFU colony forming units
- the composition can comprise 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 700CFU/g, or 600 CFU/g to about 1000 CFU/g.
- the composition may be substantially free (e.g., at least 95% free) or free of:
- Staphylococcus aureus Streptococcus pyogenes , Pseudomonas aeruginosa , Pseudomonas species, Klebsiella pneumoniae , or any combination thereof.
- a composition disclosed herein may not contain a heavy metal such as lead, bithionol, chlorofluorocarbon propellants, nitrosamines, chloroform, halogenated salicylanilides, hexachlorophene, mercury compounds, 1,4-dioxane, methylene chloride, prohibited cattle materials, sunscreen compounds, vinyl chloride, zirconium- containing complexes, or any combination thereof.
- the prohibited cattle materials can comprise the brain, skull, eyes, trigeminal ganglia, spinal cord, vertebral column, dorsal root ganglia, tonsils, distal ileum of the small intestine or any combination thereof.
- the composition may comprise lead at levels of 10 (parts per million) ppm or less.
- a composition herein does not comprise a color additive, a fragrance, a paraben, a phthalate, an alcohol, or any combination thereof.
- the color additive, fragrance, paraben, phthalate, or alcohol is present in an insignificant level in the composition, for example less than: 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%.
- the incidental ingredient may have no technical/ structural, functional or any combination thereof effect in the composition, e.g., no an active ingredient. In some instances, the composition is free from.
- an excipient disclosed herein can comprise water, glycerol, saline, a vegetable oil (e.g., seed oil), a fruit oil, a flower extract, a mineral oil, a synthetic oil, a sugar compound, a silicate, a calcium salt, a magnesium salt, sodium chloride, potassium chloride, lactic acid, a starch, a sugar alcohol, a cellulose, an activated charcoal, a glycerin, a butter, an amino acid, a paraffin, honey, a wax, beeswax, an agar, calcium carbonate, a citric acid, tartaric acid, a steric acid, xantham gun, benzoic acid, a polyethylene glycol, a silicon, derivatives thereof, salts thereof, or any combination thereof.
- a vegetable oil e.g., seed oil
- a fruit oil e.g., a flower extract
- mineral oil e.g., a synthetic oil
- a sugar compound
- an isolated prCTB is administered to the subject intravenously, subcutaneously, percutaneously, inhalationally, orally, intramuscularly, or intratumorally.
- the subject is a mammal.
- the subject is a primate.
- the subject is a human.
- the isolated prCTB expresses interleukin 15 (IL-15).
- the isolated prCTB expresses CD4, CD 16, CD56, CD 107a, CD8, or any combination thereof.
- the isolated prCTB expresses leukocyte
- LILRB1 immunoglobulin-like receptor subfamily B member 1
- LILRB2 Leukocyte immunoglobulin like receptor subfamily B member 2
- TCR T cell receptor
- KIR2DL4 immunoglobulin-like receptor 2DL4
- PD-L1 programmed death-ligand 1
- Fas apoptosis signal receptor
- Fas Ligand Fas Ligand
- CD335 NKp46
- CDl lb CD49f
- CD335 NKp46
- the isolated prCTB further expresses beta-hormone human chorionic gonadotropin (b-hCG), soluble human leukocyte antigen G (sHLA-G), transformation growth factor b ⁇ (TGF-bI), Plasminogen activator inhibitor-1 (PAI-1), interleukin 10 (IL-10), CD105, CD146, or any combination thereof.
- the isolated prCTB lacks expression of syncytin, programmed cell death protein 1 (PD-1), or a combination thereof.
- the isolated prCTB secretes a chemokine, a cytokine, a growth factor, or any combination thereof, or an exosome carrying a chemokine, a cytokine, a growth factor, or any combination thereof.
- the cytokine comprises chemokine (C-C motif) ligand 5 (CCL5), monocyte chemoattractant protein- 1 (MCP-1), monocyte chemoattractant protein- 1 (MCP-3), chemokine (C-X-C motif) ligand 1 (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.
- CCL5 monocyte chemoattractant protein- 1
- MCP-3 monocyte chemo
- the cytokine comprises interleukin la (IL-la), interleukin 1b (IL-Ib), 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.
- the cytokine comprises interferon a (IFN-a) or interferon g (IFN-g).
- 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), a fibroblast growth factor (FGF) family protein, FMS-like tyrosine kinase 3 ligand (Flt3L), soluble CD40 ligand (sCD40L), tumor necrosis factor a (TNFa), interleukin 1b (IL-Ib), or any combination thereof.
- PDGF-AA platelet-derived growth factor homodimer AA
- PDGF-BB PDGF homodimer BB
- PDGF-AB PDGF heterodimer
- VEGF vascular endothelial growth factor
- GM-CSF granulocyte- macrophage colony- stimulating
- the isolated prCTB has a higher level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN than a progenitor cell from which the isolated prCTB is differentiated in vitro , as measured by immunoblotting.
- the isolated prCTB has a level of activated signal transducer and activator of transcription 3 (STAT3) or transcription factor c-JUN 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 a progenitor cell from which the isolated prCTB is differentiated in vitro , as measured by immunoblotting.
- the isolated prCTB expresses a higher level of SOX2 protein at least about 1.1, 1.2, 1.5, 1.5, 2, 2.2, 2.5, 2.8, 3, 3.5,
- the isolated prCTB is a human cell. In some instances, the isolated prCTB is originated from a rodent, rabbit, cow, sheep, pig, dog, cat, monkey, or ape.
- a method for killing an antigen-bearing target cell comprising administering to a subject in need thereof a precursory regulatory cytotrophoblast (prCTB), wherein the isolated prCTB expresses one or more proteins that comprises: HSP90, insulin, CD4, CD 16, CD56, CD 107a, 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), CD
- prCTB precursory regulatory cyto
- the antigen-bearing cell is not an antigen-presenting cell, for example not being a dendritic cell, macrophage, or B cell.
- the antigen-bearing target cell is a cancer cell.
- the cancer cell is a solid tumor cell.
- the cancer cell is a blood cancer cell.
- the cancer cell comprises bladder cancer cell, bone cancer cell, brain cancer cell, breast cancer cell, carcinoma of cervix, colorectal cancer cell, esophageal cancer cell, gastrointestinal cancer cell, hematopoietic malignancy, head and neck squamous cell carcinoma, leukemia, liver cancer cell, lung cancer cell, lymphoma, myeloma, nasal cancer cell, nasopharyngeal cancer cell, oral cancer cell, oropharyngeal cancer cell, ovarian cancer cell, prostate cancer cell, sarcoma, stomach cancer cell, melanoma, thyroid cancer cell, or any combination thereof.
- the antigen-bearing target cell is a pathogen.
- the pathogen comprises virus, bacterium, protozoa, prion, fungus, or any combination thereof.
- the method kills at least about 5%, at least about 10%, at least about 20%, at least about 50%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or about 100% of a population of antigen-bearing target cells.
- a method for downregulating an inflammatory pathway comprising administering to a subject in need thereof a precursory regulatory cytotrophoblast (prCTB), (i) wherein the isolated prCTB expresses one or more proteins that comprises: (a) HSP90, insulin, CD4, CD 16, CD56, CD 107a, 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
- the method treats a disease or condition that comprises transplant rejection, infection, endotoxic shock associated with infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic onset juvenile idiopathic arthritis (JIA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), asthma, pelvic inflammatory disease, Alzheimer's Disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, ankylosing spondylitis, dermatomyositis, uveitis, Peyronie's Disease, coeliac 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 system, pancreatitis, trauma from surgery, graft-versus- host disease,
- the method treats an autoimmune disease that comprises 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
- the method treats Type 1 diabetes. In some instances, the method ameliorates transplant rejection.
- a method for modulating a skin condition comprising administering to a subject in need thereof a composition (e.g., a pharmaceutical composition) that comprises a chemokine, a cytokine such as interleukin, a growth factor, or any combination thereof, or an exosome carrying a chemokine, a cytokine such as an interleukin, a growth factor, or any combination thereof.
- a composition e.g., a pharmaceutical composition
- the method improves a skin condition, so that the skin condition has one or more better characteristic after the application of the method as compared to before the application of the method.
- the chemokine tcomprises GRO, MCP-1, Fractalkine, IP- 10, MCP-3, Eotaxin, MIR-Ib, or any combination thereof.
- the composition comprises an interleukin that comprises IL-6, IL-8 IL-4, IL-1RA, IL-10, IL-12P40, IL-15, IL-la, IL-17A, or any combination thereof.
- the growth factor comprises PDGF-AA, VEGF, bFGF, G-CSF, Flt-3L, GM-CSF, or any combination thereof.
- the method provides a cosmetic application. In some instances, the method tightens skin. In some instances, the method hydrates skin. In some instances, the method rejuvenates skin. In some instances, the
- composition is a medium post-passaging a stem cell.
- the stem cell is an isolated precursory regulatory cytotrophoblast (prCTB) described herein.
- prCTB isolated precursory regulatory cytotrophoblast
- a number of the passaging is at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
- the stem cell is cultured in the medium for at least about: 1-3 days, such as 1-2 days, for example before collecting the medium.
- the stem cell is cultured in the medium for at least about 12-24 hours, for example before collecting the medium.
- a number of the passaging is 5 to 10.
- the passaging occurs about every 1, 2, or 3 days.
- the passaging occurs about every 2, 4, 6, 8, 12, 16, 20, or 24 hours.
- the medium is free from a stem cell.
- the composition is in a form of a cream, liquid, gel, lotion, mist, capsule, or mask.
- the methods can be used to treat a skin disease.
- the skin disease can be eczema, psoriasis, acne, rosacea, ichthyosis, vitiligo, hives, seborrheic dermatitis, shingles, sunburn, a burn, contact dermatitis, rash, or any combination thereof.
- the method can reduce the appearance of skin aging, photoaging, or any combination thereof.
- the method can reduce the appearance of a scar.
- the method can improve wound healing.
- the method can prevent, reduce or eliminate bruising, benign growths, age spots, cancerous growths, ulcers, infections, or any combination thereof.
- the method can prevent, reduce, or eliminate lines, wrinkles, or any combination thereof of skin.
- the lines or wrinkles can be crow’s feet, smile lines, frown lines, forehead furrows, tear troughs, bunny lines, nasolabial folds, marionette lines, mental crease, necklines, age-related wrinkles, crinkle lines, elastotic creases, expression lines, gravitational folds, dynamic wrinkles, static wrinkles, atrophic wrinkles, atrophic crinkling rhytids, or any combination thereof.
- the method can prevent, reduce or eliminate loss of volume, elasticity, or any combination thereof of skin. In some instances, the method can prevent, reduce or eliminate, sagging skin, dull skin tone, mottled discoloration, rough skin, dry skin, itchy skin, thin skin, or any combination thereof. In some cases, the method can improve or ameliorate a skin condition, skin disease or any combination thereof. In some instances, the method can moisturize, tighten, lift, or rejuvenate skin. In some instances, the method can restore or sustain a healthy, smooth, blemish-free, translucent, resilient, or any combination thereof skin. In some cases, the method can heal, treat, remedy or any combination thereof the glycosaminoglycan, the dermis, the collagen and the elastin of skin.
- the improved health of skin can be measured by a wrinkle severity rating scale, a trans- epidermal water loss measurement, a skin color measurement, a skin surface topography measurement, a viscoelastic measurement by a Cutometer®, a histological examination, or any combination thereof.
- improved skin health can be measured by a diagnostic image, such as magnetic resonance imaging (MRI).
- MRI magnetic resonance imaging
- measurements can be compared before and after administration of the composition. In some instances, measurements can be compared to a standard.
- a method of treating a condition in a subject comprising administering to a subject a pharmaceutical composition that comprises a cell herein, in an amount effective for the cells to engraft to the subject (e.g., to the subject’s liver).
- the cells are administered in a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier comprises a saline for example a phosphate buffer saline, or fetal bovine serum.
- the cells are administered in a suspension containing about lxl 0 6 to about lOOxlO 6 cells per ml, about lxl 0 6 to about 250x10 6 cells per ml, about lxl 0 6 to about 500x10 6 cells per ml, or about lOxlO 6 to about 40x10 6 cells per ml.
- 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.
- the subject is a human.
- the cells are administered in a suspension containing about lxl 0 6 to about lOOxlO 6 cells per ml, about lxl 0 6 to about 250x10 6 cells per ml, about lxl 0 6 to about 500x10 6 cells per ml, or about lOxlO 6 to about 40x10 6 cells per ml.
- the cells are administered in a volume of
- administering comprises an injection, e.g., intravenous injection.
- the injection is administered at a hepatic vein.
- the injection is administered at a hepatic artery.
- the condition is a liver-associated disease or disorder.
- the condition is a liver failure.
- the liver-associated disease or disorder comprises alagille syndrome, alpha 1 anti-trypsin deficiency, autoimmune hepatitis, benign liver tumors, biliary atresia, cirrhosis, cystic disease of the liver, fatty liver disease including alcohol-related liver disease and non-alcohol fatty liver disease (NAFLD),
- galactosemia, gallstones, Gilbert’s Syndrome hemochromatosis, liver cysts, liver cancer, liver disease in pregnancy (optionally, acute fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, preeclampsia, 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 disease, or any combination thereof.
- Modes of administration of cells disclosed herein include, but are not limited to, systemic intravenous injection and injection directly to the intended site of activity (e.g., endoscopic retrograde injection).
- the preparation can be administered by any convenient route, for example, by infusion or bolus injection, and can be administered together with other biologically active agents. In some instances, the administration is systemic localized administration.
- compositions and methods for transplanting cells disclosed herein to subjects are provided herein.
- the subject is injected by the cells (e.g., intravenously, intramuscularly, transdermally, endoscopic retrograde injection, or
- the subject is not treated with an immunosuppressive agent prior to the transplanting.
- the method further comprises treating the patient with an immunosuppressive agent, e.g., FK-506, cyclosporin, or GAD65 antibodies.
- cells described herein are delivered to a targeted site (e.g., a defect section of the liver) by a delivery system suitable for targeting cells to a particular tissue.
- a delivery system suitable for targeting cells to a particular tissue.
- the cells are encapsulated in a delivery vehicle that allows for the slow release of the cell(s) at the targeted site.
- the delivery vehicle is modified such that it is specifically targeted to a particular tissue.
- the surface of the targeted delivery system is modified in a variety of ways.
- lipid groups are incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.
- Biomarker RNA expression levels could be measured by RT-PCR, Qt-PCR, microarray, Northern blot, or other similar technologies.
- detecting expression or detecting “expression levels” is intended for determining the expression level or presence of a biomarker protein or gene in the biological sample.
- detecting expression encompasses instances where a biomarker is determined not to be expressed, not to be detectably expressed, expressed at a low level, expressed at a normal level, or overexpressed.
- the expression or presence of a biomarker described herein is determined at a nucleic acid level, using, for example, immunohistochemistry techniques or nucleic acid-based techniques such as in situ hybridization and RT-PCR.
- the expression or presence of one or more biomarkers is carried out by a means for nucleic acid amplification, a means for nucleic acid sequencing, a means utilizing a nucleic acid microarray (DNA and RNA), or a means for in situ hybridization using specifically labeled probes.
- the determining the expression or presence of a biomarker is carried out through gel electrophoresis. In some instances, the determination is carried out through transfer to a membrane and hybridization with a specific probe. In some instances, the determining the expression or presence of a biomarker is carried out by a diagnostic imaging technique. In some instances, the determining the expression or presence of a biomarker is carried out by a detectable solid substrate. In some instances, the detectable solid substrate is paramagnetic nanoparticles functionalized with antibodies.
- the expression or presence of a biomarker is at an RNA (e.g. mRNA) level.
- RNA e.g. mRNA
- techniques that detect RNA (e.g. mRNA) level include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays.
- One method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that hybridize to the mRNA encoded by the gene being detected.
- the nucleic acid probe comprises of, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to an mRNA or genomic DNA encoding a biomarker described herein. Hybridization of an mRNA with the probe indicates that the biomarker or other target protein of interest is being expressed.
- the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose.
- the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in a gene chip array.
- a skilled artisan readily adapts known mRNA detection methods for use in detecting the level of mRNA encoding the biomarkers or other proteins of interest.
- An alternative method for determining the level of an mRNA of interest in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR, ligase chain reaction, self- sustained sequence replication, transcriptional amplification system, Q-Beta Replicase, rolling circle replication or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In some instances, biomarker expression is assessed by RT-PCR, ligase chain reaction, self- sustained sequence replication, transcriptional amplification system, Q-Beta Replicase, rolling circle replication or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In some instances, biomarker expression is assessed by
- RNA of interest Expression levels of an RNA of interest are monitored using a membrane blot
- the detection of expression also comprises using nucleic acid probes in solution.
- microarrays are used to determine expression or presence of one or more biomarkers.
- Nucleic acid microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of genes. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning
- Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative gene expression levels.
- High-density oligonucleotide arrays are particularly useful for determining the gene expression profile for a large number of RNA's in a sample.
- an array is fabricated on a surface of virtually any shape or even a multiplicity of surfaces. In some instances, an array is a planar array surface. In some instances, arrays include peptides or nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate. In some instances, arrays are packaged in such a manner as to allow for diagnostics or other manipulation of an all-inclusive device.
- the expression or presence of a biomarker described herein is determined at a protein level, using, for example, antibodies that are directed against specific biomarker proteins. These antibodies are used in various methods such as Western blot, ELISA, multiplexing technologies, immunoprecipitation, or immunohistochemistry techniques. In some instances, detection of biomarkers is accomplished by ELISA. In some instances, detection of biomarkers is accomplished by electrochemiluminescence (ECL).
- ECL electrochemiluminescence
- any means for specifically identifying and quantifying a biomarker in the biological sample is contemplated.
- expression level of a biomarker protein of interest in a biological sample is detected by means of a binding protein capable of interacting specifically with that biomarker protein or a biologically active variant thereof.
- labeled antibodies, binding portions thereof, or other binding partners are used.
- label when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody.
- the label is detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, catalyzes chemical alteration of a substrate compound or composition that is detectable.
- the antibodies for detection of a biomarker protein are either monoclonal or polyclonal in origin, or are synthetically or recombinantly produced.
- the amount of complexed protein for example, the amount of biomarker protein associated with the binding protein, for example, an antibody that specifically binds to the biomarker protein, is determined using standard protein detection methodologies known to those of skill in the art. A detailed review of immunological assay design, theory and protocols are found in numerous texts in the art.
- the choice of marker used to label the antibodies will vary depending upon the application. However, the choice of the marker is readily determinable to one skilled in the art. These labeled antibodies are used in immunoassays as well as in histological applications to detect the presence of any biomarker or protein of interest.
- the labeled antibodies are either polyclonal or monoclonal. Further, the antibodies for use in detecting a protein of interest are labeled with a radioactive atom, an enzyme, a chromophoric or fluorescent moiety, or a colorimetric tag as described elsewhere herein.
- the choice of tagging label also will depend on the detection limitations desired.
- Enzyme assays typically allow detection of a colored product formed by interaction of the enzyme-tagged complex with an enzyme substrate.
- Radionuclides that serve as detectable labels include, for example, 1-131, 1-123, 1-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, and Pd-109.
- Examples of enzymes that serve as detectable labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and glucose-6-phosphate dehydrogenase.
- Chromophoric moieties include, but are not limited to, fluorescein and rhodamine.
- the antibodies are conjugated to these labels by methods known in the art.
- enzymes and chromophoric molecules are conjugated to the antibodies by means of coupling agents, such as dialdehydes, carbodiimides, dimaleimides, and the like.
- conjugation occurs through a ligand-receptor pair.
- suitable ligand-receptor pairs are biotin-avidin or biotin-streptavidin, and antibody- antigen.
- expression or presence of one or more biomarkers or other proteins of interest within a biological sample is determined by radioimmunoassays or enzyme-linked immunoassays (ELISAs), competitive binding enzyme-linked immunoassays, dot blot, Western blot, chromatography such as high performance liquid chromatography (HPLC), or other assays known in the art.
- ELISAs enzyme-linked immunoassays
- HPLC high performance liquid chromatography
- a prCTB herein is derived in vitro from a pluripotent stem cell, e.g., a chorionic villi-derived progenitor cell.
- a prCTB is differentiated from a pluripotent stem cell in a culture medium supplemented with one or more differentiation factors.
- the stem cells are chorionic villi-derived progenitor cells.
- a chorionic villi-derived progenitor cell comprises a mammalian trophoblast stem cell, e.g, a human trophoblast stem cell.
- a method of obtaining precursory regulatory cytotrophoblasts comprising: differentiating pluripotent stem cells in vitro by contacting the stem cells with a fibroblast growth factor in a culture medium.
- the culture medium comprises nucleosides, L-glutamine, a dipeptide comprising L-glutamine, platelet lysate, or a combination thereof.
- the culture medium comprises nucleosides, the dipeptide, and platelet lysate.
- the culture medium comprises from about 2mM to about 200 mM of L-glutamine.
- a pluripotent stem cell e.g, a human trophoblast stem cells is contacted with a fibroblast growth factor for about 24 hours to 48 hours, thereby generating a prCTB.
- the contacting is at least about 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 horns, 36 hours, 40 hours, or 44 hours.
- the contacting is at most about 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 horns, 36 hours, 40 hours, 44 hours, or 48 hours.
- a pluripotent stem cell e.g ., a human trophoblast stem cells is contacted with a fibroblast growth factor when the stem cell is at passage 5 to 10.
- pluripotent stem cell is contacted with a fibroblast growth factor when the stem cell is at passage 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
- the method comprises culturing the stem cells with the culture medium before contacting them with the fibroblast growth factor to differentiate into the prCTBs.
- the culture medium for obtaining and/or maintaining prCTBs is free from an antibiotic, for instance, penicillin, streptomycin, or any combination thereof. In some cases, the culture medium for obtaining and/or maintaining prCTBs is free from retinoic acid. In some cases, the culture medium for obtaining and/or maintaining prCTBs is free from
- the culture medium for obtaining and/or maintaining prCTB is free from dexamethasone, recombinant human oncostatin M, BMP4, HGF, or any combination thereof.
- the culture medium for obtaining and/or maintaining prCTB is xeno-free, e.g. , free from an animal component.
- the culture medium for obtaining and/or maintaining prCTB is free from a human-derived component and an animal-derived component, e.g., being a chemically defined medium.
- the culture medium for obtaining and/or maintaining prCTB is free from a serum.
- the culture medium for obtaining and/or maintaining prCTB is free from fetal bovine serum.
- the fibroblast growth factor is basic fibroblast growth factor (bFGF).
- the stem cell is contacted with about 1 ng/ml to about 100 ng/ml bFGF. In some cases, the stem cell is contacted with about 2 ng/ml to about 50 ng/ml, about 4 ng/ml to about 30 ng/ml, about 6 ng/ml to about 15 ng/ml, or about 8 ng/ml to about 12 ng/ml bFGF.
- the stem cell is 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 cell is contacted with about 10 ng/ml bFGF.
- a mammalian trophoblast stem cell herein e.g., a human trophoblast stem hTS cell
- a mammalian trophoblast stem cell herein can be isolated from umbilical cord, amniotic fluid, amniotic membrane, the Wharton's jelly, the chorionic villi, placenta, or ectopic pregnancy, in a manner that is not disturbing nor destructive to an embryo.
- a mammalian trophoblast stem cell described herein is cultured in a culture medium free from an antibiotic, for instance, penicillin, streptomycin, or any combination thereof.
- the culture medium for obtaining the mammalian trophoblast stem cell is free from retinoic acid. In some cases, the culture medium obtaining and/or passaging the mammalian trophoblast stem cell is free from mercaptoethanol, nicotinamide, or a combination thereof. In some cases, the culture medium obtaining and/or passaging the mammalian trophoblast stem cell is free from dexamethasone, recombinant human oncostatin M, BMP4, HGF, or any combination thereof. In some cases, the culture medium obtaining and/or passaging the mammalian trophoblast stem cell is xeno-free, e.g., free from an animal component.
- the culture medium for obtaining and/or passaging the mammalian trophoblast stem cell is free from a human-derived component and an animal- derived component, e.g., being a chemically defined medium. In some cases, the culture medium obtaining and/or passaging the mammalian trophoblast stem cell is free from a serum.
- the culture medium obtaining and/or passaging the mammalian trophoblast stem cell is free from fetal bovine serum.
- a mammalian trophoblast stem cell herein e.g., an hTS cell
- amniocentesis can be a procedure used to obtain a small sample of the amniotic fluid that surrounds the fetus during pregnancy.
- an amniocentesis can be offered to women between the 15th and 20th weeks of pregnancy who are at increased risk for chromosome abnormalities, e.g., women who are over 35 years of age at delivery, or those who have had an abnormal maternal serum (blood) screening test indicating an increased risk for a chromosomal abnormality or neural tube defect.
- a needle e.g., a long, thin, hollow needle, can be used with ultrasound guide through your abdomen, into the uterus and the amniotic sac.
- a predetermined amount of amniotic fluid e.g. one ounce, can be drawn into a syringe.
- a mammalian trophoblast stem cell herein e.g., an hTS cell
- PPD preimplantation genetic diagnosis
- IVF in vitro fertilization
- the cells herein can be produced by methods for biopsy of a blastocyst, wherein the remainder of the blastocyst is implanted and results in a pregnancy and later in a live birth, e.g., the zona pellucida is removed from the blastocyst and then the blastocyst is biopsied.
- a mammalian trophoblast stem cell herein can be obtained from prenatal chorionic villus sampling (CVS).
- CVS can be a prenatal test that involves taking a sample of tissue from the placenta to test for chromosomal abnormalities and certain other genetic problems.
- CVS can be performed between the 10th and 12th weeks of pregnancy.
- the CVS procedure is transcervical, e.g., a catheter is inserted through the cervix into the placenta to obtain the tissue sample.
- the CVS procedure is transabdominal, e.g., a needle is inserted through the abdomen and uterus into the placenta to obtain the tissue sample.
- a mammalian trophoblast stem cell herein e.g., an hTS cell
- a mammalian trophoblast stem cell herein can be obtained from placental biopsies after full-term pregnancies.
- a mammalian trophoblast stem cell herein e.g., an hTS cell
- a mammalian trophoblast stem cell herein e.g., an hTS cell
- first trimester chorionic villous sampling e.g., 8 +3 to 12 +0 weeks gestational age
- term placenta from caesarean section deliveries.
- 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 min).
- Cells are subsequently centrifuged (e.g., at 1500 rpm, e.g., for 5 min), counted, and/or replated (e.g., 104 cells per cm 2 ) in a medium (e.g., Dulbecco's modified Eagle's medium+10% fetal bovine serum).
- a medium e.g., Dulbecco's modified Eagle's medium+10% fetal bovine serum.
- isolated cells can be plastic adherent.
- the cells can be used at passage 4-8.
- a mammalian trophoblast stem cell herein e.g., an hTS cell
- a mammalian trophoblast stem cell herein can be isolated from term (e.g., 38-40 weeks' gestation) placentas according to the following procedure.
- Umbilical cord blood is allowed to drain from the placentas, which are then dissected carefully.
- the harvested pieces of tissue are washed several times (e.g., in phosphate-buffered saline) and then minced (e.g., mechanically) and enzymatically digested (e.g., with 0.25% trypsin-EDTA).
- the homogenate is subsequently pelleted by centrifugation and suspended in complete medium (e.g., Dulbecco's modified Eagle's medium supplemented by 10% fetal bovine serum, 100 U/ml penicillin, and/or 100 g/ml streptomycin).
- complete medium e.g., Dulbecco's modified Eagle's medium supplemented by 10% fetal bovine serum, 100 U/ml penicillin, and/or 100 g/ml streptomycin.
- Cell cultures are maintained at a suitable condition, e.g., 37°C with a water- saturated atmosphere and 5% CO2. Medium is replaced periodically, e.g., one to two times every week.
- cells When cells are reach a desired level of confluence, e.g., more than 80% confluence, they are recovered, e.g., with 0.25% trypsin/EDTA, and replated at a dilution, e.g., of 1 :3.
- a desired level of confluence e.g., more than 80% confluence
- they are recovered, e.g., with 0.25% trypsin/EDTA, and replated at a dilution, e.g., of 1 :3.
- a mammalian trophoblast stem cell herein e.g., an hTS cell
- a mammalian trophoblast stem cell herein can be isolated from human placentas following delivery according to a procedure as follows.
- the chorion is separated from the amnion by peeling them apart.
- the decidual tissue are scrapped (e.g., mechanically) and washed (e.g., in Dulbecco's phosphate-buffered saline) before being cut into small pieces (e.g., -2 x 2 cm).
- the chorion are chopped into small pieces and subjected in to an enzyme (e.g., 0.5% trypsin-EDTA, e.g., for 5 min), followed by digestion with collagenase I (e.g., at 0.3% in 37°C incubator for 20 to 30 min).
- the mobilized cells are then collected and passed through a cell strainer (e.g., 100 pm).
- the filtered cells are collected by centrifugation (e.g., at 2,500 rpm, e.g., for 5 min).
- the cells are resuspended in a medium (e.g., a-modified minimum essential medium supplemented with 10% fetal bovine serum and/or 1% penicillin- streptomycin), and cultured in a container, e.g., T25 flasks, at a suitable condition (e.g., at 37°C and/or 5% CO2).
- a suitable condition e.g., at 37°C and/or 5% CO2
- the media is changed periodically, e.g., every 3 days, until the chorionic MSCs reached a desired level of confluency, e.g., 70% confluency.
- chorionic villi can be obtained from the fallopian tubes of un ruptured pre-implantation embryos in women with ectopic pregnancy (e.g., gestational age: 5-7 weeks).
- Tiny villous tissues can be well-minced in a suitable medium (e.g., serum-free «-MEM) and identified under microscopy followed by trypsinization (e.g., with 0.025% trypsin/EDTA) for a period of time (e.g., 15 min) and by adding a medium (e.g., «-MEM containing 10% FBS) to halt the reaction.
- a suitable medium e.g., serum-free «-MEM
- trypsinization e.g., with 0.025% trypsin/EDTA
- a period of time e.g., 15 min
- a medium e.g., «-MEM containing 10% FBS
- Adherent cells can be obtained and cultured in a suitable condition (e.g., in conditioned «-MEM, 10% FBS, and 1% penicillin- streptomycin at 37°C in 5% CO2). After two passages, the level of hCG can become undetectable measured by a commercial kit (e.g., Dako, Carpinteria, CA).
- a suitable condition e.g., in conditioned «-MEM, 10% FBS, and 1% penicillin- streptomycin at 37°C in 5% CO2
- a commercial kit e.g., Dako, Carpinteria, CA.
- kits and articles of manufacture for use with one or more 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, and the like, each of the contained s) comprising one of the separate elements to be used in a method described herein.
- Suitable containers include, for example, bottles, vials, syringes, and test tubes.
- the containers are formed from a variety of materials such as glass or plastic.
- the articles of manufacture provided herein contain packaging materials.
- packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of use.
- the contained s) include hTS cells, optionally in a composition as disclosed herein.
- kits optionally include an identifying description or label or instructions relating to its use in the methods described herein.
- a kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.
- a label is on or associated with the container.
- a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert.
- a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.
- hTSCs CREB1, CaMKII, GLUT2, and MAFA, but also b-hCG, histocompatibility antigen HLA-G, and pluripotency transcription factor CDX2 but not OCT4 (octamer-binding transcription factor 4) were expressed in hTS cells ( Figure ID). Stress protein heat shock protein HSP90 was also expressed. But hTSCs also did not express proliferation marker Ki-67, protein folding activator HSP70, tumor suppressor p53, autoantigen GAD65, and cell-cell fusion protein Syncytin (Figure 2M), supporting the concept that hTSCs stand at the first position of TE-differentiated trophoblasts.
- hTS cells can have the ability to express insulin and stress protein HSP90 to cope with the external threats, wherein hTS cells can be highly susceptible to glucose stimulation, which can cause reversible cellular transformation of the hTS cells.
- bFGF Induces Epithelial-Mesenchymal Transition (EMT) via TGF-bI.
- bFGF Promotes Signatures of Definitive Endoderm (DE) via mRNA-124a.
- bFGF targeted receptor FGFR1 at the cell membrane to induce activation of PI3K/AKT/CREB1 signaling pathway.
- DIANA- mirGen 2.0 it was verified that the bFGF-induced CREB1 targeted the consensus CREB binding sequence (TGACGTCA) at the promoter of microRNA-124a (miR-124a) by qPCR analysis, while knockdown of CREB1 reduced miR-124a expression.
- TGACGTCA consensus CREB binding sequence
- miR-124a microRNA-124a
- plasmids of SMAD4, GSIOp, and CDX2 using pGL4.51 vector were constructed for luciferase reporter gene assay.
- miR-124a was shown to target the SMAD4 gene, producing the inhibitory SMAD4. Since SMAD4 can interact with SMAD2/3 to bind to the sequences in the proximal promoters ofMIXLl gene for transcription, an inhibitory SMAD4 caused an inhibitory homeodomain protein MIXL1.
- forkhead box protein A2 (FOXA2) controlled PDX1 expression, contributing to the pro-endocrine transcription factor neurogenin 3 (NGN3) to involve in the specification of endocrine cell differentiation.
- miR-124a targeted at CDX2 gene to inhibit CDX2 synthesis, verified by the pretreatments of miR-124a and anti-miR-124a antibody.
- Downregulated CDX2 resulted in upregulation of OCT4.
- OCT4 targeted at SOX17 gene for transcription to produce SOX17 (SRY-Box protein 17), supported by the imaging study.
- bFGF can promote miR- 124a to consequently get signatures of definitive endoderm (DE) by upregulation of SOX17, FOXA2, and OCT4 as well as downregulation ofMIXLl in 8 hr induction, which can contribute to further insulin expression.
- pancreatic progenitor biomarkers including PDX1, pancreas transcription factor 1 protein (PTFla), SOX9, and homeobox 1 protein NKX (NKX6.1) were noted at 8 hr of bFGF induction.
- the appearance of these molecular signals initiated the expressions ofNGN3, pro-insulin C-peptide, and insulin at 20 hr of induction.
- pancreatic progenitor- and endocrine cell-markers including PDX1, hepatocyte nuclear factor-l-beta (HNF1B), NGN3, SOX9, NKX6.1, and insulin as well as NANOG, SOX2, glucagon, somatostatin, GLUT2, and polypeptide (PP) in the bFGF-treated hTS cells ( Figure 3).
- NANOG was downregulated but SOX2 sustained upregulation after 12 hr induction toward the end of day, where insulin-expressing prCTBs were formed.
- these molecular processes are similar to the development of b-cells in pancreas but distinguish from the glucose-induced insulin expression in hTS cells as described previously.
- the spatiotemporal swift in pluripotency transcription factors is mainly attributed from reciprocal negative autoregulatory mechanisms, whereas SOX2 plays a main role in the maintenance of sternness of prCTBs.
- prCTBs extrapancreatic tissue-specific phenotype that expresses insulin
- prCTBs extrapancreatic tissue-specific phenotype that expresses insulin
- prCTBs can be maintained mainly by SOX2 during trophoblast differentiation in vitro.
- cytotrophoblasts can give rise to: 1) villous cytotrophoblasts; 2) primitive syncytiotrophoblasts (pSTBs) and later
- STBs syncytiotrophoblasts
- EVTs extravillous cytotrophoblasts
- This group included RANTES (also as CCL5), MCP-1 (also CCL2, monocyte chemoattractant protein- 1), GROa (also CXCL1, CXCL2, macrophage inflammatory protein 2- a or MIP2-a), MCP-3 (also CCL7), IL-8, Eotaxin (also CCL11, CCL24, and CCL26), MDC (also CCL22), IP- 10 (also IFN g-induced CXCL10), Fractalkine (also CX3CL1), MIP-Ib (also CCL4), and soluble CD40-ligand (also sCD40L, CD 154).
- RANTES also as CCL5
- MCP-1 also CCL2, monocyte chemoattractant protein- 1
- GROa also CXCL1, CXCL2, macrophage inflammatory protein 2- a or MIP2-a
- MCP-3 also CCL7
- IL-8 also CCL11, CCL24, and CCL26
- MDC also CCL22
- Cytokines and Growth factors included IL-6, IL-10, IL-4, IL-7, IL-15, IL-13, IL-la, IL-Ib, IL-12p40, IL- 3, and IL-2. IFN-g and IFN-a were also secreted. While growth factors included: PDGF-AA and PDGF-AB/BB (platelet-derived growth factor family), VEGF, EGF (epidermal growth factor), bFGF, GM-CSF (granulocyte-macrophage colony- stimulating factor), Flt3L (FMS-like tyrosine kinase 3 ligand), and IL-Ib.
- growth factors included: PDGF-AA and PDGF-AB/BB (platelet-derived growth factor family), VEGF, EGF (epidermal growth factor), bFGF, GM-CSF (granulocyte-macrophage colony- stimulating factor), Flt3L (FMS-like tyrosine
- hTS cells and prCTBs were able to release soluble human leukocyte antigen G (sHLA-G), transforming growth factor beta 1 (TGF-bI), plasminogen activator inhibitor-1 (PAI-1), and IL-10 by secretomic analysis (Figure 7B).
- HLA-G human leukocyte antigen G
- TGF-bI transforming growth factor beta 1
- PAI-1 plasminogen activator inhibitor-1
- IL-10 secretomic analysis
- Figure 7C shows another summary of production of cytokines IL-6 and IL-8;
- prCTBs expressed angiogentic molecules CD 105 (Endoglin, a receptor for TGF-b to function in angiogenesis) and CD146 (vascular endothelial cadherin) (Figure 7D). These results suggested that prCTBs have abilities to function in the decidual tissues at the feto- maternal interface.
- prCTBs Exhibit Immune Cell-Associated Biomarkers [00137] Expression of immune cell-associated biomarkers was examined in prCTBs with 8 independent cell lines by FACS analysis, showing a similar pattern of NK and T cell
- immunoblotting assay identified that both hTS cells and prCTBs expressed ILT-2 (leukocyte Ig-like receptor 1, also LILRB1), ILT-4 (also LILRB2), TCR (T cell receptor), and specifically, KIR2DL4 (killer cell Ig-like receptor) which is expressed by NK cells and subsets of CD8+ T cells to inhibit the cytolytic NK cell function (Figure 7E).
- ILT-2 leukocyte Ig-like receptor 1, also LILRB1
- ILT-4 also LILRB2
- TCR T cell receptor
- KIR2DL4 killer cell Ig-like receptor
- PD-L1 programmed death-ligand 1
- Fas apoptosis signal receptor, also APO-1
- FasL Fas ligand
- NKp46 a major NK cell-activating receptor
- bFGF Induces IL-6 and IL-8 via FGFR1/CREB1 Signaling Pathway in prCTBs.
- hTSCs were incubated with bFGF for 1-day mimicking the transient stay in the fallopian tube.
- bFGF activated its receptor FGFR1 at cell membrane to induce PI3K/phosphorylated (p)AKT signaling.
- pAKT interacted and phosphorylated downstream pCREBl (cAMP responsive element binding protein 1) to activate pCREBl signaling.
- pCREBl cAMP responsive element binding protein 1
- CREB1 targeted the genes to produce IL-6 in a time-dependent manner (FIGURE 7K, left panel) and IL-8 (Figure 7K, right panel) in a dose-dependent manner, confirmed by ELISA assay ( Figure 7C).
- bFGF induces transformation of hTSCs into prCTBs and productions of IL-6 and IL-8 in prCTBs via an autocrine/paracrine fashion.
- IL-6 Induces Trophoblast Marker b-hCG and IL-8 Induces NK Cell Marker CD56 in prCTBs
- prCTBs IL-6 bound to receptor IL-6R at the cell membrane to activate CREB1 signaling, resulting in the production of b-hCG by RT-qPCR assay ( Figure 7L, left panel).
- IL-6 enabled to bind another receptor GnRHR to co-incidentally activate CREB 1 signaling to consequently produce b-hCG by Western blot assay ( Figure 7M).
- IL-8 induced CD56 also known as NCAM production but did not through IL-8R by RT-qPCR assay ( Figure 7L, right panel).
- IL-8 Induce T Cell Marker CD4 via CXCR2/CREB1 Signaling in prCTBs
- IL-8 bound and activated receptor CXCR2 at the cell membrane of prCTBs via an autocrine/paracrine fashion to induce CREB1 signaling, allowing its nuclear translocation.
- CREB1 targeted CD4 gene for transcription to produce CD4 molecule by Western blot assay ( Figure 70).
- IL-8 was also able to bind the receptor CXCR2 to consequently activate STAT3 signaling, leading to nuclear translocation of STAT3.
- STAT3 targeted at different sites of CD4 gene for transcription to produce CD4 ( Figure 7P).
- prCTBs Express Factors to Promote Angiogenesis in Decidual Tissues.
- prCTBs were able to secrete VEGF and PDGF-AA by Milliplex assay ( Figure 7C) and plasminogen activator inhibitor-1 (PAI-l)and IL-10 by ELISA assay ( Figure 7B) as well as to expressCD105(+) and CD146(+) markers by FACS analysis ( Figure 7D). All expressions of those molecules suggested that prCTBs have ability to promote angiogenesis and vasculogenesis, for example, in the SA remodeling of decidual tissues at the feto-maternal interface.
- MCP-1 and CXCL2 Synergistically Drive Movement of prCTBs.
- prCTBs Induces Apoptosis of Solid Tumor Cells Upon Interaction.
- Figure 1 IB showed the interaction of two live cells, while Figure 11C revealed the apoptosis of PANC-1 upon interaction. This interaction was further evidenced by 3D fluomicroscopy, showing the apoptosis of PANC-1 ( Figure 11D).
- PANC-1 programmed cell death-ligand- 1 but not PD-1 (programmed cell death protein 1) ( Figure 1 IE, left column), while PANC-1 expressed both PD-L1 and PD-1 ( Figure 1 IE, right column).
- prCTBs enabled to deliver the PD-Ll/PD-1 cell death signaling to the target PANC-1 cells, causing PANC-1 cell apoptosis.
- PANC-1 cell’s PD-L-1 was unable to send the death signaling towards prCTBs because of the lack of PD-1 in prCTBs, explaining no apoptosis occurred in prCTBs.
- prCTBs expressed Fas while MCF-7 expressed FasL ( Figure 1 II, lower panels). This fact implied that apoptosis might occur with prCTBs, but this was not the case.
- dNK cells in chorionic tissues were obtained from aborted women with medical reason and from ectopic pregnant woman at same gestational age of 8-weeks under consent. Firstly, it was confirmed the presence of CD56 biomarker in dNK cells (CD56 also expressed in prCTBs) by immunocytochemistry ( Figure 12A, top panels). Subsequently,
- hTS Human trophoblast stem cells were derived from trophoblast tissue.
- FBS v/v fetal bovine serum
- hTS cells including 1) MesenCultTM-ACF Plus medium, with MesenCultTM-ACF PLUS 500x Supplement and L-Glutamine, with or without a substrate such as Cell Attachment Substrate, and 2) alpha-MEM containing nucleosides, GlutaMAXTM Supplement, and 10% StemulateTM Human Platelet Lysate Cell Culture Media Supplement, with or without a substrate.
- FBS was replaced by CMP grade PLUS (Compass Biochemical).
- the characteristic biomarkers, including HLA-G, b-hCG, and CDX2 with undetectable CD34 and CD45 were stably expressed.
- Induction of the hTSC cells to prCTBs was carried out by treatment with 10 ng/ml bFGF for 24 hr in hTS cells at passages 5-10.
- the seeding density was about 10,000 cells/cm 2 .
- the culture was free from penicillin, streptomycin, mercaptoethanol, and/or nicotinamide.
- the culture can also be free from an animal component, serum such as fetal bovine serum, antibiotic, retinoic acid, dexamethasone, recombinant human oncostatin M, BMP4, and/or HGF.
- serum such as fetal bovine serum, antibiotic, retinoic acid, dexamethasone, recombinant human oncostatin M, BMP4, and/or HGF.
- the regimen of differentiation was determined by empirical experience (data not shown). Stage-specific differentiation of lineages was referred to a variety of cellular biomarkers described previously. Cells were harvested at time as indicated for different analysis.
- hTS cells were transfected with either siRNA or shRNA or 3' UTR reporter plasmids using TransIT-LTl transfection reagent (Mirus Bio LLC). Transfection was performed with 2 pg siRNA or shRNA plus 4 pi transfection reagent in 100 pi OPTI-MEM (Gibco). After incubation for 10 min at room temperature, the transfection mix was gently added to cells overnight. The transfected cells then re-incubated with the a-MEM supplemented with 10% FBS for further treatment.
- the 3' UTR region was PCR amplified by using forward primer with a Psil site and reverse primer with Mfel site for 3’ UTR reporter construct were listed as followings: For Cdx2 3’ UTR region: 5’-aaattataagctgtttgggttgttggtct-3’ and 5’- aaacaattgcccccataatttctgactgc-3’; For Smad4 3’ UTR region 1 : 5’-aaattataactcccaaagtgctgggatta- 3’ and 5’-aaacaattgctgcactgttcacaggagga-3’; For Smad4 3’ UTR region 2: 5’- aaattataacagttgtcccagtgctgcta-3’ and 5’-aaacaattgatgacttgcccaaaggtcac-3’; For GSK3P 3’ UTR region: 5’-
- firefly luciferase reporter 500 ng
- empty vector without any 3’UTR co-transfected with pGL4.74 vector 500 ng
- renilla luciferase plasmid 500 ng, Promega
- non-specific control miRNA 30 pmol
- miR-124a precursor 30 pmol
- hTS cell culture media (10 ml) were harvested at 80-90% confluence followed by centrifugation (3,000 rpm, 30 min, 4°C). The supernatants were further concentrated to 1 ml by using 3 kDa Vivaspin concentrator (Sigma). The concentrated supernatant was further detected for TGF-bI, HLA-G, and PAI-1 by immunoblotting assay. IL-10 level was measured by OptEIA ELISA assay kit following the supplier’s instructions (BD Pharmingen, San Diego). The range of detectable IL-10 concentration was between 2 and 2,000 pg/ml. An aliquot of 100 m ⁇ sample was measured in triplicate.
- hTS cell-formed cellular cluster on the culture dish was dissected with wolfram needles.
- the clumps of cells were fixed in 0.1 M PBS (Merck; pH 7.4) containing 3% (w/v) paraformaldehyde (Merck),
- hTS cells were treated with bFGF and harvested at time as indicated and put into RIPA lysis solution (Millipore) supplemented with protease (Thermo Scientific) and phosphatase inhibitor (Cell Signaling Technology). After electrophoresis of 30 pg lysates on polyacrylamide gels, electroblotting onto PDVF membranes (Millipore) was performed. By blocking of 5% non-fat dry milk in PBS at room temperature (I hr), target proteins were incubated with primary antibodies. All membranes were incubated with chemiluminescent (Millipore) and imaging was captured by the ChemiDoc XRS system (Bio-RAD). Antibodies used were listed in Key Resource Table. Data were analyzed by AlphaEaseFC (version 4.0.0).
- Thermocycler (Applied Biosystems). Comparative real-time PCR was performed in triplicate or quintuple, including no-template controls, using specific primers for miR-124 or RNU6B (Applied Biosystems). U6 snRNA (RNU6B; Applied Biosystems) served as an endogenous control. Relative expression was calculated using SDS2.2.2 software (Applied Biosystems) was used for comparative ACt analysis.
- ChIP Chromatin Immunoprecipitation Assay.
- ChIP assay was performed by using ChIP-IT Express Chromatin Immunoprecipitation Kits (Active Motif) as manufacturer’s instructions. Briefly, immunoprecipitated DNA fragments were extracted from hTS cells (1x106). Antibody anti-CREBl or anti-OCT4 or anti-P-catenin was used.
- Cell culture supernatants were harvested from: 1) the hTS cell culture (1 x 1.8 6 cells/10 ml) for 24 hr and 2) the bFGF (10 ng/ml)-treated hTS cells for 24 hr (prCTBs). These supernatants were subjected for Milliplex analysis at the National Experimental Research Laboratories, Taiwan using Luminex LX 200 instrument (R&D system, USA) and data were analyzed by Milliplex analyst software (5.1.0.0.).
- hTS cells were collected by scraping or trypsinization with IX TrypLE (Thermo Fisher Scientific) and washed with PBS.
- Cells (5 x 106 cells/ml) were incubated in blocking buffer (PBST plus 5% donkey serum) on ice for 1 hr, followed by resuspension in blocking buffer with Alexa Fluor® 647 conjugated anti-insulin antibody (9008s, Cell signaling) or unconjugated anti-insulin antibody (sc-7839, Santa Cruz) at 4 oC for 30 min, Cells were washed twice in blocking buffer and strained with unconjugated antibody followed by incubation with blocking buffer with Alexa Fluor® 647 conjugated secondary antibodies on ice in dark for 30 min. After wash twice, cells were passed through polystyrene round-bottom tube with cell strainer cap (BD Falcon) before flow cytometry (LSR-II flow cytometer; BD Biosciences). Results were analyzed by FlowJo
- hTS cells were transfected with non specific shRNA or shRNAs against CDX2 or OCT4 or SOX2 or NANOG. Cells (5 x 106 cells/ml) were then incubated with specific primary antibodies for 30 min. By incubation with the appropriate fluorescent dye-conjugated primary antibodies at adjusted dilution for 1 hr at 4°C, samples were washed and re-suspended in PBS, followed by passing through polystyrene round-bottom tube with cell strainer cap (BD Falcon) before flow cytometry (FACScan, BD Biosciences, San Jose, CA). The data were analyzed with Cell-Quest software (BD
- hTS cells or prCTBs (1 xlO 5 ⁇ 1 x 10 6 ) were suspended in 240 pi of IX FCM buffer (Leinco, FI 175). Cells (30 m ⁇ ) were stained with 7-AAD (BD, 5599257), fluorescence labeled antibodies (BD multitest 6-Color TBNK (BD, 337166) +
- BV421 -labeled anti-CD107a BioLegend, 328625) or BV421 -labeled anti-CD34 (BD, 562577) only or PerCP Cy5.5-labeled anti-CD45 (BD, 340952) + APC-labeled anti-CD3 (BD, 555342) + PE-labeled anti-y5TCR (BD, 340887) or fluorescence labeled isotype control antibodies (FITC- labeled IgGlK (BD, 556649) + PE-labeled IgGlx (BD, 556650) + PE-labeled IgG2bx (BD, 556656) + PerCP-CyTM5.5 -labeled IgGlx (BD, 552834) + PE-CyTM7-labeled IgGlx (BD, 557872) + APC-labeled IgGlx (BD, 550854) + APC-Cy7-labeled IgGlx (
- BV421 -labeled IgGlx (BD, 562438) or BV421 -labeled IgGlx (BD, 562438) only. After incubation for 15 minutes at room temperature, cells were washed with 1 ml of IX FCM buffer and resuspended in 200 m ⁇ of IX FCM buffer. Eventually, cell samples were analyzed by using FACSVerse flow cytometer (BD, 651155) and FACSuite software.
- a variety of cancer cells (2,000 cells), including PANG-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 with culture medium in the 35mm glass bottom dish (ibidi; Cat# 81158) at 5% C02, 37 °C incubation. After cell adhesion overnight, bFGF-induced hTS cells (2 x 104 cells) were added for co-culture over 24 hr. For apoptosis assay, the co-cultured cells were stained by using the Apoptosis/Necrosis kit (ab 176749, Abeam, Cambridge, England) according to the
- prCTBs (1 x 10 6 cells/ml) are seeded using the 6-well transwell insert (8 pm in pore size, Corring) and incubate for 10 minutes at 37 °C and 5% CO2 to allow the cells to settle down.
- ECM extracellular matrix
- Matrigel is thawed and liquefied on ice, and then 30-50 pi of Matrigel is added to a 24-well transwell insert and solidified in a 37 °C incubator for 15-30 minutes to form a thin gel layer.
- Cell solution is added on top of the Matrigel coating to simulate invasion through the extracellular matrix.
- the transwell cell migration assay measures the chemotactic capability of cells toward a chemo-attractant.
- the transwell cell invasion assay measures both cell chemotaxis and the invasion of cells through extracellular matrix, a process that is commonly found in cancer metastasis or embryonic development.
- the quantification of migrated cells can be performed following steps 2.4 to 2.8 (steps 2.4-2.8 do not need to be performed in a sterile environment).
- steps 2.4-2.8 do not need to be performed in a sterile environment.
- the migrated cells will drop into the media in the lower chamber.
- the number of migrated cells can be counted by using hemocytometer or flow cytometer 5
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