Lethal pluripotent stem cells

Abstract

Disclosed herein is the lethal pluripotent stem cell produced in vitro from extraembryonic stem cells such as trophoblast stem cells, its composition and its use.Disclosed herein is also the method of culturing the lethal pluripotent stem cell produced in vitro with one or more inducers to produce cell population.Disclosed herein is also the method of treating disorders or conditions by using the cells disclosed herein and the cells differentiated therefrom.

Description

[Technical Field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 020,247, filed May 5, 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002]

[0002] Novel stem cells for treating various diseases or conditions are needed as alternatives to overcome certain drawbacks of current embryonic stem cells and iPS cells.

[0003] Incorporation by Reference

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. If there is a conflict between a term in this specification and a term in an incorporated reference, the term in this specification will control. Summary of the Invention

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

[0005]

[0005] In some of many embodiments, disclosed herein are populations of lethal pluripotent stem cells (MPSCs) that express HLA-G and insulin and are capable of reaching up to at least 89 population doublings within 90 days from initiation of culturing the MPSCs. In some examples, the population of MPSCs can reach about 89 to about 100 population doublings within 90 days from initiation of culturing the MPSCs. In some examples, the population of MPSCs can reach about 25 to about 30 population doublings within about 12 days, about 50 to about 55 population doublings within about 30 days, and / or about 75 to about 80 population doublings within about 63 days from initiation of culturing the MPSCs. In some examples, the population of MPSCs can doubling in about 22 to about 27 hours, e.g., about 25 hours. In some embodiments, disclosed herein are populations of lethal pluripotent stem cells (MPSCs) that express HLA-G and insulin and are pathogen-free.

[0006] In some cases, the MPSCs disclosed herein are pathogen-free. In some examples, the MPSCs are bacteria-free. In some examples, the MPSCs are virus-free, e.g., cytomegalovirus-free. In some examples, the MPSCs are pathogen-free, e.g., EBV (Epstein-Barr virus), HAdV (human adenovirus), HCMV (human cytomegalovirus), hepatitis virus (e.g., hepatitis A, hepatitis B, and / or hepatitis C), human herpes virus (e.g., HHV6 (human herpes virus 6) and / or HHV8 (human herpes virus 8)), human immunodeficiency virus (e.g., HIV1 (human immunodeficiency virus 1), HIV2), or the like. (human immunodeficiency virus type 2)), human papillomavirus (e.g., HPV16, HPV18, etc.), herpes simplex virus (e.g., HSV1 (herpes simplex type 1), HSV2 (herpes simplex type 2), etc.), human T-lymphotropic virus (e.g., HTLV1 (human T-lymphotropic virus type 1), HTLV2 (human T-lymphotropic virus type 2), etc.), VZV (varicella virus), Corynebacterium bovis, Corynebacterium sp. (HAC2), hantavirus (e.g., Hantaan, Seoul, or Sin Nombre), lymphocytic choriomeningitis virus (LCMV), Mycoplasma sp., Treponema pallidum, and any combination thereof.

[0007]

[0007] In some examples, the MPSCs disclosed herein, or populations comprising MPSCs, further express one or more of the following proteins: beta-human chorionic gonadotropin (b-HCG), heat shock protein 90 (HSP90), caudal homeobox 2 (CDX2), fibroblast growth factor receptor 1 (FGFR1), pAKT, pCREB1 (CAMP response element binding protein 1), human lymphocyte antigen A (HLA-A), HLA-B, or HLA-C. In some examples, the population of MPSCs further express one or more of the following proteins: killer cell immunoglobulin-like receptor 4 (KIR2DL4), FMS-like tyrosine kinase 3 ligand (Flt3L), NKp46, T cell receptor (TCR), immunoglobulin-like transcript 4 (ILT-4), CD49f, CD3, CD4, CD8, CD10, CD11b, CD14, CD16, CD19, CD34, CD38, CD44, CD56, CD90 / Thy-1, CD105, CD141, CD146, CD166, or CD107a. In some examples, the population of MPSCs further express one or more of the following proteins: interleukin 6 (IL-6), IL-8, monocyte chemoattractant protein-1 (MCP-1), CLXL2, platelet-derived growth factor AA (PDGF-AA), vascular endothelial growth factor (VEGF), plasminogen activator inhibitor 1 (PAI-1), or IL-10. In some examples, at least a portion of the MPSCs do not express one or more of the following proteins: Ki-67, heat shock protein 70 (HSP70), p53, or syncytin. In some examples, the population of MPSCs (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) express one or more of the following proteins: CD44, CD90, CD105, CD146, CD166, HLA-A, HLA-B, or HLA-C. In some cases, at least a portion of the MPSCs do not express one or more of the following proteins: CD19, CD45, or HLA-DR. In some cases, more than 96%, 97%, 98%, or 99% of the MPSCs do not express one or more of the following proteins: CD19, CD45, or HLA-DR.In some examples, the population of MPSCs further express one or more of CD16 or CD56, or a combination thereof. In some examples, at least a portion of the MPSCs do not express CD3. In some examples, more than 96%, 97%, 98%, or 99% of the MPSCs do not express CD3. In some examples, at least 65% or at least 70% of the population of MPSCs express HLA-G. In some examples, the HLA-G includes HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7, or any combination thereof. In some examples, the HLA-G includes HLA-G2, HLA-G4, HLA-G6, or HLA-G7, or any combination thereof. In some examples, the HLA-G includes HLA-G6 or HLA-G7, or a combination thereof. In some instances, less than 15% (eg, less than 10%) of the population of MPSCs express HLA-G1.

[0008] In some instances, at least 10% of the population of MPSCs disclosed herein are monoclonal. In some instances, about 13% to about 15% of the population of MPSCs are monoclonal. In some instances, at least about 1 x 10 6 MPSCs are present in the population.In some cases, the MPSCs have a stable karyotype when measured by array-based whole genome assay.In some cases, the MPSCs do not undergo chromosomal abnormalities resulting from population doubling when measured by array-based whole genome assay.In some cases, the MPSCs do not undergo substantial chromosomal abnormalities resulting from freezing and thawing when measured by array-based whole genome assay.

[0009] In some cases, the present disclosure provides a method of expanding a population of MPSCs disclosed herein, comprising culturing the MPSCs at about 1,000 to about 5,000 cells / cm in culture medium. 2 and culturing the cells.

[0010] In some aspects, a method for expanding a population of lethal pluripotent stem cells (MPSCs) comprises culturing the MPSCs at a density of about 1,000 to about 5,000 cells / cm in a culture medium. 2 and culturing the cells, wherein the population of MPSCs expresses HLA-G and insulin. In some examples, the culture medium is animal component-free. In some examples, the culture medium is serum-free, e.g., fetal bovine serum. In some examples, the cells are cultured for 3 days. In some examples, the cells are cultured for 4 days. In some examples, the MPSCs are cultured at a density of about 2,000 to about 4,000 cells / cm. 2 The seeds are sown at a density of

[0011]

[0011] In some aspects, disclosed herein are methods for producing cells, comprising contacting a population of MPSCs disclosed herein with one or more inducers. The cells produced may comprise ectodermal cells. The cells produced may comprise mesodermal cells. The cells produced may comprise endodermal cells. The cells produced may comprise pancreatic cells or pancreatic progenitor cells, and optionally, the inducer comprises bFGF (basic fibroblast growth factor) and may further comprise 2-mercaptoethanol and nicotinamide. In one embodiment, the PPC comprises β-HCG, CDX2, HLA-G, or any combination thereof. In some examples, the PPC comprises β-HCG and CDX2; β-HCG and HLA-G; CDX2 and HLA-G; or HCG, CDX2, and HLA-G. Optionally, in some examples, the PPC further comprises PDX1, FOXA2, SOX9, or any combination thereof. The cells produced may include neural cells or neural progenitor cells, and optionally, the inducer comprises retinoic acid. In one embodiment, the NCS cells comprise retinoic acid receptor beta (RAR-β), CDX2, HLA-G, or any combination thereof. In some examples, the NCS cells comprise RAR-β and CDX2; RAR-β and HLA-G; CDX2 and HLA-G; or RAR-β, CDX2, and HLA-G. Optionally, in some examples, the PPCs further comprise N-CAD, neuroepithelial stem cell protein (NESTIN), SRY (sex determining region Y) box 2 (SOX2), paired box 6 (PAX6), or any combination thereof. The cells produced may include hepatocytes or hepatic progenitor cells, and optionally, the inducer includes a fibroblast growth factor (FGF) such as FGF2, a steroid such as dexamethasone, and a cytokine such as oncostatin M, and may further include a bone morphogenetic protein (BMP), e.g., BMP4, and / or hepatic growth factor. In some examples, the FGF binds to FGFR1 and is FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF8, FGF10, FGF17, FGF19, FGF20, FGF21, FGF22, or FGF23.In some examples, the steroid is a glucocorticoid steroid, such as dexamethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, or triamcinolone. In some examples, the cytokine is an interleukin-6 (IL-6) group cytokine, such as oncostatin M, e.g., human oncostatin M, IL-6, interleukin-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), and cardiotrophin-like cytokine (CLC). The cells produced may include natural killer cells, and the inducer includes FGF, such as FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF8, FGF10, FGF17, FGF19, FGF20, FGF21, FGF22, or FGF23. In one embodiment, the natural killer cells are CD16+, CD56+, and CD3-. In some examples, the natural killer cells are further HLA-G+ and CDX2+.

[0012] The cells produced may include adipocytes, chondrocytes, bone cells, or any combination thereof. In one embodiment, the cells produced include adipocytes and chondrocytes. In another embodiment, the cells produced include adipocytes and bone cells. In another embodiment, the cells produced include chondrocytes and bone cells. In another embodiment, the cells produced include adipocytes, chondrocytes, and bone cells.

[0013] In another embodiment, the cells produced include adipocytes. Adipocytes can be derived from a variety of proteins, including leptin, homeobox C8 (HOXC8), homeobox C9 (HOXC9), uncoupling protein 1 (Ucp1), cell death-inducing DFFA-like effector A (CIDEA), PR domain-containing 16 (PRDM16), Zic family member 1 (Zic1), LIM homeobox 8 (Lhx8), Eva1, epithelial-stromal interaction 1 (Epsti1), Cd137, transmembrane protein 26 (Tmem26), T-box transcription factor 1 (Tbx1), Glu / Asp-rich The adipocytes may include Cbp / P300-interacting transactivator with carboxy-terminal domain 1 (Cited1), short stature homeobox 2 (Shox2), amino acid transporter ASC-1, amino acid transporter PAT2, purinergic receptor P2RX5, adipocyte triglyceride lipase (ATGL), caveolin 1 (CAV1), fatty acid binding protein 4 (FABP4), cytochrome c oxidase subunit 4 (COX4), lamin B1 (LMNB1), or a combination thereof. In one example, the adipocytes include white adipocytes, and the white adipocytes include leptin, HOXC8, HOXC9, or a combination thereof. In another example, the adipocytes include brown adipocytes, and the brown adipocytes include Ucp1, CIDEA, PRDM16, Zic1, Lhx8, Eva1, Epsti1, or a combination thereof. In another example, the adipocytes comprise beige adipocytes, and the beige adipocytes comprise Cd137, Tmem26, Tbx1, Cited1, Shox2, or a combination thereof. In another example, the adipocytes comprise beige adipocyte precursors, and the beige adipocyte precursors comprise CD137, TMEM26, or a combination thereof.

[0014] In another embodiment, the cells produced comprise chondrocytes, which may comprise annexin A6, CD44, CD151, ITM2A, family with sequence similarity member 20-B (FAM20B), forkhead box C1 (FoxC1), FoxC2, SOX5, SOX6, SOX9, aggrecan, cathepsin B, chondroadherin-like (CHADL), chondroadherin, collagen II, collagen IV, cartilage acidic protein 1 (CRTAC1), dermatan sulfate proteoglycan 3 (DSPG3), integrin-binding sialoprotein (IBSP) / sialoprotein II, matrilin-1, matrilin-3, matrilin-4, MIA, otraprin / OTOR, URB, or a combination thereof.

[0015] In another embodiment, the cells produced include bone cells. The bone cells may include pre-osteoblasts, osteoblasts, embedded osteoblasts, osteoid osteoblasts, bone mineralizing osteocytes, or mature bone cells. The bone cells may include RUNX family transcription factor 2 (RUNX2), osteocalcin (OCN), E11, dentin matrix acidic phosphoprotein 1 (DMP1), phosphate-regulated endopeptidase homolog X-linked (PHE)X, matrix extracellular phosphoglycoprotein (MEPE), sclerostin, capping actin protein, gelsolin-like protein (CapG), ORP150, or a combination thereof. In one example, the bone cells include pre-osteoblasts, and the pre-osteoblasts include RUNX2. In another example, the bone cells include pre-osteoblasts, and the pre-osteoblasts include RUNX2. In another example, the bone cells include osteoblasts, and the osteoblasts include RUNX2 and OCN. In another example, the bone cells include embedded osteoblasts, and the embedded osteoblasts include OCN, E11, DMP1, PHEX, and CapG. In another example, the bone cells include osteoid or bone mineralizing osteocytes, and the osteoid or bone mineralizing osteocytes include OCN, E11, DMP1, PHEX, MEPE, and CapG. In another example, the bone cells include mature osteocytes, and the mature osteocytes include DMP1, PHEX, MPEP, sclerostin, CapG, and ORP150.

[0016]

[0016] In another aspect, disclosed herein is a population of lethal pluripotent stem cells (MPSCs) that express HLA-G and comprise a phenotype comprising one or more of: negative for indoleamine 2-3 deoxygenase (IDO) secretion, negative for kynurenine secretion, and positive for interleukin 2 (IL-2) secretion. In one example, the population of MPSCs comprises a phenotype that is negative for indoleamine 2-3 deoxygenase (IDO) secretion, negative for kynurenine secretion, and positive for interleukin 2 (IL-2) secretion.

[0017] In any of such aspects, embodiments, and / or examples, the inventors have demonstrated that stem cells are immune privileged, chromosomally stable (non-tumorigenic), pathogen-free, and pluripotent. The inventors have also demonstrated efficient differentiation of the stem cells into programmed natural killer (NK), cartilage, bone, adipose, neuronal, pancreatic, liver, and secretome cells, along with remarkable doubling times and proliferation characteristics.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Various aspects of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 is a line graph showing a 3-day proliferation curve of MPSCs as measured by population doublings over a 90-day time frame. [Figure 2-1]

[0020] Figures 2A-2D show the whole genome view of KARYOSTAT™ analysis of four different MPSC samples at different population doublings. Figure 2A is an MPSC sample derived from 16.5 population doublings. Figure 2B is an MPSC sample derived from 44.5 population doublings. Figure 2C is an MPSC sample derived from 62.6 population doublings. Figure 2D is an MPSC sample derived from 71.5 population doublings. [Figure 2-2] Same as description for Figure 2-1. [Figure 3]

[0021] Figure 3 shows flow cytometry analysis of MPSCs stained for HLA-G isotype. [Figure 4]

[0022] FIG. 4 shows flow cytometry analysis of MPSCs stained with 4H84 antibody for HLA-G isotype and with mouse IgG1 for control. [Figure 5A]

[0023] Figures 5A-5D show the characterization of MPSCs by the expression of specific molecular biomarkers. MPSCs express molecular biomarkers such as β-hCG, HLA-G, HSP90, and CDX2 (Figure 5A), but are negative for some, such as ki67, syncytin, HSP70, and p53 (Figure 5B). (Figure 5C) FACS analysis reveals that MPSCs express HLA-A, B, and C (left panel) as well as surface and soluble HLA-G (right panel) detected by the 4H84 antibody, compared with isotype controls and unstained cells. (Figure 5D) Representative FACS analysis of HLA-G isoforms in MPSCs at the cell surface compared with those in the cell surface and intracellular compartments. [Figure 5B] Same as description for Figure 5A. [Figure 5C] Same as description for Figure 5A. [Figure 5D] Same as description for Figure 5A. [Figure 6A]

[0024] Figures 6A-6G show the expression of molecular biomarkers of immune cells in MPSCs. By imaging or FACS analysis, MPSCs express various molecular biomarkers of NK cells (Figure 6A), T cells (Figure 6B), dendritic cells (Figures 6C and 6D), macrophages (Figure 6E), and stem cell precursors (Figures 6F and 6G). Specific biomarkers are indicated on the images or plots. [Figure 6B] Same as description for Figure 6A. [Figure 6C] Same as description for Figure 6A. [Figure 6D] Same as description for Figure 6A. [Figure 6E] Same as description for Figure 6A. [Figure 6F] Same as description for Figure 6A. [Figure 6G] Same as description for Figure 6A. [Figure 7]

[0025] FIG. 7 provides exemplary growth curves of MPSC1 (upward triangles; upper line), MPSC2 (squares), MPSC3 (downward triangles), and MPSC4 (circles) over 33 passages. [Figure 8A]

[0026] FIG. 8A shows the standard curve for the IDO secretion assay. [Figure 8B]

[0027] Figure 8B shows the results of IDO secretion from the three cell lines at various concentrations of IFN-γ stimulation compared to the control. The data are "negative," indicating no effect on IDO secretion compared to the control. [Figure 9A]

[0028] FIG. 9A shows the standard curve for the kynurenine secretion assay. [Figure 9B]

[0029] Figure 9B shows the results of the effect of IFN-γ stimulation at three different concentrations on kynurenine secretion at 24, 48, and 72 hours compared to the control and medium alone. The data are "negative," indicating no effect on kynurenine secretion compared to the control. [Figure 10A]

[0030] FIG. 10A shows the standard curve for the IL-2 secretion assay. [Figure 10B]

[0031] Figure 10B shows the effect of IFN-γ stimulation on IL-2 secretion at three different concentrations at 24, 48, and 72 hours compared to the control and medium alone. The data are "positive," indicating that the cells increased IL-2 secretion compared to the control. [Figure 10C]

[0032] Figure 10C shows the effect of MPSC seeding density of approximately 3000 cells / cm2 at 24 hours of co-culture compared to control. A dose-dependent increase was observed. [Figure 10D]

[0033] Figure 10D shows the effect of MPSC seeding density of approximately 2000 cells / cm2 at 24 hours of co-culture compared to control. A dose-dependent increase was observed. [Figure 10E]

[0034] Figure 10E shows the effect of MPSC seeding density of approximately 3000 cells / cm2 at 48 hours of co-culture compared to control. A dose-dependent increase was observed. [Figure 10F]

[0035] Figure 10F shows the effect of an MPSC seeding density of approximately 2000 cells / cm2 at 48 hours of co-culture compared to control. A dose-dependent increase was observed. MPSCs increased, rather than decreased, IL-2 secretion by activated Jurkat cells. [Figure 11A]

[0036] Figure 11A is a graph showing cell numbers at 72 hours, with dead cells shown at the top and live cells shown at the bottom of each bar. [Figure 11B]

[0037] FIG. 11B is a graph showing population doublings in each type of medium. [Figure 12A]

[0038] Figure 12A is a graph showing cell counts over different days in culture. ΔD2-D6 = 44,000,000 cells. [Figure 12B]

[0039] FIG. 12B is a graph showing the % viable cells in culture. [Figure 12C]

[0040] Figure 12C is a graph showing population doublings comparing adherent cultures versus suspension cultures. ΔD2-D6 = 4.9 PD. Adherent cultures initially doubled faster than suspension cultures, but over time, suspension cultures achieved a faster rate of population doubling. DETAILED DESCRIPTION OF THE INVENTION

[0020]

[0041] Disclosed herein are novel and unique in vitro-produced lethal pluripotent stem cells (MPSCs), their compositions, and their use in generating differentiated cells of various phenotypes (e.g., pancreatic, neural, hepatic, immunoregulatory, or natural killer cell phenotypes) or in treating disorders (e.g., diabetes, neuronal loss or degeneration, liver disease, cancer, inflammation, viral infection, or autoimmune disease) or improving conditions (e.g., skin conditions). MPSCs differ from previous trophoblast stem cells and have advantages including, but not limited to, rapid and scalable population doubling; exhibiting a pathogen-free profile; being highly immune privileged and suitable for transplantation; possessing excellent chromosomal stability, e.g., possessing a stable karyotype for at least 71 population doublings; and producing a robust secretome enriched in cytokines, chemokines, and exosomes. MPSCs differ from embryonic stem cells and are ethically sourced and cultured. Although MPSCs are lethal (e.g., have a finite proliferation capacity), they can reach population doublings much more rapidly than embryonic stem cells and iPS cells. Unlike cells derived from the placenta, umbilical cord, or bone marrow, MPSCs are pluripotent and can differentiate or mature into the three major cell groups that make up humans: ectoderm (which gives rise to skin, neurons, and the nervous system), endoderm (which forms the gastrointestinal and respiratory tract, endocrine glands, liver or hepatocyte-like cells, and pancreas or pancreatic cells), and mesoderm (which forms bone (osteocytes), fat, cartilage (chondrocytes), much of the circulatory system, muscle, connective tissue, immune cells, etc.). Furthermore, MPSCs are nontumorigenic, e.g., do not induce tumors or teratomas, as shown in studies in immunocompetent rats.

[0021]

[0042] The details of one or more embodiments of the invention are set forth in the accompanying drawings, claims, and description herein. Other features, objects, and advantages of the embodiments of the invention disclosed and contemplated herein may be combined with any other embodiment, unless expressly excluded.

[0022]

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of any claimed subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the terms "including" and other forms such as "include," "includes," and "included" is not limiting.

[0023]

[0044] As used herein, ranges and amounts can be expressed by adding "about" to a particular value or range, for example, to mean ±15% of the referenced numerical value. "About" also includes the exact amount, for example, "about 5 μL" means "about 5 μL" and "5 μL." In general, the term "about" includes amounts that are expected to fall within experimental error.

[0024]

[0045] As used herein, the terms "treating," "treatment," and the like refer to obtaining a desired pharmacological and / or physiological effect. In some examples, an individual (e.g., an individual suffering from and / or suspected of being genetically predisposed to a liver-related disease or disorder) is prophylactically treated with a preparation of cells described herein, where such prophylactic treatment completely or partially prevents the liver-related disease or disorder or its signs or symptoms. In some examples, an individual is therapeutically treated (e.g., if the individual is suffering from a liver-related disease or disorder), where such therapeutic treatment causes a partial or complete cure of the disease or disorder, and / or reverses adverse effects caused by the disease or disorder, and / or stabilizes the disease or disorder, and / or delays the progression of the disease or disorder, and / or causes regression of the disease or disorder.

[0025]

[0046] Administration (e.g., transplantation) of the cells disclosed herein to the area in need of treatment can be achieved, for example, but not limited to, by local infusion during surgery, by injection, by catheter, or by implant, which can be of a porous, non-porous, or gelatinous material, including membranes such as sialastic membranes or fibers.

[0026]

[0047] "Transplanting" a composition into a mammal refers to introducing the composition into the body of a mammal by any method established in the art. The composition introduced is the "graft" and the mammal is the "recipient." The graft and recipient may be syngeneic, allogeneic, or xenogeneic. Additionally, the transplant may be autologous.

[0027]

[0048] When used in relation to a cell or cell population, the term "isolated" refers to the state of a cell or cell population that is separated from the host organism from which the cell or cell population may originate and is not present in the host organism.In some cases, the isolated cell is isolated from the same host organism or is contacted with other cells derived from it.In some cases, the isolated cell is purified and separated from any other cells.In some cases, the isolated cell is derived in vitro from a stem cell.

[0028]

[0049] An "effective amount" is the amount of a therapeutic agent sufficient to achieve its intended purpose. An effective amount of a composition for treating or ameliorating a disorder is the amount of the composition sufficient to reduce or eliminate the symptoms of the disorder.

[0029]

[0050] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0030] Cells and compositions

[0051] Disclosed herein, in some of many embodiments, are populations of lethal pluripotent stem cells (MPSCs) that express HLA-G and insulin and can reach a maximum of at least 89 population doublings within 90 days from the initiation of culturing the MPSCs. In some examples, the population of MPSCs can reach a maximum of at least 89-100 population doublings within 90 days from the initiation of culturing the MPSCs. In some examples, the population of MPSCs can reach about 25 to about 30 population doublings within about 12 days, about 50 to about 55 population doublings within about 30 days, and / or about 75 to about 80 population doublings within about 63 days from the initiation of culturing the MPSCs. In some examples, the population of MPSCs can doubling in about 22 to about 27 hours, e.g., about 25 hours. Disclosed herein, in some embodiments, are populations of lethal pluripotent stem cells (MPSCs) that express HLA-G and insulin and are pathogen-free. In some examples, the MPSCs lack expression of p53, syncytin, Ki67, heat shock protein 70 (HSP70), or any combination thereof. In some examples, the MPSCs are human cells. In some examples, the MPSCs originate from or are derived from rodents, rabbits, cows, sheep, pigs, dogs, cats, monkeys, or apes.

[0031]

[0052] In another aspect, disclosed herein is a population of lethal pluripotent stem cells (MPSCs) that express HLA-G and comprise a phenotype comprising one or more of: negative for indoleamine 2-3 deoxygenase (IDO) secretion, negative for kynurenine secretion, and positive for interleukin 2 (IL-2) secretion. In one example, the population of MPSCs comprises a phenotype that is negative for indoleamine 2-3 deoxygenase (IDO) secretion, negative for kynurenine secretion, and positive for interleukin 2 (IL-2) secretion. This phenotype is the opposite of what one skilled in the art would expect for MPSCs. Although the surface phenotypic markers make the cells appear to be mesenchymal stem cells, they behave functionally differently.

[0032]

[0053] In some cases, the MPSCs disclosed herein are pathogen-free. In some examples, the MPSCs are bacteria-free. In some examples, the MPSCs are virus-free, e.g., cytomegalovirus-free. In some examples, the MPSCs are virus-free, e.g., EBV (Epstein-Barr virus), HAdV (human adenovirus), HCMV (human cytomegalovirus), hepatitis virus (e.g., hepatitis A, hepatitis B, hepatitis C), herpes virus (e.g., HHV6 (human herpes virus 6), HHV8 (human herpes virus 8), etc.), human immunodeficiency virus (e.g., HIV1 (human immunodeficiency virus 1), HIV2 (human immunodeficiency virus 2)), human papillomavirus (HPV; e.g., HPV16, HPV18, etc.), herpes simplex virus (e.g., HS), The present invention does not include a pathogen selected from the group consisting of herpes simplex virus type 1 (HSV1), herpes simplex virus type 2 (HSV2), human T-lymphotropic viruses (e.g., HTLV1 (human T-lymphotropic virus type 1), HTLV2 (human T-lymphotropic virus type 2)), varicella virus (VZV), Corynebacterium bovis, Corynebacterium species (HAC2), hantavirus (e.g., Hantaan, Seoul, or Sin Nombre), LCMV (lymphocytic choriomeningitis virus), Mycoplasma species, Treponema pallidum, cytomegalovirus (CMV), and combinations thereof.

[0033]

[0054] In some examples, the MPSCs or populations comprising MPSCs disclosed herein further express one or more of the following proteins: b-HCG, HSP90, CDX2, FGFR1, pAKT, pCREB1, HLA-A, HLA-B, or HLA-C. In some examples, the population of MPSCs further express one or more of the following proteins: KIR2DL4, Flt3L, NKp46, TCR, ILT-4, CD49f, CD3, CD4, CD8, CD10, CD11b, CD14, CD16, CD19, CD34, CD38, CD44, CD56, CD90 / Thy-1, CD105, CD141, CD146, CD166, or CD107a. In some examples, the population of MPSCs further express one or more of the following proteins: IL-6, IL-8, MCP-1, CLXL2, PDGF-AA, VEGF, PAI-1, or IL-10. In some examples, at least a portion of the MPSCs do not express one or more of the following proteins: Ki-67, HSP70, p53, or syncytin. In some examples, the population of MPSCs (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) express one or more of the following proteins: CD44, CD90, CD105, CD146, CD166, HLA-A, HLA-B, or HLA-C. In some examples, at least a portion of the MPSCs do not express one or more of the following proteins: CD19, CD45, or HLA-DR. In some cases, more than 96%, 97%, 98%, or 99% of the MPSCs do not express one or more of the proteins CD19, CD45, or HLA-DR. In some cases, the population of MPSCs further express one or more of the proteins CD16 or CD56, or a combination thereof. In some cases, at least a portion of the MPSCs do not express CD3. In some cases, more than 96%, 97%, 98%, or 99% of the MPSCs do not express CD3. In some cases, at least 65% or at least 70% of the population of MPSCs express HLA-G. In some cases, the HLA-G includes HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7, or any combination thereof.In some examples, HLA-G includes HLA-G2, HLA-G4, HLA-G6, or HLA-G7, or any combination thereof. In some examples, HLA-G includes HLA-G6 or HLA-G7, or a combination thereof. In some examples, less than 15% (e.g., less than 10%) of the population of MPSCs express HLA-G1.

[0034]

[0055] In some instances, at least 10% of the population of MPSCs disclosed herein are monoclonal. In some instances, about 13% to about 15% of the population of MPSCs are monoclonal. In some instances, at least about 1 x 10 6 MPSCs are present in the population.In some cases, the MPSCs have a stable karyotype when measured by array-based whole genome assay.In some cases, the MPSCs do not undergo chromosomal abnormalities resulting from population doubling when measured by array-based whole genome assay.In some cases, the MPSCs do not undergo substantial chromosomal abnormalities resulting from freezing and thawing when measured by array-based whole genome assay.

[0035]

[0056] In some cases, the cells provided herein, e.g., MPSCs, are genetically modified. In some examples, the cells are genetically modified to express an exogenous gene, e.g., a transgene. As used herein, the term "transgene" and its grammatical equivalents may refer to a gene or genetic material that is transferred into an organism. For example, a transgene may be a stretch or segment of DNA containing a gene that is transferred into an organism. When a transgene is transferred into an organism, the organism is then referred to as a transgenic organism. A transgene can retain its ability to produce RNA or a polypeptide (e.g., a protein) in the transgenic organism. A transgene may be composed of different nucleic acids, e.g., RNA or DNA. A transgene may encode a genetically engineered T cell receptor, e.g., a TCR transgene. A transgene may include a TCR sequence. A transgene may include an oncogene. A transgene may include an immune oncogene. A transgene may include a recombination arm. A transgene may include a genetically engineered site. In some examples, a transgene is an oncogene. In some examples, the transgene is an immune oncogene. In some examples, the transgene is a tumor suppressor gene. In some examples, the transgene encodes a protein that directly or indirectly promotes protein degradation. In some examples, the transgene is an oncolytic gene. In some examples, the transgene can assist lymphocytes in targeting tumor cells. In some examples, the transgene is a T cell enhancer gene. In some examples, the transgene is an oncolytic virus gene. In some examples, the transgene inhibits tumor cell growth. In some examples, the transgene is an anti-cancer receptor. In some examples, the transgene is an anti-angiogenic factor. In some examples, the transgene is a cytotoxic gene.Exemplary transgenes include, but are not limited to, one or more genes encoding CD28, inducible costimulatory factor (ICOS), CD27, 4-1BB (CD137), ICOS-L, CD70, 4-1BBL, signal 3, cytokines such as IL-2, IL-7, IL-12, IL-15, IL-21, ICAM-1 (CD54), LFA-3 (CD58), HLA class I genes, B7, CD80, CD83, CD86, CD32, CD64, 4-1BBL, CD3, CD1d, CD2, membrane-bound IL-15, membrane-bound IL-17, membrane-bound IL-21, membrane-bound IL-2, truncated CD19, VEGF, caspases, chemokines, or antibodies (e.g., monoclonal antibodies) against any of the above, or any combination thereof. In some examples, the transgene encodes a protein involved in cell or tissue repair (e.g., a protein associated with DNA repair, immune response (e.g., interferons and interleukins), and structural proteins). In some examples, the transgene encodes a growth factor receptor. In some examples, the MPSCs disclosed herein comprise a transgene encoding a TCR, a B cell receptor (BCR), a chimeric antigen receptor (CAR), or any combination thereof. In some examples, the MPSCs described herein comprise a transgene encoding an oncogene receptor.

[0036]

[0057] In some cases, the composition comprising the cells disclosed herein is formulated as a pharmaceutical composition for intravenous administration to mammals, including humans.In some cases, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer.If necessary, the composition also contains a local anesthetic to alleviate pain at the injection site.When the composition is to be administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline.When the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0037]

[0058] In one aspect, disclosed herein is a composition (e.g., a pharmaceutical composition) comprising the cells disclosed herein. In some examples, the composition further comprises a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, and combinations thereof. In other examples, a colloidal dispersion system is used. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0038] How to use

[0059] In some aspects, disclosed herein are methods for producing cells, comprising contacting a population of MPSCs disclosed herein with one or more inducers. In some examples, the produced cells are ectodermal cells. In some examples, the produced cells are mesodermal cells. In some examples, the produced cells are endodermal cells. In some examples, the produced cells are pancreatic cells or pancreatic progenitor cells, and optionally, the inducer comprises bFGF (basic fibroblast growth factor), and in some examples, may further comprise 2-mercaptoethanol and nicotinamide. In one embodiment, the PPC comprises β-HCG, CDX2, HLA-G, or any combination thereof. In some examples, the PPC comprises β-HCG and CDX2; β-HCG and HLA-G; CDX2 and HLA-G; or HCG, CDX2, and HLA-G. Optionally, in some examples, the PPC further comprises PDX1, FOXA2, SOX9, or any combination thereof. In some examples, the cells produced are neural cells or neural progenitor cells, and optionally, the inducer comprises retinoic acid. In one embodiment, the NCS cells comprise RAR-β, CDX2, HLA-G, or any combination thereof. In some examples, the NCS cells comprise RAR-β and CDX2; RAR-β and HLA-G; CDX2 and HLA-G; or RAR-β, CDX2, and HLA-G. Optionally, in some examples, the PPCs further comprise N-CAD, NESTIN, SOX2, PAX6, or any combination thereof. In some examples, the cells produced are hepatocytes or hepatic progenitor cells, and optionally, the inducer comprises a fibroblast growth factor (FGF) such as FGF2, a steroid such as dexamethasone, and a cytokine such as oncostatin M, and in some examples, may further comprise a bone morphogenetic protein (BMP), e.g., BMP4, and / or hepatic growth factor. In some examples, the FGF binds to FGFR1 and is FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF8, FGF10, FGF17, FGF19, FGF20, FGF21, FGF22, or FGF23.In some examples, the steroid is a glucocorticoid steroid, such as dexamethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, or triamcinolone. In some examples, the cytokine is an interleukin 6 group cytokine, such as oncostatin M, for example, human oncostatin M, interleukin-6, interleukin-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), and cardiotrophin-like cytokine (CLC). In some examples, the cell is a natural killer cell, and the inducer comprises FGF, such as FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF8, FGF10, FGF17, FGF19, FGF20, FGF21, FGF22, or FGF23. In some instances, MPSCs can be differentiated into neural progenitor cells in 1 day using a 1-step protocol, compared to 30 days (or 3-4 weeks) and 4-step differentiation for embryonic stem cells or iPS cells. In some instances, MPSCs can be differentiated into insulin-producing pancreatic progenitor cells in 1 day using a 1-step protocol, compared to 8-15 days and 4-5 steps for embryonic stem cells or iPS cells. In some instances, MPSCs can be differentiated into hepatocyte-like cells in 6 days using a 2-step protocol, compared to 12-21 days and 3-step differentiation for embryonic stem cells or iPS cells.

[0039]

[0060] In one aspect, the cells produced include mesenchymal stromal cells, including adipocytes, chondrocytes, osteocytes, or any combination thereof. In one embodiment, the cells produced include adipocytes and chondrocytes. In another embodiment, the cells produced include adipocytes and osteocytes. In another embodiment, the cells produced include chondrocytes and osteocytes. In another embodiment, the cells produced include adipocytes, chondrocytes, and osteocytes.

[0040]

[0061] In another embodiment, the cells produced include adipocytes. The adipocytes may include leptin, HOXC8, HOXC9, Ucp1, CIDEA, PRDM16, Zic1, Lhx8, Eva1, Epsti1, Cd137, Tmem26, Tbx1, Cited1, Shox2, the amino acid transporter ASC-1, the amino acid transporter PAT2, the purinergic receptor P2RX5, ATGL, CAV1, FABP4, COX4, LMNB1, or a combination thereof. In one example, the adipocytes include white adipocytes, and the white adipocytes include leptin, HOXC8, HOXC9, or a combination thereof. In another example, the adipocytes include brown adipocytes, and the brown adipocytes include Ucp1, CIDEA, PRDM16, Zic1, Lhx8, Eva1, Epsti1, or a combination thereof. In another example, the adipocytes comprise beige adipocytes, and the beige adipocytes comprise Cd137, Tmem26, Tbx1, Cited1, Shox2, or a combination thereof. In another example, the adipocytes comprise beige adipocyte precursors, and the beige adipocyte precursors comprise CD137, TMEM26, or a combination thereof.

[0041]

[0062] In another embodiment, the cells produced comprise chondrocytes, which may comprise annexin A6, CD44, CD151, ITM2A, FAM20B, FoxC1, FoxC2, SOX5, SOX6, SOX9, aggrecan, cathepsin B, CHADL, chondroadherin, collagen II, collagen IV, CRTAC1, DSPG3, IBSP / sialoprotein II, matrilin-1, matrilin-3, matrilin-4, MIA, otraprin / OTOR, URB, or a combination thereof.

[0042]

[0063] In another embodiment, the cells produced include bone cells. The bone cells may include pre-osteoblasts, osteoblasts, embedded osteoblasts, osteoid osteoblasts, bone mineralizing osteocytes, or mature bone cells. The bone cells may include RUNX2, OCN, E11, DMP1, PHEX, MEPE, sclerostin, CapG, ORP150, or a combination thereof. In one example, the bone cells include pre-osteoblasts, and the pre-osteoblasts include RUNX2. In another example, the bone cells include pre-osteoblasts, and the pre-osteoblasts include RUNX2. In another example, the bone cells include osteoblasts, and the osteoblasts include RUNX2 and OCN. In another example, the bone cells include embedded osteoblasts, and the embedded osteoblasts include OCN, E11, DMP1, PHEX, and CapG. In another example, the bone cells include osteoid or mineralizing bone cells, and the osteoid or mineralizing bone cells include OCN, E11, DMP1, PHEX, MEPE, and CapG. In another example, the bone cells include mature bone cells, and the mature bone cells include DMP1, PHEX, MPEP, sclerostin, CapG, and ORP150.

[0043]

[0064] In some cases, the cells disclosed herein are administered to a subject intravenously, subcutaneously, transdermally, by inhalation, orally, intramuscularly, or intratumorally. In some cases, the subject is a mammal. In some cases, the subject is a primate. In some cases, the subject is a human.

[0044]

[0065] In some cases, disclosed herein are methods for killing antigen-bearing target cells, the method comprising administering the cells disclosed herein to a subject in need thereof. In some examples, the antigen-bearing target cells are cancer cells. In some examples, the cancer cells are solid tumor cells. In some examples, the cancer cells are blood cancer cells. In some examples, the cancer cells include bladder cancer cells, osteosarcoma cells, brain cancer cells, breast cancer cells, colorectal cancer cells, esophageal cancer cells, gastrointestinal cancer cells, liver cancer cells, lung cancer cells, nasal cancer cells, nasopharyngeal cancer cells, oral cancer cells, oropharyngeal cancer cells, ovarian cancer cells, prostate cancer cells, stomach cancer cells, skin cancer cells, thyroid cancer cells, or any combination thereof. In some cases, the cancer cells are derived from cancers including hematopoietic malignancies, head and neck squamous cell carcinoma, leukemia, lymphoma, myeloma, sarcoma, melanoma, bladder cancer, osteosarcoma, brain tumors, breast cancer, cervical cancer (e.g., carcinoma of the cervix), colorectal cancer, esophageal cancer, gastrointestinal cancer, liver cancer, lung cancer, nasal cavity cancer, nasopharyngeal cancer, oral cancer, oropharyngeal cancer, ovarian cancer, prostate cancer, stomach cancer, skin cancer, thyroid cancer, or any combination thereof. The cancer includes primary cancer. Alternatively, the cancer includes metastatic cancer. In some examples, the antigen-bearing target cells are pathogens. In some examples, the pathogen includes viruses, bacteria, protozoa, prions, fungi, or any combination thereof. In some examples, the method kills at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of a population of target cells bearing the antigen. In some examples, the method kills about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of a population of target cells bearing the antigen.

[0045]

[0066] In some cases, disclosed herein are methods for downregulating inflammatory pathways, comprising administering to a subject in need thereof the cells disclosed herein. In some cases, the methods include methods for treating graft rejection, infection, endotoxic shock associated with infection, arthritis, rheumatoid arthritis, psoriatic arthritis, systemic juvenile idiopathic arthritis (JIA), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), asthma, pelvic inflammatory disease, Alzheimer's disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, multiple sclerosis, ankylosing spondylitis, dermatomyositis, uveitis, Peyronie's disease, and the like. The compounds are used to treat diseases or conditions including cerebrovascular disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke, type 1 diabetes, Lyme disease, arthritis, meningoencephalitis, immune-mediated inflammatory diseases of the central and peripheral nervous system, pancreatitis, trauma from surgery, graft-versus-host disease, heart disease, bone resorption, burn patients, myocardial infarction, Paget's disease, osteoporosis, sepsis, hepatic or pulmonary fibrosis, periodontitis, or hypochlorhydria. In some examples, the method treats autoimmune diseases including type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, Sjögren's syndrome, scleroderma, polymyositis, chronic active hepatitis, mixed connective tissue disease, primary biliary cirrhosis, pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease, vitiligo, gluten-sensitive enteropathy, Graves' disease, myasthenia gravis, autoimmune neutropenia, idiopathic thrombocytopenic purpura, rheumatoid arthritis, cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's disease, bullous pemphigoid, discoid lupus, ulcerative colitis, dense deposit disease, inflammatory bowel disease, psoriasis, or any combination thereof. In some examples, the method treats type 1 diabetes. In some examples, the method improves transplant rejection.

[0046]

[0067] In another aspect, disclosed herein is a method of treating a condition in a subject, the method comprising administering to the subject a pharmaceutical composition comprising the cells described herein in an amount effective to engraft the cells into the subject (e.g., the liver of the subject). In some examples, the cells are administered in a pharmaceutically acceptable carrier. In some examples, the pharmaceutically acceptable carrier comprises saline, e.g., phosphate buffered saline, or fetal bovine serum. In some examples, the cells are administered at a concentration of about 1x10 per ml. 6 ~about 100x10 6 cells, approximately 1 x 10 per ml 6 ~about 250x10 6 cells, approximately 1x10 6 ~about 500x10 6 cells, or approximately 10x10 6 ~ approx. 40x10 6 The cells are administered in a suspension containing cells. In some examples, the cells are administered in a volume of about 1-5 ml, 1-10 ml, 1-50 ml, 1-100 ml, or 10-150 ml. In some examples, the subject is a human. In some examples, the administration includes injection, for example, intravenous injection. In some examples, the injection is administered into the hepatic vein. In some examples, the injection is administered into the hepatic artery. In some examples, the condition is a liver-related disease or disorder, for example, acute liver disease. In some examples, the condition is liver failure. In some examples, the liver-related disease or disorder comprises Alagille syndrome, alpha-1 antitrypsin deficiency, autoimmune hepatitis, benign liver tumors, biliary atresia, cirrhosis, cystic diseases of the liver, fatty liver disease including alcohol-related liver disease and non-alcoholic fatty liver disease (NAFLD), galactosemia, gallstones, Gilbert's syndrome, hemochromatosis, liver cysts, liver cancer, liver disease in pregnancy (optionally acute fatty liver of pregnancy, intrahepatic cholestasis of pregnancy, preeclampsia, or HELLP syndrome (e.g., hemolysis, elevated liver function tests, thrombocytopenia)), neonatal hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, porphyria, Reye's syndrome, sarcoidosis, toxic hepatitis, glycogen storage disease type 1, tyrosinemia, viral hepatitis, Wilson's disease, or any combination thereof.

[0047]

[0068] The administration mode of the cells disclosed herein includes, but is not limited to, systemic intravenous injection and direct injection into the intended site of action (for example, 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 bioactive agents.In some cases, administration is systemic local administration.

[0048]

[0069] In some aspects, the present specification provides compositions and methods for transplanting cells disclosed herein into subjects.In some examples, the subject is injected with cells (for example, intravenously, intramuscularly, percutaneously, endoscopic retrograde injection, or intraperitoneally).In some examples, the subject is not treated with immunosuppressants before transplantation.In some examples, the method further comprises treating the patient with an immunosuppressant, for example, FK-506, cyclosporine, or anti-glutamic acid decarboxylase 65 kilodalton isoform (GAD65) antibody.

[0049]

[0070] In some examples, the cells described herein are delivered to target site (for example, defective part of liver) by a suitable delivery system that targets cells to specific tissue.For example, cells are encapsulated in a delivery vehicle that allows cells to be released gradually at target site.Delivery vehicle can be modified so that it is specifically targeted to specific tissue.The surface of the targeted delivery system can be modified in various ways.In the case of liposome-targeted delivery system, lipid group can be contained in the lipid bilayer of liposome, so that targeting ligand can be maintained in stable association with the liposome bilayer.

[0050]

[0071] Administration of the cells described herein can be tailored to each individual, as needed, by (1) increasing or decreasing the amount of cells infused; (2) varying the number of infusions; or (3) varying the method of delivery of the cells.

[0051] Detection Method

[0072] Methods for determining the expression or presence of the above biomarkers are well known in the art and can be measured, for example, by flow cytometry, immunohistochemistry, Western blot, immunoprecipitation, magnetic bead selection, and quantification of cells expressing any of these cell surface markers. RNA expression levels of biomarkers can be measured, for example, using RT-PCR, Qt-PCR, microarray, Northern blot, or other similar techniques.

[0052]

[0073] "Detecting expression" or detecting "expression levels" is intended to determine the expression level or presence of a biomarker protein or gene in a biological sample. Thus, "detecting expression" encompasses instances where a biomarker is determined to be not expressed, not detectably expressed, expressed at low levels, expressed at normal levels, or overexpressed.

[0053]

[0074] In some examples, the expression or presence of the biomarkers described herein is determined at the nucleic acid level using, for example, immunohistochemistry techniques or nucleic acid-based techniques such as in situ hybridization and RT-PCR. In some examples, the expression or presence of one or more biomarkers is performed by means for nucleic acid amplification, nucleic acid sequencing, utilizing nucleic acid microarrays (DNA and RNA), or by means for in situ hybridization using specifically labeled probes.

[0054]

[0075] In some cases, the determination of the expression or presence of a biomarker is performed by gel electrophoresis. In some cases, the determination is performed by transfer to a membrane and hybridization with a specific probe. In some cases, the determination of the expression or presence of a biomarker is performed by a diagnostic imaging technique. In some cases, the determination of the expression or presence of a biomarker is performed by a detectable solid substrate. In some cases, the detectable solid substrate is a paramagnetic nanoparticle functionalized with an antibody.

[0055]

[0076] In some examples, the expression or presence of a biomarker is at the RNA (e.g., mRNA) level. In some examples, techniques for detecting RNA (e.g., mRNA) levels include, but are not limited to, Southern or Northern analysis, polymerase chain reaction analysis, and probe arrays.

[0056]

[0077] One method for detecting mRNA levels involves contacting isolated mRNA with a nucleic acid molecule (probe) that hybridizes to the mRNA encoded by the gene to be detected. The nucleic acid probe comprises, for example, a full-length cDNA or a portion thereof, such as an oligonucleotide that is at least 7, 15, 30, 50, 100, 250, or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to the mRNA or genomic DNA encoding the biomarker described herein. Hybridization of the mRNA with the probe indicates that the biomarker or other target protein of interest is expressed.

[0057]

[0078] In some examples, for example, by running isolated mRNA on agarose gel, and transferring mRNA from gel to membrane, for example, nitrocellulose, mRNA is immobilized on solid surface and contacted with probe.In some examples, probe is immobilized on solid surface, and for example, in gene chip array, mRNA is contacted with probe.Those skilled in the art can easily adapt known mRNA detection methods to be used for detecting the level of mRNA that codes for biomarker or other proteins of interest.

[0058]

[0079] Alternative methods for determining the level of target mRNA in a sample include, for example, RT-PCR, ligase chain reaction, self-sustained sequence replication, transcription amplification system, Q-beta replicase, rolling circle replication, or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when such molecules are present in very small numbers. In some cases, biomarker expression is evaluated by quantitative fluorescent T-PCR (e.g., TAQMAN® system).

[0059]

[0080] Expression levels of the RNA of interest are monitored using membrane blots (e.g., those used in hybridization analyses such as Northerns, dots, etc.), or microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acid). Detection of expression also includes using nucleic acid probes in solution.

[0060]

[0081] In some cases, microarrays are used to determine the expression or presence of one or more biomarkers.Nucleic acid microarrays provide a method for simultaneously measuring the expression levels of a large number of genes.Each array consists of a reproducible pattern of capture probes attached to a solid support.Labeled RNA or DNA is hybridized to complementary probes on the array, and then detected by laser scanning, and the hybridization intensity for each probe on the array is determined and converted into a quantitative value that represents relative gene expression levels.High-density oligonucleotide arrays are particularly useful for determining the gene expression profile of a large number of RNAs in a sample.

[0061]

[0082] In some cases, the array is fabricated on a surface of virtually any shape, or even on multiple surfaces. In some cases, the array is a planar array surface. In some cases, the array comprises peptides or nucleic acids on beads, gels, polymer surfaces, fibers such as fiber optics, glass, or any other suitable substrate. In some cases, the array is packaged in a manner that allows for diagnostic or other operation of the comprehensive device.

[0062]

[0083] In some examples, the expression or presence of the biomarkers described herein is determined at the protein level, for example, using antibodies against specific biomarker proteins. These antibodies are used in various methods, such as Western blot, ELISA, multiplexing techniques, immunoprecipitation, or immunohistochemistry techniques. In some examples, the detection of the biomarkers is achieved by ELISA. In some examples, the detection of the biomarkers is achieved by electrochemiluminescence (ECL).

[0063]

[0084] Any means for specifically identifying and quantifying a biomarker in a biological sample is contemplated. Thus, in some examples, the expression level of a biomarker protein of interest in a biological sample is detected by a binding protein that can specifically interact with the biomarker protein or its biologically active variant. In some examples, a labeled antibody, its binding portion, or other binding partner is used. The word "label" as used herein refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody to produce a "labeled" antibody. In some examples, the label is detectable itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzyme label, catalyzes a chemical change in a substrate compound or composition that is detectable.

[0064]

[0085] Antibodies for the detection of biomarker proteins can be either monoclonal or polyclonal in origin, or synthetically or recombinantly produced. The amount of complexed protein, e.g., the amount of biomarker protein associated with a binding protein, e.g., an antibody that specifically binds to the biomarker protein, is determined using standard protein detection methods known to those skilled in the art. Detailed reviews of the design, theory, and protocols of immunological assays can be found in several textbooks in the field.

[0065]

[0086] The choice of marker used to label the antibody will vary depending on the application. However, the choice of marker can be easily determined by one skilled in the art. These labeled antibodies are used in immunoassays and histological applications to detect the presence of any biomarker or protein of interest. Labeled antibodies can be either polyclonal or monoclonal. Furthermore, antibodies for use in detecting proteins of interest are labeled with radioactive atoms, enzymes, chromogenic or fluorescent moieties, or colorimetric tags as described elsewhere herein. The choice of tagging label will also depend on the desired detection limit. Enzyme assays (e.g., ELISA) typically allow for the detection of colored products formed by the interaction of an 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, 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. Chromogenic moieties include, but are not limited to, fluorescein and rhodamine. Antibodies are conjugated to these labels by methods known in the art. For example, enzymes and chromogenic moieties are conjugated to antibodies using coupling agents such as dialdehydes, carbodiimides, and dimaleimides. Alternatively, conjugation occurs via a ligand-receptor pair. Examples of suitable ligand-receptor pairs include, but are not limited to, biotin-avidin or biotin-streptavidin, and antibody-antigen.

[0066]

[0087] In some examples, the expression or presence of one or more biomarkers or other proteins of interest in a biological sample is determined by radioimmunoassay or enzyme-linked immunoassay (ELISA), competitive binding enzyme-linked immunoassay, dot blot, Western blot, chromatography such as high performance liquid chromatography (HPLC), or other assays known in the art. Thus, detection assays include steps such as, but not limited to, immunoblotting, immunodiffusion, immunoelectrophoresis, and immunoprecipitation.

[0067] How to obtain cells

[0088] In some cases, extraembryonic mammalian stem cells (e.g., trophoblast stem cells) may be the source cells for generating the lethal pluripotent stem cells (MPSCs) disclosed herein. In some examples, mammalian stem cells are isolated from amniotic fluid, amniotic membrane, Wharton's jelly, chorionic villi, or ectopic pregnancy in a manner that does not disturb or destroy the embryo.

[0068]

[0089] In some examples, MPSCs are obtained in a culture medium that does not contain antibiotics, such as penicillin, streptomycin, or any combination thereof. In some examples, the culture medium for obtaining mammalian stem cells does not contain retinoic acid. In some examples, the culture medium for obtaining and / or passaging mammalian stem cells does not contain mercaptoethanol, nicotinamide, or a combination thereof. In some examples, the culture medium for obtaining and / or passaging mammalian stem cells does not contain dexamethasone, recombinant human oncostatin M, BMP4, HGF, or any combination thereof. In some examples, the culture medium for obtaining and / or passaging mammalian stem cells is xeno-free, e.g., does not contain animal components. In some examples, the culture medium for obtaining and / or passaging mammalian stem cells is free of human-derived components and animal-derived components, e.g., a chemically defined medium. In some examples, the culture medium for obtaining and / or passaging mammalian stem cells does not contain serum. In some examples, the culture medium for obtaining and / or passaging mammalian stem cells does not contain fetal bovine serum.

[0069]

[0090] In some cases, the present disclosure provides a method of expanding a population of MPSCs disclosed herein, comprising culturing the MPSCs at about 1,000 to about 5,000 cells / cm in culture medium. 2 and culturing the cells.

[0070]

[0091] In some embodiments, a method for expanding a population of lethal pluripotent stem cells (MPSCs) comprises culturing MPSCs at a density of about 1,000 to about 5,000 cells / cm in a culture medium. 2 and culturing the cells, wherein the population of MPSCs expresses HLA-G and insulin. In some examples, the culture medium is animal component-free. In some examples, the culture medium is serum-free, e.g., fetal bovine serum. In some examples, the cells are cultured for about 3 days. In some examples, the cells are cultured for about 4 days. In some examples, the MPSCs are cultured at a density of about 2,000 to about 4,000 cells / cm. 2 The seeds are sown at a density of

[0071]

[0092] In some examples, mammalian stem cells can be isolated from amniocentesis biopsies or amniotic fluid. In one example, amniocentesis can be a procedure used to obtain a small sample of amniotic fluid surrounding a pregnant fetus. In one example, amniocentesis can be offered to women between the 15th and 20th weeks of pregnancy who are at high risk for chromosomal abnormalities, such as women over 35 years of age at delivery, or women who have had abnormal maternal serum (blood) screening test results indicating an increased risk of chromosomal abnormalities or neural tube defects. In one example, a needle, such as a long, thin, hollow needle, can be used with ultrasound guidance through the abdomen into the uterus and amniotic sac. A predetermined amount of amniotic fluid, such as 28.35 g (1 ounce), can be drawn into a syringe.

[0072]

[0093] In some examples, the mammalian stem cells herein can be obtained from a blastomere biopsy during preimplantation genetic diagnosis (PGD), for example, in conjunction with a reproductive therapy such as in vitro fertilization (IVF). In one example, the cells herein can be produced by a method for blastocyst biopsy, in which the remaining blastocyst is implanted, resulting in pregnancy and later in a live birth, for example, by removing the zona pellucida from the blastocyst and then biopsying the blastocyst.

[0073]

[0094] In some examples, the mammalian stem cells herein can be obtained from prenatal chorionic villus sampling (CVS). In one example, CVS can be a prenatal test that involves collecting tissue samples from the placenta to test for chromosomal abnormalities and certain other genetic problems. In one example, CVS can be performed between the 10th and 12th weeks of pregnancy. In one example, the CVS procedure is transcervical, for example, a catheter is inserted through the cervix into the placenta to obtain a tissue sample. In one example, the CVS procedure is transabdominal, for example, a needle is inserted through the abdomen and uterus into the placenta to obtain a tissue sample.

[0074]

[0095] In some instances, the mammalian stem cells herein are obtained from first trimester chorionic villus sampling (e.g., from the eighth trimester of pregnancy). +3 ~12 +0 Villous tissue can be isolated from term placentas derived from 14-week (or 18-week) or cesarean section deliveries. The chorionic villus tissue can be separated from the amniotic membrane, minced, and / or enzymatically digested (e.g., with about 3 ml of TRYPLE® Select Enzyme, for about 15 min). Cells can then be centrifuged (e.g., at about 150 x g + / - 10%, for about 5 min), counted, and / or replated (e.g., in 1 cm) in culture medium (e.g., α-MEM with STEMULATE™ Human Platelet Lysate Cell Culture Media Supplement or MESENCULT™-ACF Plus Culture Kit). 2(Approximately 100 cells per culture). In one example, the isolated cells may be plastic-adherent. In one example, the cells may be used at about 4 to about 8 passages.

[0075]

[0096] In some cases, chorionic villi can be obtained from the fallopian tubes of unruptured preimplantation embryos in women with ectopic pregnancies (e.g., gestational ages: about 5 to about 8 weeks, about 6 to about 8 weeks, or about 4 to about 8 weeks after fertilization). A small amount of chorionic villi can be thoroughly minced in a suitable medium (e.g., serum-free α-MEM) and identified under a microscope. Then, trypsinization (e.g., using about 3 ml of TRYPLE® Select Enzyme) can be performed for a period of time (e.g., about 15 minutes). The reaction can be stopped by adding medium (e.g., α-MEM containing STEMULATE™ Human Platelet Lysate Cell Culture Media Supplement or MESENCULT™-ACF Plus Culture Kit). Adherent cells can be obtained and cultured under suitable conditions (e.g., in conditioned α-MEM containing STEMULATE™ Human Platelet Lysate Cell Culture Media Supplement or MESENCULT™-ACF Plus Culture Kit at 37°C in 5% CO2).

[0076] Kits / Products

[0097] Disclosed herein are kits and products 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, etc., each of which contains one of the separate components used in the methods described herein.Suitable containers include, for example, bottles, vials, syringes, test tubes, etc.In some cases, the container is made of various materials, such as glass or plastic.

[0077]

[0098] The products provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for the selected formulation and intended mode of use.

[0078]

[0099] For example, a container optionally contains cells, in a composition disclosed herein. Such kits optionally include an identifying description or label or instructions for their use in the methods described herein.

[0079]

[0100] Kits typically include a label listing the contents and / or instructions for use, as well as a package insert containing the instructions. A set of instructions will also typically be included.

[0080]

[0101] In some cases, label is on container or connected to container.In some cases, label is on container when the letter, number or other symbol that forms label is attached, molded or etched on container itself; label is connected to container when it is in the receptacle or carrier that also holds container, for example, as package insert.In some cases, label is used to indicate that content is used for specific therapeutic application.Label also indicates the instruction for using content, such as in the method described herein.

[0081] Agents, compositions, and uses thereof

[0102] Disclosed herein are compositions (e.g., in vitro compositions, pharmaceutical compositions, etc.) and agents containing cells produced by the methods described herein. Compositions or agents having the desired purity, along with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington, The Science and Practice of Pharmacy, 20th ed., Mack Publishing (2000)), can be prepared in the form of lyophilized formulations or aqueous solutions. As used herein, "pharmaceutically acceptable carriers" or "pharmaceutically acceptable excipients" include any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. A preferred diluent for aerosol or parenteral administration is phosphate-buffered saline solution (PBS) or normal (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., A. Gennaro (ed.), Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th ed., Mack Publishing, 2000).

[0082]

[0103] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers, such as phosphates, citrates, and other organic acids; salts such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as , serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0083]

[0104] The use of the cells produced herein described for in vitro culture or assay is described herein.For example, cells can be used in immunofluorescence or fluorescence-activated cell sorting (FACS) assay.In some cases, the cells produced can be neural stem cells (NCS), pancreatic progenitor cells (PPC), ectodermal cells, mesodermal cells, endodermal cells, hepatocytes or hepatic progenitor cells.

[0084]

[0105] Described herein is the use of neural stem cells (NSCs) produced by the methods described herein to test new drugs for safety and efficacy. For example, a test agent can be contacted with a culture containing the produced cells and the effect determined. If the test agent is toxic to the cells, the proliferation of the culture may decrease and / or die. If the test agent is effective, the proliferation of the culture may increase. In the case of neural stem cells, the test agent may induce the production of motor neurons. Described herein is the use of neural stem cells (NSCs) produced by the methods described herein to produce motor neurons in vitro or in vivo. Described herein is the use of neural stem cells (NSCs) produced by the methods described herein in the manufacture of a medicament for the treatment of motor neuron disease. Described herein is the use of neural stem cells (NSCs) produced by the methods described herein in the manufacture of a medicament for the treatment of spinal cord injury. Described herein is the use of neural stem cells (NSCs) produced by the methods described herein to produce artificial tissues or organs in vitro.

[0085]

[0106] Described herein is the use of pancreatic progenitor cells (PPCs) produced by the methods described herein to test new drugs for safety and efficacy. For example, a test agent can be contacted with a culture containing the produced cells and the effect determined. If the test agent is toxic to the cells, the growth of the culture may decrease and / or die. If the test agent is effective, the growth of the culture may increase. In the case of PPCs, the test agent may induce the production of endocrine and / or exocrine cells. Described herein is the use of PPCs produced by the methods described herein to produce endocrine and / or exocrine cells in vitro or in vivo. Described herein is the use of PPCs produced by the methods described herein in the manufacture of a medicament for the treatment of a disease or disorder caused by pancreatic trauma. Described herein is the use of PPCs produced by the methods described herein in the manufacture of a medicament for the treatment of pancreatic trauma. Described herein is the use of PPCs produced by the methods described herein in the manufacture of a medicament for the treatment of pancreatic trauma. Described herein is the use of PPCs produced by the methods described herein to produce artificial tissues or organs (e.g., pancreas) in vitro. [Example]

[0086]

[0107] The present application can be better understood by reference to the following non-limiting examples, which are provided as illustrative embodiments of the present application. The following examples are presented to more fully illustrate the embodiments, but should not be construed as in any way limiting the broad scope of the present application.

[0087] Example 1: MPSCs reached 89 population doublings

[0108] Extraembryonic stem cells (e.g., trophoblast stem cells) were derived from human donors as source cells. As shown in Table 1 below, several non-limiting culture media were tested to culture and expand the cells into lethal pluripotent stem cells (MPSCs).

[0088] [Table 1]

[0089]

[0110] Seeding density: 10,000 cells / cm 2 to 2,000-4,000 cells / cm 2 Lowering the cell density to 3000-5000 cells / cm improved the population doubling rate of MPSCs. 2 Three-day subcultures of cells seeded at a density of 4000 / 3000 cells / cm produce similar numbers of PDs. 2 Alternating 3 day / 4 day subcultures of cells seeded at 4000 cells / cm for earlier passages. 2 The culture environment can be 21% O2 / 5% CO2 or 2% O2 / 5% CO2 / 93% N2.

[0090]

[0111] Some of the growth results over 90 days, i.e., 30 passages of 3-day subculture, are shown in Figure 1, which is a line graph showing the growth curve of 3-day subculture of MPSCs as measured by population doublings (PD) over a 90-day time frame. MPSCs reached a maximum of 25 PDs by approximately 12 days, 50 PDs by approximately 30 days, 75 PDs by approximately 63 days, and 89 PDs by approximately 90 days.

[0091]

[0112] MPSCs have an extended population doubling capacity when cultured in xeno-free medium, with a doubling time of approximately 27 hours, resulting in approximately 70-80 doublings. On average, it takes approximately 27 hours for cells to double in population, which can be calculated using the formula T = td / log2[(2-y) / (1-y)], where T is the duration of the cell cycle, td is the average time it takes for cells to double in number, and y is the percentage of cells in the G0 phase.

[0092]

[0113] This extended doubling capacity makes MPSCs an ideal cell population for industrial-scale expansion, eliminating the need for repeated isolation from donors or biological sources. The large batch size capability of MPSCs will generate sufficient MPSCs from a single source to streamline therapeutic development and manufacturing by reducing the product variability associated with multiple cell banks and the costs of manufacturing and releasing equivalent products from different donors, accelerating the realization of stem cell-based therapeutics in the clinic.

[0093] Example 2: MPSCs do not have chromosomal abnormalities

[0114] Figures 2A-2D show the KARYOSTAT™ whole genome view of four different MPSC samples derived from different population doublings. The KARYOSTAT™ assay allows for digital imaging of chromosomal abnormalities. The size of structural abnormalities that can be detected is greater than approximately 2 Mb (megabases) for chromosomal gains and greater than approximately 1 Mb for chromosomal losses. Genomic DNA was purified from cells, and the genomic DNA was added to the GENECHIP® for KARYOTATE™. The GENECHIP® can determine chromosomal copy number variants. Figures 2A-2D show the whole genome view, showing all somatic and sex chromosomes in one pane. Figure 2A is an MPSC sample derived from 16.5 population doublings, Figure 2B is an MPSC sample derived from 44.5 population doublings, Figure 2C is an MPSC sample derived from 62.6 population doublings, and Figure 2D is an MPSC sample derived from 71.5 population doublings. The smoothed signal plot (right y-axis) is a smoothed log2 ratio depicting the signal intensity of the probes on the microarray. A value of 2 may represent a normal copy number status (CN=2). A value of 3 may represent a chromosomal gain (CN=3). A value of 1 may represent a chromosomal loss (CN=1). The gray signals represent the raw signal for each individual chromosomal probe, while the black signals represent the normalized probe signal used to identify copy number and abnormalities (if any). No observable chromosomal abnormalities were seen in Figure 2A-D. MPSC cells can undergo multiple population doublings without exhibiting chromosomal abnormalities. For example, monoclonal populations can be expanded and then frozen for future use. Once cells are propagated from monoclonal cryopreserved cultures, they can be expanded without substantial chromosomal abnormalities. The chromosomal stability of MPSCs offers another advantage over human embryonic stem cells and iPSCs, which often exhibit genetic abnormalities or mutations associated with immortality.

[0094] Example 3: Characterization of MPSCs by expressing specific molecular biomarkers

[0115] MPSCs express HLA-G, a marker of immune privilege. Unlike adult or postnatal human mesenchymal stromal cells, the MPSCs herein express human leukocyte antigen-G (HLA-G), a major histocompatibility complex class I antigen found exclusively in the placenta, which binds to HLA-G receptors on leukocytes and suppresses immune function through several mechanisms, including inducing apoptosis in activated T cells, modulating the activity of natural killer (NK) cells and dendritic cells, and inhibiting T cell proliferation. Referring to Figure 3, this figure shows MPSCs stained with the primary antibody 4H84. For Figure 3, MPSCs were harvested from culture in MESENCULT® ACF Plus Medium. Cells were resuspended in flow cytometry wash buffer (Gibco DPBS, substantially free of calcium chloride or magnesium chloride, approximately 2% fetal bovine serum, and approximately 0.1% sodium azide) and collected at approximately 0.25–0.5 × 10 per sample. 6 Cells were aliquoted into flow cytometry tubes and centrifuged. Cells were fixed with 4% paraformaldehyde solution at room temperature for approximately 15 minutes. In some cases, after fixation, the cells were permeabilized with approximately 500 μl (microliters) of cold Perm Buffer III (BD Biosciences) and then incubated on ice. Permeabilization allowed for staining of intracellular material. After incubation, the cells were washed with flow cytometry wash buffer, centrifuged, and resuspended in flow cytometry staining buffer (R&D Systems). Primary antibody staining occurred when a dilution of HLA-G primary antibody (e.g., 4H84 antibody) was added to the cells and incubated at room temperature. The cells were washed several times with flow cytometry wash buffer. After the primary antibody bound to the cells, the secondary antibody was added. The secondary antibody procedure was performed in a dark room. The secondary antibody was added in flow cytometry staining buffer at a dilution of approximately 1:2000. The cells were resuspended in approximately 100 μl of diluted secondary antibody solution and incubated for approximately 30 minutes. The cells were washed several times in flow cytometry wash buffer. After washing the cells, the cells were resuspended in flow cytometry staining buffer and diluted to approximately 0.5×10 6The cells were resuspended to a concentration of 1000 cells / ml and then sampled by flow cytometry. The primary antibody MEM-G / 11 recognizes the membrane-bound HLA-G1 isoform. The primary antibody 4H84 recognizes the alpha domain of the seven isoforms of HLA-G. Figure 3 shows MPSCs that were permeabilized and stained with the primary antibody HLA-G 4H84. The staining indicates the presence of HLA-G isoforms in or on MPSCs. At a primary antibody dilution of 1:50, approximately 76% of the cells were positive compared to the isotype control. Figure 4 shows cells stained with the primary antibody 4H84 (lower panel) and the primary isotype control antibody mouse IgG1 (upper panel). The cells showed limited staining with the IgG1 antibody, with 99.64% of events staining with the 4H84 antibody, indicating that this antibody is specific for MPSCs. The expression of HLA-G in MPSCs shown in Figure 3 allows the cells to have access to immune-privileged sites, such as the fetus.

[0095]

[0116] The MPSCs herein exhibited the phenotype and morphology of human MSCs by expressing characteristic markers as measured by fluorescence-activated cell sorting (FACS), see Table 2 below.

[0096] [Table 2]

[0097]

[0118] MPSCs, as described herein, may offer an alternative solution to mesenchymal stromal cells (MSCs). Human MSCs exhibit immunosuppressive effects, trilineage differentiation in vitro, and have been safely delivered to patients for a variety of indications, including approval for treating niche indications such as autoimmune perianal fistulas and graft-versus-host disease. However, widespread adoption of MSC-based therapies has been hindered by the inability to manufacture large batches of MSCs due to a population doubling limit of approximately 30–40 doublings before reaching cellular senescence.

[0098]

[0119] The MPSCs herein exhibited a natural killer cell phenotype as determined by FACS, see Table 3 below.

[0099] [Table 3]

[0100]

[0121] Immunocytochemically, MPSCs expressed various cellular biomarkers, including β-hCG, HLA-G, heat shock protein 90 (HSP90), and CDX2 (Figure 5A). However, MPSCs did not express the proliferation marker Ki-67, HSP70, tumor suppressor p53, or the cell-cell fusion protein syncytin (Figure 5B), supporting the notion that MPSCs are the primary trophoblast for TE differentiation. Specifically, MPSCs expressed HLA-A, -B, and -C as well as surface and intracellular HLA-G by flow cytometry analysis (FACS) using various antibodies (Figure 5C; Figure 5D). However, they did not express HLA-DR.

[0101]

[0122] Figure 5D shows representative FACS images of HLA-G isoforms in MPSCs. Using Ab 4H84, all seven isoforms were barely detected on the cell surface (top left column), whereas 68.7% of all seven HLA-G isoforms were detected in permeabilized MPSCs (bottom left column). Ab MEM-G / 11 detected only a small amount of HLA-G G1 on the cell surface (top middle column), whereas 8.1% of HLA-G G1 was detected (top middle column). Similarly, Ab MEM-G9 detected HLA-G G1, G3, and G5 on the cell surface (top right column), whereas none of HLA-G was detected.

[0102]

[0123] Human MPSCs express immune cell-associated biomarkers. Cells were characterized using immunocytochemistry and FACS analysis. MPSCs express NK cell cluster of differentiation (CD) 56, CD16, and CD16.dim They were shown to express a variety of biomarkers associated with immune cells, including the inhibitory receptors KIR2DL4, CD11b, and the activating receptors NKp46 and CD10 (Figure 6A); TCR, CD49f, ILT-4, CD3, CD4, CD8, CD44, CD90 / Thy-1, CD44, and CD166 on T cells (Figure 6B); CD19 and CD141 on dendritic cells (Figures 6C and 6D); CD14 on macrophages (Figure 6E); Flt3L (Figure 6F) and CD34 on hematopoietic stem cells (Figure 6G); and CD38 on lymphocytes (Figure 6G). The expression of these biomarkers in MPSCs was subsequently analyzed using eight independent cell lines, which showed similar patterns of NK and T cell biomarkers (CD16+CD56). + CD107(+) cells and CD107(+) cells showed the highest expression in MPSCs. FACS analysis showed that NK cell and T cell biomarkers accounted for the majority of immune cells in MPSCs, but CD107(+)CD(16+56)(+) cells and CD8(+)CD(16+56)(+) cells accounted for the majority of cell populations in MPSCs.

[0103] Example 4: A significant fraction of MPSCs are monoclonal

[0124] MPSC single clones were obtained from the MPSC cultures. The MPSCs were grown on an inverted microscope and the cell types were recorded. The old medium was removed, and the cells were washed with sterile PBS. TRYPLE® solution was added to the MPSC cultures. The cells were incubated at 37°C and 5% CO2 for approximately 6 minutes. After incubation, the cells were detached. Culture medium was added to the cells to stop the TRYPLE® reaction. The cells were harvested and the cell number was calculated using a cell counter. Approximately 200 cells were removed and placed in a centrifuge tube. The medium was replenished, and the cells were split into 96-well plates using a multichannel pipette. The 96-well plates were grown at 37°C and 5% CO2 and incubated for approximately 14 days. During this process, the medium was changed every 2-3 days. The old medium was washed away, and TRYPLE® solution was added to the cells. After a short incubation, culture medium was added to stop the TRYPLE® reaction, and the cells were transferred to 6-well plates and grown at 37°C and 5% CO2. The culture medium was changed every 2-3 days until the cells were subcultured in 100 mm dishes and grown at 37°C and 5% CO2. The medium was changed every 2-3 days. When the monoclonal cells reached 80-95% thickness, they were frozen. From the donor ectopic tissue, cells were expanded either as wild-type passages mixed with cells or as monoclonal expansions. The monoclonal expansion provided multiple doses. For example, for every 1 million cells derived from the donor ectopic tissue, approximately 125,000 monoclonals could be cultured. Each monoclonal cell was 7x10 28 At 100 million cells per dose, each monoclone may have 7x10 20 The total potential from each ectopic tissue collected is: 125,000 x 7 x 10 20 Dose=8.8x10 25 Dosage: Each MPSC monoclone is capable of supporting a complete product cycle.

[0104]

[0125] Every vial of 1M cells can potentially yield approximately 130,000 single clones. See Table 4 below.

[0105] [Table 4]

[0106] Example 5: MPCS are pathogen-free

[0127] Regardless of whether the source cells were pathogen-infected or pathogen-free, the human MPCS obtained herein are pathogen-free. Nine cell lines of MPSCs were tested. PCR assays were performed to detect Corynebacterium bovis, Corynebacterium species (HAC2), EBV, HAdV, Hantaan hantavirus, HCMV, hepatitis A, hepatitis B, hepatitis C, HHV6, HHV8, HIV1, HIV2, HPV16, HPV18, HSV1, HSV2, HTLV1, HTLV2, LCMV, Mycoplasma species, Seoul hantavirus, Sin Nombre hantavirus, Treponema pallidum, and VZV. All cell lines were found to be negative for all 25 pathogens in the h-IMPACT I panel.

[0107] Example 6: Optimization of HLA-G FACS staining (surface vs. intracellular)

[0128] MPSC wild-type (WT) cell line 1 (MPSC1) was cultured in nutrient medium plus cell attachment substrate and passaged 12 times at 37°C and 5% CO2. Fixed and permeabilized cells were prepared for cell surface and intracellular staining. The staining conditions are shown in Table 5 below.

[0108] [Table 5]

[0109]

[0130] The primary antibody histogram results are shown in Table 6 below for dilutions of 1:25, 1:50, 1:100, and 1:200.

[0110] [Table 6]

[0111]

[0132] The primary antibody dot plot results (A-FL1 vs. SSC) are shown below in Table 7 for dilutions of 1:25, 1:50, 1:100, and 1:200.

[0112] [Table 7]

[0113]

[0134] The primary antibody dot plot results (FL1 vs. FL2) are shown below in Table 8 for dilutions of 1:25, 1:50, 1:100, and 1:200.

[0114] [Table 8]

[0115]

[0136] MPSC1 were cultured in nutrient medium plus cell attachment substrate and passaged 7 times (19.8 population doublings) at 37°C, 5% CO2. Fixed and permeabilized cells were prepared for cell surface and intracellular staining using HLA-G 4H84 and HLA-G MEM-G / 11 antibodies. Cells fixed after surface staining were analyzed using HLA-G MEM-G / 11 antibody. The results are shown in Table 9 below.

[0116] [Table 9]

[0117]

[0138] HLA-G staining of MPSC1 is shown in Table 10, along with the results of the FLA-G 4H86 dot plot (FL1 vs. SSC) compared to the control.

[0118] [Table 10]

[0119] Example 7: Generation of a developmental cell bank and its evaluation

[0140] Four extraembryonic stem cell lines (e.g., human trophoblast stem cells) were used as source cells: lethal pluripotent stem cell line 1 (MPSC1), MPSC2, MPSC3, and MPSC4. Cell banks were developed by culturing the cell lines separately in nutrient medium (e.g., MESENCULT™ + cell attachment substrate). Subcultures of the cells were performed at 3000-4000 cells / cm. 2 The cells were seeded at a density of 10, 35, 55, and 70 PD and cultured for 3 or 4 days. The study endpoints were 10, 35, 55, and 70 PD. The phenotype was then assessed by FACS characterization, MPSC / NK markers, and HLA-G. The functionality of the resulting cells was further evaluated.

[0120] Example 8: Experiments on MPSC / NK production, phenotype and function Lethal pluripotent stem cell line 1 (MPSC1)

[0141] Lethal pluripotent stem cell line 1 (MPSC1) was used as the source cell. Cultures were grown at 3000 / 4000 cells / cm. 2 The cells were seeded at 1000 x g / ml, cultured in nutrient medium (e.g., MESENCULT+ cell attachment substrate), and expanded. Two cell banks (CB) were frozen: CB2:31.3PD and CD3:53.1PD. Phenotypic and functional assays were performed.

[0121]

[0142] Characterization of C1 was as follows: MPSC1 was evaluated for function (3-lineage differentiation and secretome analysis).

[0122]

[0143] Characterization of C2 was as follows: MPSC1 P13, 40.3PD were assessed for phenotyping and for function (tri-lineage differentiation and secretome analysis) by FACS for MSC / NK markers and HLA-G.

[0123] Lethal pluripotent stem cell line 2

[0144] Lethal pluripotent stem cell line 2 (MPSC2) was used as the source cell. Cultures were maintained at 3000 / 4000 cells / cm. 2 The cells were seeded at 100°C, cultured in nutrient medium (e.g., MESENCULT™ + cell attachment substrate), and expanded. Three cell banks (CB) were frozen: CB1: 8.1 PD; CB2: 29.3 PD; and CB3: 47.4 PD. Phenotypic and functional assays were performed. The average doubling time (P4–P6) was 26.9 h.

[0124]

[0145] Characterization of C1 was as follows: MPSC2 passage 5 (P5), 11.2 passage doubling (PD), assessed for phenotyping and for function (tri-lineage differentiation and secretome analysis) by FACS for MSC / NK markers and HLA-G.

[0125]

[0146] Characterization of C2 was as follows: MPSC2 P16, 36.9PD were assessed for phenotyping and for function (tri-lineage differentiation and secretome analysis) by FACS for MSC / NK markers and HLA-G.

[0126] Lethal pluripotent stem cell line 3

[0147] Lethal pluripotent stem cell line 3 (MPSC3) was used as the source cell. Cultures were maintained at 3000 / 4000 cells / cm. 2 The cells were seeded at 100°C, cultured in nutrient medium (e.g., MESENCULT™ + cell attachment substrate), and expanded. Three cell banks (CB) were frozen: CB1: 8.1 PD; CB2: 25.5 PD; and CB3: 37.3 PD. Phenotypic and functional assays were performed. The average doubling time (P4–P6) was 32.7 h.

[0127]

[0148] Characterization of C1 was as follows: MPSCs 3 passage 5 (P5), 10.2 passage doubling (PD) were assessed for phenotyping and for function (tri-lineage differentiation and secretome analysis) by FACS for MSC / NK markers and HLA-G.

[0128]

[0149] Characterization of C2 was as follows: MPSC3 P18, 33.3PD was assessed for phenotyping and for function (tri-lineage differentiation and secretome analysis) by FACS for MPSC / NK markers and HLA-G.

[0129] Lethal pluripotent stem cell line 4

[0150] A lethal pluripotent stem cell line (MPSC4) was used as the source cell. Cultures were maintained at 3000 / 4000 cells / cm. 2 Cells were seeded at 1000 x g for 10 min, cultured in nutrient medium (e.g., MESENCULT™ + cell attachment substrate), and expanded for a total of approximately 6.5 PDs. Cells were either frozen or characterized by FACS analysis for mesenchymal stem cell (MSC) / natural killer NK markers and HLA-G. The average doubling time from P4 to P5 was approximately 43.7 h.

[0130] Growth curve

[0151] Growth curves of the four MPSC cell lines are shown in Figure 7. MPSC1 for this experiment was subjected to two freeze / thaw cycles, likely reducing the maximum number of PDs in culture.

[0131]

[0152] Results from a second experiment of growth curves and collection of cell banks (CB) 1, 2, 3, and 4 for MPSC1, MPSC2, MPSC3, and MPSC4 were obtained. MPSC4 doubling was slow, and experiments for that cell line were discontinued.

[0132] [Table 11]

[0133]

[0153] Phenotypic characterization was performed for each cell line at the following passage doubling (PD) times:

[0134] [Table 12]

[0135]

[0154] Cell surface antigen expression of MPSC / NK markers and HLA-G was assessed by FACS, as was adherence to plastic under standard culture conditions and the ability to differentiate multipotently into osteoblasts, adipocytes, and chondroblasts.

[0136] [Table 13]

[0137]

[0155] FACS characterization of the MPSC marker, HLA-G, for C1 and C2 is as follows.

[0138] [Table 14]

[0139] [Table 15]

[0140] [Table 16]

[0141] [Table 17]

[0142]

[0156] Functional: Trilineage differentiation was assessed using the following differentiation protocol:

[0143] [Table 18]

[0144]

[0157] MPSC3 did not differentiate into adipocytes even after 7 weeks in adipocyte differentiation medium. In contrast, MPSC1 and MPSC2 showed the ability to differentiate into adipocytes at 5.5 and 7 weeks, respectively (data not shown).

[0145]

[0158] At 3 weeks, MPSC1, MPSC2, and MPSC3 C1 cells demonstrated bone formation using Alizarin Red staining compared to controls (data not shown).

[0146] Example 9: Hypoxia-induced differentiation

[0159] Extraembryonic stem cell lines (e.g., human trophoblast stem cells) are cultured until confluence is reached (e.g., approximately 3000 cells / cm). 2 ~about 9000 cells / cm 2 , or approximately 6000 cells / cm 2 ), and grown in nutrient medium (e.g., MESENCULT™ + cell attachment substrate). Cells were washed, and the medium was replaced without supplements. Hypoxia was induced in a chamber (e.g., culture in a 2% O2 gas mixture for approximately 24 hours). The medium was collected and frozen until use. Media from all three cell lines was tested using the QUANTIBODY™ Human Kiloplex Array (RAYBIOTECH™ Life, Inc.) to quantitatively analyze 1,000 proteins. The experiment was repeated for MPSC1 and MPSC2. Briefly, samples were processed, and analyte concentrations (pg / mL) were determined and compared to a standard curve. Data were determined as the % of samples below the limit of detection (LOD), the % of samples above the LOD but less than three times the LOD, the % of samples within the best confidence interval, and the % of samples above the maximum value.

[0147]

[0160] The population doubling and doubling time of MPSCs are as follows: DT time (h) * : Average doubling time calculated over three consecutive passages upon thawing, excluding the first two passages immediately after thawing to allow for complete cell recovery. ** PD: maximum number of population doublings achieved in culture for the corresponding culture conditions.

[0148] [Table 19]

[0149]

[0161] FACS characterization of C1 cells for MPSC-negative markers is as follows:

[0150] [Table 20]

[0151]

[0162] FACS characterization of C1 cells for MPSC-positive markers is as follows:

[0152] [Table 21]

[0153]

[0163] FACS characterization of C1 cells for NK positive markers is as follows:

[0154] [Table 22]

[0155]

[0164] The differentiation of MPSCs into pancreatic progenitor cells and neural stem cells was determined. Samples were collected at three different concentrations and three different time points for FACS analysis and mRNA analysis.

[0156] [Table 23]

[0157] Example 10: Generation of immunosuppressive cells and evaluation of IDO secretion

[0165] Three extraembryonic stem cell lines (e.g., human trophoblast stem cells) were used as source cells: lethal pluripotent stem cell line 1 (MPSC1) P5 cells, MPSC2 P8 cells, and MPSC3 P8 cells. Cells were cultured separately in nutrient medium (e.g., MESENCULT™ + cell attachment substrate). Subcultures of cells were performed at approximately 5,000 cells / cm. 2 The cells were seeded at a density of 0 ng / mL (control), cultured for approximately 3 days, and then treated with 0 ng / mL (control), approximately 20 ng / mL, approximately 50 ng / mL, or approximately 100 ng / mL interferon gamma (IFN-γ) for 24 hours. Cells and supernatants were then collected. The immunosuppressive capacity of the resulting MPSCs was assessed by ELISA.

[0158]

[0166] Indoleamine 2,3-dioxygenase (IDO) secretion was assessed upon IFN-γ stimulation. Figure 8A shows the standard curve for the assay. Figure 8B shows the results of three cell lines stimulated with various concentrations of IFN-γ on IDO secretion compared to the control. IFN-γ-primed MPSCs were not observed to statistically increase IDO secretion.

[0159] Example 11: Evaluation of immunosuppressive cell generation and kynurenine secretion

[0167] Human trophoblast stem cell line 1 (MPSC1) P5 cells were cultured in nutrient medium (e.g., MESENCULT™ + cell attachment substrate). Subculture of cells was performed at approximately 5,000 cells / cm. 2 The cells were seeded at a density of 0 ng / mL (control), 20 ng / mL, 50 ng / mL, or 100 ng / mL interferon gamma (IFN-γ) for 24, 48, and 72 hours, after which the cells and supernatants were harvested.

[0160]

[0168] Kynurenine secretion was assessed upon IFN-γ stimulation. Figure 9A shows the standard curve for the assay. Figure 9B shows the results of the effect of IFN-γ stimulation at three different concentrations on kynurenine secretion for 24, 48, and 72 hours compared to the control and medium alone. MPSCs primed with IFN-γ were not observed to statistically increase kynurenine secretion.

[0161] Example 12: Generation of immunosuppressive cells and evaluation of IL-2 secretion

[0169] Lethal pluripotent stem cell line 1 (MPSC1) P5 cells were cultured in nutrient medium (e.g., MESENCULT™ + cell attachment substrate). Jurkat cells (approximately 100,000 cells / mL) were activated with 1 μg / mL GIBCO® phytohemagglutinin, type M (PHA-M) + 50 ng / mL phorbol 12-myristate 13-acetate (PMA) for 24 hours.

[0162]

[0170] Co-cultures of MPSC1 cells and Jurkat cells were established. Samples included: (1) MPSCs alone, (2) MPSC1 + resting Jurkat cells, (3) MPSC1 + activated Jurkat cells, and (4) activated Jurkat cells alone. MPSCs were cultured at approximately 2000 to 3000 cells / cm. 2 Jurkat cells were seeded at a density of approximately 50,000 to approximately 500,000 cells / well. The cells were co-cultured for 24 or 48 hours, and the supernatant was collected. IL-2 secretion was assessed by ELISA. Figure 10A shows the standard curve for the assay. Figure 10B shows the results of the effect of IFN-γ stimulation on IL-2 secretion at 24 and 48 hours. Co-culture of MPSC1 with activated Jurkat cells induced the highest IL-2 secretion. A dose-dependent increase was observed. Figure 10C shows the difference between approximately 3,000 cells / cm at 24 hours of co-culture compared to the control. 2 Figure 10D shows the effect of MPSC seeding density on the IL-16 / ... 2 Figure 10E shows the effect of MPSC seeding density on the 48-hour co-culture of approximately 3000 cells / cm compared to the control. A dose-dependent increase was observed. 2 Figure 10F shows the effect of MPSC seeding density on the 48-hour co-culture of approximately 2000 cells / cm compared to the control. A dose-dependent increase was observed. 2Figure 1 shows the effect of MPSC seeding density on IL-2 secretion in activated Jurkat cells. A dose-dependent increase was observed. MPSCs increased, rather than decreased, IL-2 secretion by activated Jurkat cells. Samples were FACS phenotyped and results at 24 and 48 hours (h) are provided below.

[0163] [Table 24]

[0164] Example 13: Differentiation of stem cells into pancreatic progenitor cells (PPCs) or neural stem cells (NSCs) MPSC1

[0171] Approximately 1x10 6 Stem cells (e.g., MPSC1 P4) were thawed in nutrient medium (e.g., MESENCULT™ + cell attachment substrate, or MEM-alpha + STEMULATE). Cells were then expanded at P5: 4000 cells / cm. 2 of MESENCULT™ + cell attachment substrate or 4000 cells / cm 2 MEM-alpha + STEMULATE. PPC or NSC differentiation began at P6. Culture and differentiation conditions were as follows:

[0165] [Table 25]

[0166]

[0172] Cells were assessed for adhesion to microcarriers and suspension expansion. Expansion was performed in 100 mL bioreactors.

[0167] MPSC2

[0173] Approximately 1x10 6 Stem cells (e.g., MPSC2 P4) were thawed in nutrient medium (e.g., MESENCULT™ + cell attachment substrate, or MEM-alpha + STEMULATE). Cells were then expanded at P5: 5000 cells / cm. 2 of MESENCULT™ + cell attachment substrate or 5000 cells / cm 2MEM-alpha + STEMULATE. PPC or NSC differentiation began at P6. Culture and differentiation conditions were as follows:

[0168] [Table 26]

[0169] Differentiation experimental setup

[0170] [Table 27]

[0171] FACS characterization of neural stem cells MPSC1 culture condition 1: neural stem cell markers

[0172] [Table 28]

[0173] [Table 29]

[0174] MPSC1 culture condition 2: neural stem cell markers

[0175] [Table 30]

[0176] [Table 31]

[0177] MPSC2 culture condition 1: neural stem cell markers

[0178] [Table 32]

[0179] [Table 33]

[0180] MPSC2 culture condition 2: neural stem cell markers

[0181] [Table 34]

[0182] [Table 35]

[0183]

[0174] The NCS markers of the cells produced include one or more of NCAD, NESTIN, SOX2, PAX6, or any combination thereof. In one example, the NCS cells include one of N-CAD, NESTIN, SOX2, and PAX6. In one example, the NCS cells include two of NCAD, NESTIN, SOX2, and PAX6 (e.g., NCAD and NESTIN, NCAD and SOX2, NCAD and PAX6, NESTIN and SOX2, NESTIN and PAX6, or SOX2 and PAX6). In one example, the NCS cells include three of NCAD, NESTIN, SOX2, and PAX6 (e.g., CAD / NESTIN / SOX2, CAD / NESTIN / PAX6, NESTIN / SOX2 / PAX6). In one example, the NCS cells include all of NCAD, NESTIN, SOX2, and PAX6.

[0184] MPSC1 culture condition 1: pancreatic progenitor cell markers

[0185] [Table 36]

[0186] [Table 37]

[0187] MPSC1 culture condition 2: pancreatic progenitor cell markers

[0188] [Table 38]

[0189]

[0175] Bold indicates markers that were consistently increased or decreased after FBF treatment.

[0190] [Table 39]

[0191] MPSC2 culture condition 1: pancreatic progenitor cell markers

[0192] [Table 40]

[0193] [Table 41]

[0194] MPSC2 culture condition 2: pancreatic progenitor cell markers

[0195] [Table 42]

[0196] [Table 43]

[0197]

[0176] The PPC markers of the produced cells include one or more of PDX1, FOXA2, SOC9, or any combination thereof. In one example, the PPC cells include one of PDX1, FOXA2, and SOC. In another example, the PPC cells include two of PDX1, FOXA2, and SOC. For example, the PPC may include PDX1 and FOXA2, PDX1 and SOC, or FOXA2 and SOC. In another example, the PPC cells include all of PDX1, FOXA2, and SOC.

[0198] Example 14: Scale-up of MPSC manufacturing

[0199] [Table 44]

[0200]

[0177] MPSC1 cells (approximately 10,000 cells / cm 2 ) were cultured with microcarriers on a shaker. After 3 days of expansion in (1) MESENCULT™, (2) MESENCULT™ + BSA, (3) MESENCULT™ + PLU, or (4) Rooster medium, cells were analyzed by trypan blue exclusion and live / dead cell imaging.

[0201]

[0178] Figure 11A is a graph showing cell number at 72 hours. In each bar, dead cells are shown at the top and live cells are shown at the bottom. Figure 11B is a graph showing population doublings in each type of culture.

[0202] Example 15: Expansion of MPSCs in bioreactors

[0179] MPSC (approximately 4,600 cells / cm 2 ) were cultured with microcarriers in Rooster MXC XF medium in 100 mL PBS bioreactors at 25 rpm for approximately 7 days. Cell counts and viability were determined. FACS characterization of cell markers was performed comparing adherent versus suspension culture.

[0203]

[0180] Figure 12A is a graph showing cell counts at different days in culture. D2-D6 = 44,000,000 cells. Figure 12B is a graph showing the % of viable cells in culture. Figure 12C is a graph showing population doublings comparing adherent vs. suspension cultures. D2-D6 = 4.9 PD. Adherent cultures initially doubled faster than suspension cultures, but over time, suspension cultures achieved a faster rate of population doubling.

[0204]

[0181] MPSC and NK markers for adherent and suspension cell cultures are as shown below.

[0205] [Table 45]

[0206] [Table 46]

[0207] [Table 47]

[0208] [Table 48]

[0209] Further optimization

[0210] [Table 49]

[0211] Summary of findings

[0182] Proliferation curve analysis shows different proliferation characteristics among different MPSC lines: MPSC4 < MPSC3 < MPSC2 < MPSC1. FACS characterization of MSC / NK markers shows a heterogeneous cell population for the MPSC4 cell line; the MPSC2, MPSC3, and MPSC1 cell lines are homogeneous and express core MSC markers. Short-term differentiation into PPC and NSC using MESENCULT™ and MEM-α + STEMULATE media shows media-dependent marker expression; both the MPSC1 and MPSC2 cell lines express a combination of PPC, NSC, and MPSC markers after differentiation. In co-culture with cell lines such as activated T cells (e.g., Jurkat cells), MPSC was able to stimulate activated IL-2 secretion in Jurkat cells.

[0212]

[0183] Some embodiments have been shown and described herein, but such embodiments are provided as an example only. Some modifications, variations, and substitutions can be made without departing from the present invention. It should be understood that various alternative means to the embodiments of the present invention described herein can be used in the implementation of the present invention. Scope of claims at the time of international application [Section 1] A lethal pluripotent stem cell (MPSC) population that expresses HLA-G and insulin and can reach at least 89 population doublings within approximately 90 days of initiating culture of MPSCs. [Section 2] 2. The population of MPSCs of claim 1, which can reach about 25 to about 30 population doublings within about 12 days, about 50 to about 55 population doublings within about 30 days, and / or about 75 to about 80 population doublings within about 63 days from the initiation of culturing the MPSCs. [Section 3] 3. The population of MPSCs of claim 1 or 2, which is capable of doubling in about 22 to about 27 hours. [Section 4] 4. The population of MPSCs of claim 3, which is capable of doubling in about 25 hours. [Section 5] A population of lethal pluripotent stem cells (MPSCs) that express HLA-G and insulin and are pathogen-free. [Section 6] 6. The population of MPSCs of any one of claims 1 to 5, wherein the MPSCs are bacteria-free. [Section 7] 7. The population of MPSCs of any one of claims 1 to 6, wherein the MPSCs are virus-free. [Section 8] 8. The population of MPSCs of any one of claims 1 to 7, wherein the MPSCs are cytomegalovirus-free. [Section 9] 9. The population of MPSCs of any one of claims 1 to 8, wherein the MPSCs are free of a pathogen that is EBV (Epstein-Barr virus), human adenovirus (HAdV), human cytomegalovirus (HCMV), hepatitis virus, human immunodeficiency virus (HIV), human papillomavirus (HPV), herpes simplex virus (HSV), human T-lymphotropic virus (HTLV), varicella virus (VZV), Corynebacterium, Hantavirus, lymphocytic choriomeningitis virus (LCMV), mycoplasma, treponema, or any combination thereof. [Section 10] 10. The population of MPSCs of claim 9, wherein the MPSCs are free of hepatitis viruses, and the hepatitis viruses include hepatitis A, hepatitis B, hepatitis C, or a combination thereof. [Section 11] 10. The population of MPSCs of claim 9, wherein the MPSCs do not comprise herpes simplex virus (HSV), and the herpes simplex virus (HSV) comprises human herpes virus 6 (HHV6), human herpes virus 8 (HHV8), or a combination thereof. [Section 12] 10. The population of MPSCs of claim 9, wherein the MPSCs do not comprise human immunodeficiency virus, and the human immunodeficiency virus comprises human immunodeficiency virus 1 (HIV1), human immunodeficiency virus 2 (HIV2), or a combination thereof. [Section 13] 10. The population of MPSCs of claim 9, wherein the MPSCs do not comprise human papillomavirus, and the human papillomavirus comprises HPV16, HPV18, or a combination thereof. [Section 14] 10. The population of MPSCs of claim 9, wherein the MPSCs do not comprise herpes simplex virus, and the herpes simplex virus comprises herpes simplex virus (HSV1), herpes simplex virus (HSV2), or a combination thereof. [Section 15] 10. The population of MPSCs of claim 9, wherein the MPSCs do not comprise human T-lymphotropic viruses, and the human T-lymphotropic viruses comprise human T-lymphotropic virus (HTLV1), human T-lymphotropic virus (HTLV2), or a combination thereof. [Section 16] 10. The population of MPSCs of claim 9, wherein the MPSCs do not comprise Corynebacterium, and the Corynebacterium comprises Corynebacterium bovis, Corynebacterium species (HAC2), or a combination thereof. [Section 17] 10. The population of MPSCs of claim 9, wherein the MPSCs do not comprise a hantavirus, and the hantavirus comprises a Hantaan hantavirus, a Seoul hantavirus, a Sin Nombre hantavirus, or a combination thereof. [Section 18] 18. The population of MPSCs of any one of claims 1 to 17, further expressing one or more proteins of b-HCG, HSP90, CDX2, FGFR1, pAKT, pCREB1, HLA-A, HLA-B, HLA-C, or any combination thereof. [Section 19] 19. The population of MPSCs of any one of claims 1 to 18, further expressing one or more proteins: KIR2DL4, Flt3L, NKp46, TCR, ILT-4, CD49f, CD3, CD4, CD8, CD10, CD11b, CD14, CD16, CD19, CD34, CD38, CD44, CD56, CD90 / Thy-1, CD105, CD141, CD146, CD166, CD107a, or any combination thereof. [Section 20] 20. The population of MPSCs of any one of claims 1 to 19, further expressing one or more of the following proteins: IL-6, IL-8, MCP-1, CLXL2, PDGF-AA, VEGF, PAI-1, IL-10, or any combination thereof. [Section 21] 21. A population of MPSCs according to any one of claims 1 to 20, wherein at least a portion of the MPSCs do not express one or more of the following proteins: Ki-67, HSP70, p53, syncytin, or a combination thereof. [Section 22] 22. A population of MPSCs according to any one of claims 1 to 21, which express one or more of the following proteins: CD44, CD90, CD105, CD146, CD166, HLA-A, HLA-B, HLA-C, or a combination thereof. [Section 23] 23. A population of MPSCs according to any one of claims 1 to 22, wherein at least a portion of the MPSCs do not express one or more of the following proteins: CD19, CD45, HLA-DR, or a combination thereof. [Section 24] 24. A population of MPSCs according to any one of claims 1 to 23, wherein more than 96% of the MPSCs do not express one or more of the following proteins: CD19, CD45, HLA-DR, or a combination thereof. [Section 25] 25. A population of MPSCs according to any one of claims 1 to 24, which expresses CD16, CD56 or a combination thereof. [Section 26] 26. A population of MPSCs according to any one of claims 1 to 25, wherein at least a portion of the MPSCs do not express CD3. [Section 27] 27. The population of MPSCs of any one of claims 1 to 26, wherein more than 96% of the MPSCs do not express CD3. [Section 28] 28. The population of MPSCs of any one of claims 1 to 27, wherein at least 65% of the population of MPSCs express HLA-G. [Section 29] 29. The population of MPSCs of claim 28, wherein the HLA-G comprises HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, HLA-G7, or any combination thereof. [Section 30] 30. The population of MPSCs of claim 28 or 29, wherein the HLA-G comprises HLA-G2, HLA-G4, HLA-G6, HLA-G7, or any combination thereof. [Section 31] 31. The population of MPSCs of any one of claims 28 to 30, wherein the HLA-G comprises HLA-G6, HLA-G7, or a combination thereof. [Section 32] 32. The population of MPSCs of any one of claims 1 to 31, wherein less than 15% of the population of MPSCs express HLA-G1. [Section 33] 33. A population of MPSCs according to any one of claims 1 to 32, wherein at least 10% are monoclonal. [Section 34] 26. The population of MPSCs of claim 25, wherein about 13% to about 15% are monoclonal. [Section 35] At least 1x10 6 35. The population of MPSCs of any one of claims 1 to 34, comprising MPSCs. [Section 36] 36. The population of MPSCs of any one of claims 1 to 35, wherein the MPSCs have a stable karyotype as measured by an array-based whole genome assay. [Section 37] 37. The population of MPSCs of any one of claims 1 to 36, wherein the MPSCs do not exhibit chromosomal abnormalities resulting from population doubling as measured by an array-based whole genome assay. [Section 38] 38. The population of MPSCs of any one of claims 1 to 37, wherein the MPSCs do not exhibit substantial chromosomal abnormalities resulting from freezing and thawing as measured by an array-based whole genome assay. [Section 39] A method for expanding a population of lethal pluripotent stem cells (MPSCs), comprising culturing the cells in a culture medium at a density of about 1,000 to about 5,000 cells / cm. 2 and culturing the cells. [Section 40] A method for expanding a population of lethal pluripotent stem cells (MPSCs), comprising culturing the cells in a culture medium at a density of about 1,000 to about 5,000 cells / cm. 2 and culturing the cells, wherein the population of MPSCs expresses HLA-G and insulin. [Section 41] 41. The method of claim 39 or 40, wherein the culture medium is animal component-free. [Section 42] 42. The method of any one of claims 39 to 41, wherein the culture medium is serum-free. [Section 43] 39. The method of claim 38, wherein the culture medium does not contain fetal bovine serum. [Section 44] 44. The method of any one of claims 39 to 43, wherein the MPSCs are cultured for about 3 days. [Section 45] 45. The method of any one of claims 39 to 44, wherein the MPSCs are cultured for about 4 days. [Section 46] The subculture of MPSCs was approximately 2,000 to 4,000 cells / cm. 2 46. ​​The method of any one of claims 39 to 45, wherein the seeds are seeded at a density of [Section 47] 47. A population of cells produced by the method of any one of claims 39 to 46. [Section 48] 39. A method of producing cells comprising contacting a population of MPSCs according to any one of claims 1 to 38 with one or more inducers. [Section 49] 49. The method of claim 48, wherein the cells produced are ectodermal cells. [Section 50] 49. The method of claim 48, wherein the cells produced are mesodermal cells. [Section 51] 49. The method of claim 48, wherein the cells produced are endodermal cells. [Section 52] 49. The method of claim 48, wherein the cells produced are pancreatic cells or pancreatic progenitor cells (PPCs). [Section 53] 53. The method of claim 52, wherein the one or more inducers comprise bFGF (basic fibroblast growth factor). [Section 54] 54. The method of claim 53, wherein the one or more inducers further comprise 2-mercaptoethanol and nicotinamide. [Section 55] 55. The method of any one of claims 52 to 54, wherein the PPC comprises β-HCG, CDX2, HLA-G, or any combination thereof. [Section 56] 56. The method of claim 55, wherein the PPC comprises β-HCG and CDX2; β-HCG and HLA-G; CDX2 and HLA-G; or HCG, CDX2, and HLA-G. [Section 57] 57. The method of claim 55 or 56, wherein the PPC further comprises PDX1, FOXA2, SOX9, or any combination thereof. [Section 58] 49. The method of claim 48, wherein the cells produced are neural cells (NCS) or neural progenitor cells. [Section 59] 59. The method of claim 58, wherein the one or more inducers comprise retinoic acid. [Section 60] 60. The method of claim 58 or 59, wherein the NCS cells comprise RAR-β, CDX2, HLA-G, or any combination thereof. [Section 61] 61. The method of claim 60, wherein the NCS cells comprise RAR-β and CDX2; RAR-β and HLA-G; CDX2 and HLA-G; or RAR-β, CDX2 and HLA-G. [Section 62] 62. The method of claim 60 or 61, wherein the NCS cells further comprise N-CAD, NESTIN, SOX2, PAX6, or any combination thereof. [Section 63] 49. The method of claim 48, wherein the cells produced are hepatocytes or hepatic progenitor cells. [Section 64] 64. The method of claim 63, wherein the one or more inducers include fibroblast growth factors (FGFs), steroids, and cytokines. [Section 65] 49. The method of claim 48, wherein the cells produced are natural killer cells and the inducing agent is FGF. [Section 66] 66. The method of claim 65, wherein the natural killer cells are CD16+, CD56+, and CD3-. [Section 67] The method of claim 66, wherein the natural killer cells are further HLA-G+ and CDX2+. [Section 68] 49. The method of claim 48, wherein the cells produced comprise adipocytes, chondrocytes, osteocytes, or any combination thereof. [Section 69] 69. The method of claim 68, wherein the cells produced include adipocytes and chondrocytes. [Section 70] 69. The method of claim 68, wherein the cells produced include adipocytes and bone cells. [Section 71] 69. The method of claim 68, wherein the cells produced include chondrocytes and osteocytes. [Section 72] 69. The method of claim 68, wherein the cells produced include adipocytes, chondrocytes, and osteocytes. [Section 73] 69. The method of claim 68, wherein the cells produced comprise adipocytes. [Section 74] 69. The method of claim 68, wherein the adipocytes contain leptin, HOXC8, HOXC9, Ucp1, CIDEA, PRDM16, Zic1, Lhx8, Eva1, Epsti1, Cd137, Tmem26, Tbx1, Cited1, Shox2, amino acid transporter ASC-1, amino acid transporter PAT2, purinergic receptor P2RX5, ATGL, CAV1, FABP4, COX4, LMNB1, or a combination thereof. [Section 75] 75. The method of claim 73 or 74, wherein the adipocytes comprise white adipocytes. [Section 76] 76. The method of claim 75, wherein the white adipocytes contain leptin, HOXC8, HOXC9, or a combination thereof. [Section 77] 75. The method of claim 73 or 74, wherein the adipocytes comprise brown adipocytes. [Section 78] 78. The method of claim 77, wherein the brown adipocytes contain Ucp1, CIDEA, PRDM16, Zic1, Lhx8, Eva1, Epsti1, or a combination thereof. [Section 79] 75. The method of claim 73 or 74, wherein the adipocytes comprise beige adipocytes. [Section 80] 80. The method of claim 79, wherein the beige adipocytes comprise Cd137, Tmem26, Tbx1, Cited1, Shox2, or a combination thereof. [Section 81] 75. The method of claim 73 or 74, wherein the adipocytes comprise beige adipocyte precursors. [Section 82] 82. The method of claim 81, wherein the beige adipocyte precursors comprise CD137, TMEM26, or a combination thereof. [Section 83] 69. The method of claim 68, wherein the cells produced comprise chondrocytes. [Section 84] 84. The method of claim 83, wherein the chondrocytes comprise annexin A6, CD44, CD151, ITM2A, FAM20B, FoxC1, FoxC2, SOX5, SOX6, SOX9, aggrecan, cathepsin B, CHADL, chondroadherin, collagen II, collagen IV, CRTAC1, DSPG3, IBSP / sialoprotein II, matrilin-1, matrilin-3, matrilin-4, MIA, otraprin / OTOR, URB, or a combination thereof. [Section 85] 69. The method of claim 68, wherein the cells produced comprise bone cells. [Section 86] 86. The method of claim 85, wherein the bone cells comprise pre-osteoblasts, osteoblasts, embedded osteoblasts, osteoid osteocytes, bone mineralizing osteocytes, or mature bone cells. [Section 87] 87. The method of claim 85 or 86, wherein the bone cells contain RUNX2, OCN, E11, DMP1, PHEX, MEPE, sclerostin, CapG, ORP150, or a combination thereof. [Section 88] 87. The method of claim 85 or 86, wherein the bone cells comprise pre-osteoblasts, and the pre-osteoblasts comprise RUNX2. [Section 89] 87. The method of claim 85 or 86, wherein the bone cells comprise pre-osteoblasts, and the pre-osteoblasts comprise RUNX2. [Section 90] 87. The method of claim 85 or 86, wherein the bone cells comprise osteoblasts, and the osteoblasts comprise RUNX2 and OCN. [Section 91] The method of claim v, wherein the bone cells comprise embedded osteoblasts, and the embedded osteoblasts comprise OCN, E11, DMP1, PHEX, and CapG. [Section 92] 87. The method of claim 85 or 86, wherein the bone cells comprise osteoid or bone mineralizing bone cells, and the osteoid or bone mineralizing bone cells comprise OCN, E11, DMP1, PHEX, MEPE, and CapG. [Section 93] 87. The method of claim 85 or 86, wherein the bone cells comprise mature bone cells, and the mature bone cells comprise DMP1, PHEX, MPEP, sclerostin, CapG, and ORP150. [Section 94] A population of lethal pluripotent stem cells (MPSCs) that express HLA-G and comprise a phenotype that includes one or more of: negative for indoleamine 2-3 deoxygenase (IDO) secretion, negative for kynurenine secretion, and positive for interleukin 2 (IL-2) secretion. [Section 95] 95. The population of MPSCs of claim 94, comprising a phenotype that is negative for indoleamine 2-3 deoxygenase (IDO) secretion, negative for kynurenine secretion, and positive for interleukin 2 (IL-2) secretion.

Claims

1. A population of lethal pluripotent stem cells (MPSCs), comprising: (a) The MPSC population was detected to express HLA-G and β-HCG proteins. (b) at least 90% of the population of MPSCs has detectable protein expression of CD44, CD90, CD105, CD146, CD166, HLA-A, HLA-B, and HLA-C; (c) the MPSC population had undetectable protein expression of Ki-67, HSP70, p53, and syncytin; and (d) at least 96% of the population of MPSCs have undetectable protein expression of CD19, CD45, HLA-DR, and CD3; and (e) A population of MPSCs that can reach at least 89 population doublings within 90 days of initiating culture of the MPSCs.

2. The MPSCs can reach 25-30 population doublings within 12 days, 50-55 population doublings within 30 days, and / or 75-80 population doublings within 63 days from the initiation of culture; and / or It can double in 25 hours or 22-27 hours. A population of MPSCs according to claim 1.

3. The population of MPSCs of claim 1 that is pathogen-free.

4. The population of MPSCs of claim 3 , wherein the MPSCs are free of at least one bacterium or virus.

5. 4. The population of MPSCs of claim 3, wherein the pathogen is EBV (Epstein-Barr virus), human adenovirus (HAdV), human cytomegalovirus (HCMV), hepatitis virus, human immunodeficiency virus (HIV), human papillomavirus (HPV), herpes simplex virus (HSV), human T-lymphotropic virus (HTLV), varicella virus (VZV), Corynebacterium, Hantavirus, lymphocytic choriomeningitis virus (LCMV), mycoplasma, treponema, or any combination thereof.

6. 5. The population of MPSCs of claim 4, wherein the MPSCs are free of at least one virus, wherein the virus is one or more of the following: (a) hepatitis viruses, including hepatitis A, hepatitis B, hepatitis C, or a combination thereof; (b) herpes simplex virus (HSV), including human herpes virus 6 (HHV-6), human herpes virus 8 (HHV8), herpes simplex virus (HSV-1), herpes simplex virus (HSV-2), or a combination thereof; (c) human immunodeficiency virus, including human immunodeficiency virus 1 (HIV1), human immunodeficiency virus 2 (HIV2), or a combination thereof; (d) human papillomavirus, including HPV16, HPV18, or a combination thereof; (e) human T-lymphotropic viruses, including human T-lymphotropic virus (HTLV-1), human T-lymphotropic virus (HTLV-2), or a combination thereof; (f) Hantaviruses, including Hantaan hantavirus, Seoul hantavirus, Sin Nombre hantavirus, or combinations thereof.

7. 6. The population of MPSCs of claim 5, wherein the MPSCs do not comprise Corynebacterium, and the Corynebacterium comprises Corynebacterium bovis, Corynebacterium sp. (HAC2), or a combination thereof.

8. A population of MPSCs as described in claim 1, further expressing one or more proteins of HSP90, CDX2, or any combination thereof.

9. 2. The population of MPSCs of claim 1, further expressing one or more of the following proteins: KIR2DL4, FLT3L, NKp46, TCR, ILT-4, CD49f, CD8, CD10, CD11b, CD16, CD38, CD44, CD56, CD90 / Thy-1, CD105, CD141, CD146, CD166, CD107a, or any combination thereof.

10. 2. The population of MPSCs of claim 1, wherein at least 65% of the population of MPSCs express HLA-G, wherein the HLA-G comprises HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, HLA-G7, or any combination thereof.

11. 2. The population of MPSCs of claim 1, wherein less than 15% of the population of MPSCs express HLA-G1. Claim 12: A population of MPSCs of at least 1 x 10 ６ 2. The population of MPSCs of claim 1, comprising MPSCs.

13. 2. The population of MPSCs of claim 1, wherein the MPSCs exhibit one or more of the following: (a) exhibit a stable karyotype as measured by an array-based whole-genome assay; (b) does not exhibit chromosomal abnormalities resulting from population doubling as measured by an array-based whole-genome assay; or (b) do not exhibit substantial chromosomal aberrations resulting from freezing and thawing as measured by an array-based whole-genome assay;

14. 10. An in vitro method for expanding a population of lethal pluripotent stem cells (MPSCs) according to claim 1, comprising culturing the MPSCs at a density of 1,000-5,000 cells / cm in a culture medium. ２ and culturing the cells.

15. The method of claim 14, further comprising: (a) the culture medium does not contain animal components, serum, or fetal bovine serum; and / or (b) the MPSCs are cultured for at least 3 or 4 days; and / or (c) contacting the population of MPSCs with one or more inducers, thereby producing a population of cells obtained by the method.

16. 15. The method of claim 14, further comprising contacting the population of MPSCs with one or more inducers, wherein the population of cells produced is an ectodermal cell, a mesodermal cell, an endodermal cell, a pancreatic cell, a pancreatic progenitor cell (PPC), a neural stem cell (NSC), a neural progenitor cell, a hepatocyte, a hepatic progenitor cell, a natural killer cell, an adipocyte, a chondrocyte, or a bone cell.

17. 17. The method of claim 16, wherein the one or more inducers include: (a) bFGF (basic fibroblast growth factor); (b) 2-mercaptoethanol and nicotinamide; (c) retinoic acid; (d) fibroblast growth factor (FGF), a steroid, and a cytokine; or (e) FGF.

Images