Lethal pluripotent stem cells

Abstract

To overcome certain shortcomings of current embryonic stem cells and iPS cells, we provide novel stem cells for treating various diseases or conditions. [Solution] Lethal pluripotent stem cells produced in vitro from extraembryonic stem cells such as trophoblast stem cells, compositions thereof, and their use are disclosed herein. A method for producing a cell population by culturing lethal pluripotent stem cells produced in vitro with one or more inducers is also disclosed herein. A method for treating a disorder or condition by utilizing the cells disclosed herein and cells differentiated therefrom is also disclosed herein.

Description

[Technical Field]

[0001] cross reference

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

[0002]

[0002] Novel stem cells are needed to treat various diseases or conditions as an alternative to overcome certain shortcomings of current embryonic stem cells and iPS cells.

[0003] Embedding by reference

[0003] All publications, patents, and patent applications described herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually incorporated by reference. In the event of any inconsistency between the terminology used herein and the terminology used in the incorporated references, the terminology used herein shall prevail. [Overview of the project]

[0004]

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

[0005]

[0005] In some embodiments, populations of lethal pluripotent stem cells (MPSCs) expressing HLA-G and insulin and capable of reaching a population doubling of at least 89 within 90 days from the start of culture of the MPSCs. In some examples, the population of MPSCs can reach a population doubling of about 89 to about 100 within 90 days from the start of culture of the MPSCs. In some examples, the population of MPSCs can reach a population doubling of about 25 to about 30 within about 12 days from the start of culture of the MPSCs, a population doubling of about 50 to about 55 within about 30 days, and / or a population doubling of about 75 to about 80 within about 63 days. In some examples, the population of MPSCs can double in about 22 to about 27 hours, for example, about 25 hours. In some embodiments, populations of lethal pluripotent stem cells (MPSCs) expressing HLA-G and insulin and free of pathogens are disclosed herein.

[0006]

[0006] In some cases, the MPSCs disclosed herein are pathogen-free. In some cases, the MPSCs are bacteria-free. In some cases, the MPSCs are viruses-free, such as cytomegaloviruses. In some cases, the MPSCs are EBV (Epstein-Barr virus), HAdV (human adenovirus), HCMV (human cytomegalovirus), hepatitis viruses (e.g., hepatitis A, hepatitis B, and / or hepatitis C), human herpesviruses (e.g., HHV6 (human herpesvirus type 6) and / or HHV8 (human herpesvirus type 8)), human immunodeficiency viruses (e.g., HIV1 (human immunodeficiency virus type 1), HIV2 (Human immunodeficiency virus type 2), human papillomavirus (e.g., HPV16, HPV18, etc.), herpes simplex virus (e.g., HSV1 (herpes simplex virus type 1), HSV2 (herpes simplex virus 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., Hantavirus, It does not contain pathogens selected from the group consisting of Mycoplasma sp., Treponema pallidum, and any combination thereof.

[0007]

[0007] In some cases, the MPSCs, or populations comprising MPSCs, disclosed herein further express one or more proteins from among beta-human chorionic gonadotropin (b-HCG), heat shock protein 90 (HSP90), tail-type 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 cases, the MPSC population further expresses one or more proteins from among 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 cases, the MPSC population further expresses one or more proteins from among interleukin-6 (IL-6), IL-8, monocyte chemotactic protein-1 (MCP-1), CLXL2, platelet-derived growth factor AA (PDGF-AA), vascular endothelial growth factor (VEGF), plasminogen-activating inhibitor 1 (PAI-1), or IL-10. In some cases, at least a portion of the MPSCs do not express one or more proteins from among Ki-67, heat shock protein 70 (HSP70), p53, or syncytin. In some cases, the MPSC population (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) expresses one or more proteins from among CD44, CD90, CD105, CD146, CD166, HLA-A, HLA-B, or HLA-C. In some cases, at least a portion of MPSCs do not express one or more of the CD19, CD45, or HLA-DR proteins. In some cases, more than 96%, 97%, 98%, or 99% of MPSCs do not express one or more of the CD19, CD45, or HLA-DR proteins.In some examples, the population of MPSCs further expresses one or more proteins 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 expresses HLA-G. In some examples, 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 expresses HLA-G1.

[0008]

[0008] In some examples, at least 10% of the population of MPSCs disclosed herein is monoclonal. In some examples, about 13% to about 15% of the population of MPSCs is monoclonal. In some examples, at least about 1x10 6 individual MPSCs are present in the population. In some examples, MPSCs have a stable karyotype when measured by an array-based whole genome assay. In some examples, MPSCs do not experience chromosomal abnormalities resulting from population doubling when measured by an array-based whole genome assay. In some examples, MPSCs do not experience substantial chromosomal abnormalities resulting from freezing and thawing when measured by an array-based whole genome assay.

[0009]

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

[0010]

[0010] In some embodiments, a method for growing a population of lethal pluripotent stem cells (MPSCs) is provided, with approximately 1,000 to approximately 5,000 cells / cm³ in the culture medium. 2 A method is disclosed herein comprising the steps of seeding a subculture of MPSCs at a density and culturing the cells, wherein the population of MPSCs expresses HLA-G and insulin. In some cases, the culture medium does not contain animal components. In some cases, the culture medium does not contain serum, e.g., fetal bovine serum. In some cases, the cells are cultured for 3 days. In some cases, the cells are cultured for 4 days. In some cases, the MPSCs are cultured at a density of approximately 2,000 to approximately 4,000 cells / cm³. 2 It is sown at this density.

[0011]

[0011] In some embodiments, a method for producing cells is disclosed herein, comprising the step of contacting a population of MPSCs disclosed herein with one or more inducers. The cells produced may include ectoderm cells. The cells produced may include mesoderm cells. The cells produced may include endoderm cells. The cells produced may include pancreatic cells or pancreatic progenitor cells, and optionally the inducers may include bFGF (basic fibroblast growth factor), and further include 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 nerve cells or neural progenitor cells, and the inducer may optionally include retinoic acid. In one embodiment, the NCS cells include retinoic acid receptor beta (RAR-β), CDX2, HLA-G, or any combination thereof. In some examples, the NCS cells include 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 PPC further includes 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 may include fibroblast growth factor (FGF) such as FGF2, steroids such as dexamethasone, and cytokines such as oncostatin M, and may further include bone morphogenetic proteins (BMPs), e.g., BMP4, and / or hepatic growth factors. In some examples, the FGF is bound to FGFR1 and is FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF8, FGF10, FGF17, FGF19, FGF20, FGF21, FGF22, or FGF23.In some cases, the steroids are glucocorticoid steroids, such as dexamethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, or triamcinolone. In some cases, the cytokines are interleukin-6 (IL-6) group cytokines, such as oncostatin M, such as human oncostatin M, IL-6, interleukin-11, leukemia suppressor factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), and cardiotrophin-like cytokines (CLC). The cells produced may include natural killer cells, and the inducer includes FGF, for example, 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+. That is the case.

[0012]

[0012] The cells produced may include 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 yet another embodiment, the cells produced include chondrocytes and osteocytes. In yet another embodiment, the cells produced include adipocytes, chondrocytes, and osteocytes.

[0013]

[0013] In another embodiment, the cells produced include adipocytes. Adipocytes contain 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), and Glu / Asprit. The adipocytes may include a Cbp / P300 interaction transactivator having a carboxyl-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, which contain leptin, HOXC8, HOXC9, or a combination thereof. In another example, the adipocytes include brown adipocytes, which contain Ucp1, CIDEA, PRDM16, Zic1, Lhx8, Eva1, Epsti1, or a combination thereof. In another example, adipocytes include beige adipocytes, which contain Cd137, Tmem26, Tbx1, Cited1, Shox2, or a combination thereof. In yet another example, adipocytes include beige adipocyte precursors, which contain CD137, TMEM26, or a combination thereof.

[0014]

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

[0015]

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

[0016]

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

[0017] In any such aspect, embodiment, and / or example, the inventors have demonstrated that the stem cells are immune privileged, chromosomally stable (non-tumor-forming), pathogen-free, and pluripotent. The inventors have also demonstrated the efficient differentiation of their stem cells into programmed natural killer (NK), cartilage, bone, adipose, neuronal, pancreatic, liver, and secretome cells, along with remarkable doubling time and proliferative properties.

[0018] Brief explanation of the drawing

[0018] Various aspects of the present invention are described in particular in conjunction with the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description, which describes exemplary embodiments utilizing the principles of the present invention, and to the appended drawings. [Brief explanation of the drawing]

[0019] [Figure 1]

[0019] Figure 1 is a line graph showing the 3-day growth curve of MPSCs measured by population doubling over a 90-day timeframe. [Figure 2-1]

[0020] Figures 2A–2D show whole-genome views of KARYOSTAT® analysis of four different MPSC samples from different population doublings. Figure 2A is an MPSC sample from a population doubling of 16.5. Figure 2B is an MPSC sample from a population doubling of 44.5. Figure 2C is an MPSC sample from a population doubling of 62.6. Figure 2D is an MPSC sample from a population doubling of 71.5. [Figure 2-2] Same as the explanation in Figure 2-1. [Figure 3]

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

[0022] Figure 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 expressing 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 shows that MPSCs express HLA-A, B, C (left panel) and surface and soluble HLA-G detected by 4H84 antibody (right panel) compared to isotype control and unstained cells. (Figure 5D) Representative FACS analysis of HLA-G isoforms in MPSCs on the cell surface compared to intracellular levels. [Figure 5B] The explanation is the same as in Figure 5A. [Figure 5C] The explanation is the same as in Figure 5A. [Figure 5D] The explanation is the same as in Figure 5A. [Figure 6A]

[0024] Figures 6A–6G show the expression of molecular biomarkers of immune cells in MPSCs. Imaging or FACS analysis reveals that 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 image or plot. [Figure 6B] The explanation is the same as in Figure 6A. [Figure 6C] The explanation is the same as in Figure 6A. [Figure 6D] The explanation is the same as in Figure 6A. [Figure 6E] The explanation is the same as in Figure 6A. [Figure 6F] The explanation is the same as in Figure 6A. [Figure 6G] The explanation is the same as in Figure 6A. [Figure 7]

[0025] Figure 7 provides illustrative growth curves for MPSC1 (upward triangle; top line), MPSC2 (square), MPSC3 (downward triangle), and MPSC4 (circle) over 33 passages. [Figure 8A]

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

[0027] Figure 8B shows the results of IDO secretion in three cell lines induced by various concentrations of IFN-γ stimulation, compared to a control. This data is "negative," indicating no effect on IDO secretion compared to the control. [Figure 9A]

[0028] Figure 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 on kynurenine secretion at three different concentrations at 24, 48, and 72 hours, compared to the control and culture medium alone. This data is "negative," indicating no effect on kynurenine secretion compared to the control. [Figure 10A]

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

[0031] Figure 10B shows the results of 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 culture medium alone. This data is "positive," indicating that cells increased IL-2 secretion compared to the control. [Figure 10C]

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

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

[0034] Figure 10E shows the effect of an MPSC seeding density of approximately 3000 cells / cm2 in 48 hours of co-culture compared to the 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 in 48 hours of co-culture compared to a 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 the number of cells after 72 hours. In each bar, dead cells are shown at the top and living cells at the bottom. [Figure 11B]

[0037] Figure 11B is a graph showing population doubling for each type in the culture medium. [Figure 12A]

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

[0039] Figure 12B is a graph showing the percentage of live cells in culture. [Figure 12C]

[0040] Figure 12C is a graph showing population doubling comparing adherent culture versus suspension culture. ΔD2-D6 = 4.9PD. Adherent culture initially doubled faster than suspension culture, but over time, suspension culture achieved a faster rate of population doubling. [Modes for carrying out the invention]

[0020]

[0041] Novel and unique lethal pluripotent stem cells (MPSCs) produced in vitro, their compositions, and the generation or impairment of differentiated cells with various phenotypes (e.g., pancreatic, nerve, liver, immunomodulatory, or natural killer cell phenotypes) (e.g., diabetes, neuropathy). The use of MPSCs in the treatment of or improvement of conditions (e.g., skin conditions) (or degeneration, liver disease, cancer, inflammation, viral infection, or autoimmune disease) is disclosed herein. MPSCs are different from, but not limited to, previous trophoblast stem cells, and have advantages such as rapid, scalable population duplication; exhibiting a pathogen-free profile; possessing high immune privileges and suitability for transplantation; having excellent chromosomal stability, e.g., possessing a stable karyotype up to at least 71 population duplications; and producing a robust secretome rich in cytokines, chemokines, and exosomes. MPSCs are different from embryonic stem cells and are ethically sourced and cultured. Although MPSCs are lethal (e.g., have finite proliferative capacity), they can reach population duplication much faster 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 main cell groups that make up humans: ectoderm (which gives rise to skin, neurons, and the nervous system), endoderm (which forms the gastrointestinal tract and respiratory system, 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 non-tumor-forming, as demonstrated in studies of immunocompetent rats, and do not induce tumors or teratomas, for example.

[0021]

[0042] Details of one or more embodiments of the present invention are described in the accompanying drawings, claims, and this description. Other features, purposes, and advantages of embodiments of the present invention disclosed and intended herein may be combined with any other embodiments unless expressly excluded.

[0022]

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the field to which the claimed subject matter pertains. It should be understood that the above general description and the following detailed description are illustrative and descriptive, and not limitations on any claimed subject matter. In this application, the use of singular forms includes plural forms unless otherwise specifically stated. It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple references unless otherwise explicitly stated in the text. In this application, the use of "or" means "and / or" unless otherwise stated. Furthermore, the use of the term "including" and other forms such as "include," "includes," and "included" is not limited.

[0023]

[0044] As used herein, ranges and quantities may be expressed with the prefix "approximately" to a specific value or range, for example, meaning ±15% of the referenced number. "Approximately" can also include exact quantities; for example, "approximately 5 μL" means both "approximately 5 μL" and "5 μL". Generally, the term "approximately" includes quantities that are expected to fall within the range of experimental error.

[0024]

[0045] As used herein, terms such as “to treat” and “treatment” mean to obtain a desired pharmacological and / or physiological effect. In some cases, an individual (e.g., an individual suffering from and / or suspected to be genetically predisposed thereto) is treated prophylactically with the cell preparations described herein, and such prophylactic treatment completely or partially prevents the liver-related disease or disorder or its signs or symptoms. In some cases, an individual is treated therapeutically (e.g., if the individual has a liver-related disease or disorder), and such therapeutic treatment causes a partial or complete cure of the disease or disorder and / or reverses adverse effects resulting from the disease or disorder and / or stabilizes the disease or disorder and / or slows the progression of the disease or disorder and / or leads to regression of the disease or disorder. Wake up.

[0025]

[0046] The administration (e.g., transplantation) of cells disclosed herein to an area requiring treatment may be achieved, for example, by local infusion during surgery, by injection, by catheter, or by implantation, the implantation being of a porous, non-porous, or gelatinous material, including a membrane such as a sialastic membrane or fibers.

[0026]

[0047] "Transplanting" a composition into a mammal means introducing a composition into the body of a mammal by any method established in the art. The composition to be introduced is the "graft," and the mammal is the "recipient." The graft and recipient may be syngeneic, homogeneous, or heterogeneous. Furthermore, the transplant may be autotransplantation.

[0027]

[0048] When used in relation to cells or cell populations, the term “isolated” refers to a state in which cells or cell populations are separated from a host organism from which they may originate and are not present in the host organism. In some cases, isolated cells are isolated from the same host organism or in contact with other cells derived from it. In some cases, isolated cells are purified and isolated from any other cells. In some cases, isolated cells are induced in vitro from stem cells.

[0028]

[0049] An "effective dose" is the amount of therapeutic agent sufficient to achieve the intended purpose. An effective dose of a composition for treating or improving 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 should not be construed as limiting the subjects described.

[0030] Cells and compositions

[0051] In some embodiments, populations of lethal pluripotent stem cells (MPSCs) expressing HLA-G and insulin and capable of reaching population doubling of at least 89 within 90 days from the start of MPSC culture are disclosed herein. In some examples, the population of MPSCs can reach population doubling of at least 89–100 within 90 days from the start of MPSC culture. In some examples, the population of MPSCs can reach population doubling of about 25–30 within about 12 days, about 50–55 within about 30 days, and / or about 75–80 within about 63 days from the start of MPSC culture. In some examples, the population of MPSCs can double in about 22–27 hours, for example, about 25 hours. In some embodiments, populations of lethal pluripotent stem cells (MPSCs) expressing HLA-G and insulin and free of pathogens are disclosed herein. In some cases, MPSCs lack expression of p53, syncytin, Ki67, heat shock protein 70 (HSP70), or any combination thereof. In some cases, MPSCs are human cells. In some cases, MPSCs originate from or are derived from rodents, rabbits, cattle, sheep, pigs, dogs, cats, monkeys, or apes.

[0031]

[0052] In another embodiment, a population of lethal pluripotent stem cells (MPSCs) expressing HLA-G and comprising a phenotype including one or more of the following: negative for indoleamine 2-3 deoxygenase (IDO) secretion, negative for kynurenine secretion, and positive for interleukin-2 (IL-2) secretion is disclosed herein. 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 contrary to what a person skilled in the art might expect of MPSCs. Depending on the surface phenotype marker, cells may resemble mesenchymal stem cells, but they behave functionally differently.

[0032]

[0053] In some cases, the MPSCs disclosed herein are pathogen-free. In some cases, the MPSCs are bacteria-free. In some cases, the MPSCs are viruses-free, such as cytomegalovirus. In some cases, the MPSCs are EBV (Epstein-Barr virus), HAdV (human adenovirus), HCMV (human cytomegalovirus), hepatitis viruses (e.g., hepatitis A, hepatitis B, hepatitis C), herpesviruses (e.g., HHV6 (human herpesvirus 6), HHV8 (human herpesvirus 8), etc.), human immunodeficiency viruses (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 It does not contain pathogens selected from the group consisting of V1 (herpes simplex virus type 1), HSV2 (herpes simplex virus type 2), 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., Hantan, Saul, or Sinnonble), LCMV (lymphocytic choriomeningitis virus), Mycoplasma species, Treponema pallidum, cytomegalovirus (CMV), and combinations thereof.

[0033]

[0054] In some cases, the MPSCs disclosed herein, or populations comprising MPSCs, further express one or more proteins from among β-HCG, HSP90, CDX2, FGFR1, pAKT, pCREB1, HLA-A, HLA-B, or HLA-C. In some cases, populations of MPSCs further express one or more proteins from among β-HCG, HSP90, CDX2, FGFR1, pAKT, pCREB1, HLA-A, HLA-B, or HLA-C. In some cases, the MPSC population further expresses one or more proteins from among IL-6, IL-8, MCP-1, CLXL2, PDGF-AA, VEGF, PAI-1, or IL-10. In some cases, at least a portion of the MPSCs do not express one or more proteins from among Ki-67, HSP70, p53, or syncytin. In some cases, the MPSC population (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) expresses one or more proteins from among 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 proteins from among CD19, CD45, or HLA-DR. In some cases, more than 96%, 97%, 98%, or 99% of MPSCs do not express one or more proteins from CD19, CD45, or HLA-DR. In some cases, a population of MPSCs further expresses one or more proteins from CD16 or CD56 or a combination thereof. In some cases, at least a portion of MPSCs do not express CD3. In some cases, more than 96%, 97%, 98%, or 99% of MPSCs do not express CD3. In some cases, at least 65% or at least 70% of a population of MPSCs express HLA-G. In some cases, HLA-G includes HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, or HLA-G7, or any combination thereof.In some cases, HLA-G includes HLA-G2, HLA-G4, HLA-G6, or HLA-G7, or any combination thereof. In some cases, HLA-G includes HLA-G6, HLA-G7, or a combination thereof. In some cases, less than 15% (e.g., less than 10%) of the MPSC population express HLA-G1.

[0034]

[0055] In some cases, at least 10% of the population of MPSCs disclosed herein is monoclonal. In some cases, about 13% to about 15% of the population of MPSCs is monoclonal. In some cases, at least about 1 x 10 6 A number of MPSCs exist within the population. In some cases, MPSCs have a stable karyotype when measured by array-based whole-genome assays. In some cases, MPSCs do not experience chromosomal abnormalities originating from population doubling when measured by array-based whole-genome assays. In some cases, MPSCs do not experience substantial chromosomal abnormalities originating from freezing and thawing when measured by array-based whole-genome assays.

[0035]

[0056] In some cases, cells provided herein, e.g., MPSCs, are genetically modified. In some examples, cells are genetically modified to express exogenous genes, e.g., transgenes. As used herein, the term “transgene” and its grammatical equivalents may refer to a gene or genetic material that is introduced into an organism. For example, a transgene may be a stretch or segment of DNA containing a gene that is introduced into an organism. When a transgene is introduced into an organism, that organism is subsequently referred to as a transgenic organism. A transgene may retain its ability to produce RNA or polypeptides (e.g., proteins) in the transgenic organism. A transgene may consist of different nucleic acids, e.g., RNA or DNA. A transgene may encode a genetically modified T cell receptor, e.g., a TCR transgene. A transgene may contain a TCR sequence. A transgene may contain an oncogene. A transgene may contain an immunooncogene. A transgene may contain a recombinant arm. A transgene may contain a genetically modified site. In some examples, a transgene is an oncogene. In some cases, the transgene is an immunooncogene. In some cases, the transgene is a tumor suppressor gene. In some cases, the transgene codes for a protein that directly or indirectly promotes proteolysis. In some cases, the transgene is an oncolytic gene. In some cases, the transgene can help lymphocytes target tumor cells. In some cases, the transgene is a T-cell enhancer gene. In some cases, the transgene is an oncolytic virus gene. In some cases, the transgene inhibits tumor cell proliferation. In some cases, the transgene is an anti-cancer receptor. In some cases, the transgene is an anti-angiogenic factor. In some cases, the transgene is a cytotoxic gene.Examples of transgenes include, but are not limited to, one or more genes encoding CD28, inducible costimulator (ICOS), CD27, 4-1BB (CD137), ICOS-L, CD70, 4-1BBL, signal 3, 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 against any of the above (e.g., monoclonal antibodies), or any combination thereof. In some examples, the transgene encodes a protein involved in cell or tissue repair (e.g., DNA repair, immune responses (e.g., interferons and interleukins), and proteins associated with structural proteins). In some examples, the transgene encodes a growth factor receptor. In some examples, the MPSCs disclosed herein include transgenes encoding TCRs, B cell receptors (BCRs), chimeric antigen receptors (CARs), or any combination thereof. In some examples, the MPSCs described herein include transgenes encoding oncogene receptors.

[0036]

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

[0037]

[0058] In one embodiment, a composition comprising cells disclosed herein (e.g., a pharmaceutical composition) is 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, colloidal dispersions are used. Colloidal dispersions include polymer 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 embodiments, a method for producing cells is disclosed herein, comprising the step of contacting a population of MPSCs disclosed herein with one or more inducers. In some examples, the cells produced are ectoderm cells. In some examples, the cells produced are mesoderm cells. In some examples, the cells produced are endoderm cells. In some examples, the cells produced are pancreatic cells or pancreatic progenitor cells, and optionally, the inducers include bFGF (basic fibroblast growth factor), and in some examples, further include 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 nerve cells or neural progenitor cells, and the inducer, if necessary, includes retinoic acid. In one embodiment, the NCS cells include RAR-β, CDX2, HLA-G, or any combination thereof. In some examples, the NCS cells include RAR-β and CDX2; RAR-β and HLA-G; CDX2 and HLA-G; or RAR-β, CDX2, and HLA-G. If necessary, in some examples, the PPC further includes N-CAD, NESTIN, SOX2, PAX6, or any combination thereof. In some examples, the cells produced are hepatocytes or hepatic progenitor cells, and the inducer, if necessary, includes fibroblast growth factor (FGF) such as FGF2, steroids such as dexamethasone, and cytokines such as oncostatin M, and in some examples, further may include bone morphogenetic proteins (BMPs), e.g., BMP4, and / or hepatic growth factors. In some cases, FGF binds to FGFR1, resulting in FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF8, FGF10, FGF17, FGF19, FGF20, FGF21, FGF22, or FGF23.In some cases, the steroid is a glucocorticoid steroid, e.g., dexamethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, or triamcinolone. In some cases, the cytokine is an interleukin-6 group cytokine, e.g., oncostatin M, e.g., human oncostatin M, interleukin-6, interleukin-11, leukemia suppressor factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), and cardiotrophin-like cytokine (CLC). In some cases, the cell is a natural killer cell, and the inducer includes FGF, e.g., FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF8, FGF10, FGF17, FGF19, FGF20, FGF21, FGF22, or FGF23. In some cases, MPSCs can differentiate into neural progenitor cells in one day using a one-step protocol, compared to the 30-day (or 3-4 week) and four-step differentiation process for embryonic stem cells or iPS cells. Compared to the 8-15 day and 4-5 step differentiation of embryonic stem cells or iPS cells, MPSCs can differentiate into insulin-producing pancreatic progenitor cells in 1 day using a 1-step protocol. In some cases, MPSCs differentiate into hepatocyte-like cells in 6 days using a 2-step protocol, compared to the 12-21 day and 3 step differentiation of embryonic stem cells or iPS cells.

[0039]

[0060] In one embodiment, 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 yet another embodiment, the cells produced include chondrocytes and osteocytes. In yet another embodiment, the cells produced include adipocytes, chondrocytes, and osteocytes.

[0040]

[0061] In another embodiment, the cells produced include adipocytes. Adipocytes may 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. In one example, the adipocytes include white adipocytes, which contain leptin, HOXC8, HOXC9, or a combination thereof. In another example, the adipocytes include brown adipocytes, which contain Ucp1, CIDEA, PRDM16, Zic1, Lhx8, Eva1, Epsti1, or a combination thereof. In another example, adipocytes include beige adipocytes, which contain Cd137, Tmem26, Tbx1, Cited1, Shox2, or a combination thereof. In yet another example, adipocytes include beige adipocyte precursors, which contain CD137, TMEM26, or a combination thereof.

[0041]

[0062] In another embodiment, the cells produced include chondrocytes. Chondrocytes may include 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, otraprine / OTOR, URB, or a combination thereof.

[0042]

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

[0043]

[0064] In some cases, the cells disclosed herein are administered to a subject intravenously, subcutaneously, percutaneously, by inhalation, or 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, methods for killing antigen-carrying target cells, comprising the step of administering the cells disclosed herein to a subject requiring such treatment, are disclosed herein. In some examples, the antigen-carrying target cells are cancer cells. In some examples, the cancer cells are solid tumor cells. In some examples, the cancer cells are hematopoietic cancer cells. In some examples, the cancer cells include bladder cancer cells, osteosarcoma cells, brain tumor cells, breast cancer cells, colorectal cancer cells, esophageal cancer cells, gastrointestinal cancer cells, liver cancer cells, lung cancer cells, nasal cavity cancer cells, nasopharyngeal cancer cells, oral cancer cells, oropharyngeal cancer cells, ovarian cancer cells, prostate cancer cells, gastric cancer cells, skin cancer cells, thyroid cancer cells, or any combination thereof. In some cases, cancer cells originate from cancers including hematopoietic malignancies, head and neck squamous cell carcinoma, leukemia, lymphoma, myeloma, sarcoma, melanoma, bladder cancer, osteosarcoma, brain tumor, breast cancer, cervical cancer (e.g., cervical carcinoma), 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. Cancer includes primary cancer. Or, cancer includes metastatic cancer. In some cases, the target cells carrying the antigen are pathogens. In some cases, pathogens include viruses, bacteria, protozoa, prions, fungi, or any combination thereof. In some cases, 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 the population of antigen-carrying target cells. In some cases, the method kills approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the population of antigen-carrying target cells.

[0045]

[0066] In some cases, methods for downmodulating inflammatory pathways are disclosed herein, comprising the step of administering the cells disclosed herein to subjects in need thereof. In some cases, the methods are for graft rejection, infection, infection-associated endotoxin shock, 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, and Peyronie's disease. Treat diseases or conditions including 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 systems, pancreatitis, surgical trauma, 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 cases, 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 bowel disease, Graves' disease, myasthenia gravis, autoimmune neutropenia, idiopathic thrombocytopenic purpura, rheumatoid arthritis, cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's disease, bullous pemphigoid, lupus discoid, ulcerative colitis, dense deposit disease, inflammatory bowel disease, psoriasis, or any combination thereof. In some cases, the method treats type 1 diabetes. In some cases, the method improves graft rejection.

[0046]

[0067] In another embodiment, a method for treating a condition in a subject is disclosed herein, comprising the step of administering to the subject an amount of a pharmaceutical composition containing the cells described herein that is effective in causing the cells to engraft in 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 includes physiological saline, such as phosphate buffered saline, or fetal bovine serum. In some examples, the cells are 6 ~ about 100 x 10 6 cells, about 1 x 10 6 ~ about 250 x 10 6 cells, about 1 x 10 6 ~ about 500 x 10 6 cells, or about 10 x 10 6 ~ about 40 x 10 6 cells and are administered in a suspension. 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 an injection, such as an intravenous injection. In some examples, the injection is administered via the hepatic vein. In some examples, the injection is administered via the hepatic artery. In some examples, the condition is a liver-related disease or disorder, such as acute liver disease. In some examples, the condition is liver failure. In some examples, the liver-related disease or disorder includes Alagille syndrome, alpha1-antitrypsin deficiency, autoimmune hepatitis, benign liver tumors, biliary atresia, cirrhosis, cystic diseases of the liver, fatty liver diseases including alcohol-related liver disease and non-alcoholic fatty liver disease (NAFLD), galactosemia, gallstones, Gilbert syndrome, hemochromatosis, liver cysts, liver cancer, liver diseases during pregnancy (if necessary, 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 syndrome, sarcoidosis, toxic hepatitis, type 1 glycogenosis, tyrosinemia, viral hepatitis, Wilson disease, or any combination thereof.

[0047]

[0068] Modes of administration of cells disclosed herein include, but are not limited to, systemic intravenous infusion and direct infusion to the intended site of activity (e.g., endoscopic retrograde infusion). Preparations can be administered by any convenient route, for example, by infusion or bolus infusion, and can be administered together with other bioactive agents. In some cases, administration is systemic or local.

[0048]

[0069] In some embodiments, compositions and methods for transplanting cells disclosed herein are provided herein. In some examples, the subjects are injected by cells (e.g., intravenously, intramuscularly, percutaneously, endoscopic retrograde injection, or intraperitoneally). In some examples, the subjects are not treated with immunosuppressants prior to transplantation. In some examples, the method further includes the step of treating the patient with an immunosuppressant, such as FK-506, cyclosporine, or an anti-glutamate decarboxylase 65 kilodalton isoform (GAD65) antibody.

[0049]

[0070] In some examples, the cells described herein are delivered to a target site (e.g., a defective area of ​​the liver) by a delivery system suitable for targeting the cells to a specific tissue. For example, the cells are encapsulated in a delivery vehicle that allows for sustained release of the cells at the target site. The delivery vehicle can be modified to specifically target a particular tissue. The surface of the targeted delivery system can be modified in various ways. In the case of liposome-targeted delivery systems, lipid groups can be incorporated into the lipid bilayer of the liposome to maintain the targeting ligand during stable association with the liposome bilayer.

[0050]

[0071] The administration of cells described herein can be adjusted for each individual as needed by (1) increasing or decreasing the amount of cells injected; (2) changing the number of injections; or (3) changing the method of cell delivery.

[0051] Detection method

[0072] Methods for determining the expression or presence of the above biomarkers are well known in the industry, for example, flow cytometry, immunohistochemistry, Western blotting, immunoprecipitation, magnetic bead selection, and quantification of cells expressing any of these cell surface markers. Therefore, it can be measured. The RNA expression level of a biomarker can be measured using, for example, RT-PCR, Qt-PCR, microarrays, Northern blotting, 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" includes examples of cases in which a biomarker is determined to be not expressed, not detected, expressed at low levels, expressed at normal levels, or overexpressed.

[0053]

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

[0054]

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

[0055]

[0076] In some cases, the expression or presence of a biomarker is at the RNA (e.g., mRNA) level. In some cases, techniques for detecting RNA (e.g., mRNA) levels include, but are not limited to, Southern or Northern DNA 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 includes, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250, or 500 nucleotides in length, sufficient to specifically hybridize to the mRNA or genomic DNA encoding the biomarker described herein under stringent conditions. Hybridization of mRNA and the probe indicates that the biomarker or other target protein of interest is being expressed.

[0057]

[0078] In some cases, the mRNA is immobilized on a solid surface and brought into contact with a probe, for example, by flowing the isolated mRNA onto an agarose gel and then transferring the mRNA from the gel to a membrane, such as nitrocellulose. In some cases, the probe is immobilized on a solid surface and the mRNA is brought into contact with the probe, for example, in a gene chip array. Those skilled in the art can easily adapt known mRNA detection methods for use in detecting levels of mRNA encoding biomarkers or other proteins of interest.

[0058]

[0079] Alternative methods for determining the level of target mRNA in a sample include, for example, nucleic acid amplification processes using RT-PCR, ligase chain reaction, self-sustaining sequence replication, transcription amplification systems, Q-beta replicase, rolling circle replication, or any other nucleic acid amplification method. Next, the detection of the amplified molecule is performed 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 fluorescence T-PCR (e.g., the TAQMAN® system).

[0059]

[0080] The expression level of the target RNA is monitored using membrane blotting (e.g., those used in hybridization analyses such as Northern or Dot blotting) or microwells, sample tubes, gels, beads, or fibers (or any solid support containing the bound nucleic acid). Expression detection also includes the use of 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 offer a method for simultaneously measuring the expression levels of multiple genes. Each array consists of a reproducible pattern of capture probes bound to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array, then detected by laser scanning to determine the hybridization intensity for each probe on the array, which is then converted into a quantitative value representing the relative gene expression level. High-density oligonucleotide arrays are particularly useful for determining gene expression profiles for multiple RNAs in a sample.

[0061]

[0082] In some examples, the array is manufactured on a surface of substantially any shape, or even on multiple surfaces. In some examples, the array is a planar array surface. In some examples, the array contains peptides or nucleic acids on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. In some examples, the array is packaged in a manner that allows for other operations of the diagnostic or comprehensive device.

[0062]

[0083] In some cases, 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 blotting, ELISA, multiplexing techniques, immunoprecipitation, or immunohistochemistry. In some cases, biomarker detection is achieved by ELISA. In some cases, biomarker detection is achieved by electrochemiluminescence (ECL).

[0063]

[0084] Any means are intended to specifically identify and quantify biomarkers in a biological sample. Thus, in some cases, the expression level of a biomarker protein of interest in a biological sample is detected by a binding protein that can specifically interact with that biomarker protein or a biologically active variant. In some cases, a labeled antibody, its binding site, or other binding partner is used. As used herein, the word “labeled” means a detectable compound or composition that is directly or indirectly conjugated to an antibody to produce a “labeled” antibody. In some cases, the label is detectable by itself (e.g., radioisotope labeling or fluorescent labeling) or, in the case of enzymatic labeling, catalyzes a chemical change in a detectable substrate compound or composition.

[0064]

[0085] Antibodies for detecting biomarker proteins are either monoclonal or polyclonal, or produced by synthesis or recombination. 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 outlines of immunological assay design, theory, and protocols can be found in several textbooks in the art.

[0065]

[0086] The choice of markers used to label antibodies will vary depending on the application. However, the selection of markers is readily apparent to those 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 polyclonal or monoclonal. Furthermore, antibodies used to detect proteins of interest are labeled with radioactive atoms, enzymes, chromogenic or fluorescent moieties, or colorimetric tags as described elsewhere in this specification. The choice of label to tag will also depend on the desired detection limit. Enzyme assays (e.g., ELISA) typically allow for the detection of colored products formed by the interaction of enzyme-tagged complexes with enzyme substrates. Examples of radionuclides that act as detectable labels include 1-131, 1-123, 1-125, Y-90, Re-188, At-211, Cu-67, Bi-212, and Pd-109. Examples of enzymes that act as detectable labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and glucose-6-phosphate dehydrogenase. Examples of 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, the enzyme and chromogenic moiety are conjugated to the antibody with coupling agents such as dialdehyde, carbodiimide, and dimaleimide. Alternatively, conjugation occurs via ligand-receptor pairs. Examples of preferred ligand-receptor pairs include, but are not limited to, biotin-avidin or biotin-streptavidin, and antibody-antigen.

[0066]

[0087] In some cases, the expression or presence of one or more biomarkers or other proteins of interest in a biological sample is determined by radioimmunoassay or enzyme-conjugated immunoassay (ELISA), competitive enzyme-conjugated immunoassay, dot blotting, Western blotting, chromatography such as high-performance liquid chromatography (HPLC), or other assays known in the art. Thus, detection assays include, but are not limited to, steps such as 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 source cells for producing lethal pluripotent stem cells (MPSCs) as disclosed herein. In some cases, mammalian stem cells are isolated from amniotic fluid, amniotic membrane, Wharton's jelly, chorionic villi, or ectopic pregnancy in a manner that does not inhibit or destroy the embryo.

[0068]

[0089] In some cases, MPSCs are obtained in culture media that do not contain antibiotics, such as penicillin, streptomycin, or any combination thereof. In some cases, the culture media for obtaining mammalian stem cells do not contain retinoic acid. In some cases, the culture media for obtaining and / or subculturing mammalian stem cells do not contain mercaptoethanol, nicotinamide, or any combination thereof. In some cases, the culture media for obtaining and / or subculturing mammalian stem cells do not contain dexamethasone, recombinant human oncostatin M, BMP4, HGF, or any combination thereof. In some cases, the culture media for obtaining and / or subculturing mammalian stem cells are xeno-free, for example, free of animal components. In some cases, the culture media for obtaining and / or subculturing mammalian stem cells do not contain human-derived and animal-derived components, for example, known-composition media. In some cases, the culture media for obtaining and / or subculturing mammalian stem cells do not contain serum. In some cases, the culture media for obtaining and / or subculturing mammalian stem cells do not contain fetal bovine serum.

[0069]

[0090] In some cases, this disclosure is a method for growing a population of MPSCs disclosed herein, in a culture medium of about 1,000 to about 5,000 cells / cm³. 2 The present invention provides a method comprising the steps of seeding a subculture of MPSCs at a density and culturing the cells.

[0070]

[0091] In some embodiments, a method for growing a population of lethal pluripotent stem cells (MPSCs) is provided, with approximately 1,000 to 5,000 cells / cm³ in the culture medium. 2 A method is disclosed herein comprising the steps of seeding a subculture of MPSCs at a density and culturing the cells, wherein the population of MPSCs expresses HLA-G and insulin. In some cases, the culture medium does not contain animal components. In some cases, the culture medium does not contain serum, e.g., fetal bovine serum. In some cases, the cells are cultured for about 3 days. In some cases, the cells are cultured for about 4 days. In some cases, the MPSCs are cultured at a density of about 2,000 to about 4,000 cells / cm³. 2 It is sown at this density.

[0071]

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

[0072]

[0093] In some cases, mammalian stem cells as defined herein can be obtained, for example, from blastomere biopsies during preimplantation genetic diagnosis (PGD) in conjunction with reproductive therapies such as in vitro fertilization (IVF). In one example, the cells as defined herein can be produced by a method for blastocyst biopsy, in which the remaining blastocyst is implanted, resulting in pregnancy and subsequent live birth, and the blastocyst is biopsied after, for example, the zona pellucida is removed from the blastocyst.

[0073]

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

[0074]

[0095] In some cases, mammalian stem cells as used herein are obtained from chorionic villus sampling in early pregnancy (e.g., the 8th stage of pregnancy). +3 ~12 +0 It can be isolated from a full-term placenta derived from a cesarean section or cesarean delivery. The chorionic tissue can be separated from the amnion, subdivided, and / or enzymatically digested (e.g., digested for about 15 minutes using about 3 ml of TRYPLE® Select Enzyme). The cells can then be centrifuged (e.g., at about 150 x g + / - 10%, for about 5 minutes), counted, and / or re-seeded in culture medium (e.g., α-MEM containing STEMULATE® Human Platelet Lysate Cell Culture Media Supplement or MESENCULT®-ACF Plus Culture Kit) (e.g., 1 cm 2(Approximately 100 cells per cell). In one example, the isolated cells may be plastic-adherent. In one example, the cells can be used for approximately 4 to 8 passages.

[0075]

[0096] In some cases, chorionic villi can be obtained from the fallopian tubes of unruptured, pre-implantation embryos in women with ectopic pregnancies (e.g., gestational age: approximately 5–8 weeks, 6–8 weeks, or 4–8 weeks after fertilization). A small amount of chorionic tissue can be finely fragmented in a suitable culture medium (e.g., serum-free α-MEM), identified under a microscope, and then triedpsinized for a set period of time (e.g., about 15 min) using approximately 3 ml of TRYPLE® Select Enzyme. The reaction can then be stopped by adding culture medium (e.g., STEMULATE® Human Platelet Lysate Cell Culture Media Supplement or α-MEM containing MESENCULT®-ACF Plus Culture Kit). Adherent cells can be obtained and cultured under suitable conditions (for example, 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] Kit / Product

[0097] Kits and products for use with one or more methods and compositions described herein are disclosed herein. Such kits include a carrier, package, or container partitioned to receive one or more containers, such as vials and tubes, each containing one of the separate elements used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In some examples, the containers are formed from a variety of materials, such as glass or plastic.

[0077]

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

[0078]

[0099] For example, the container may optionally contain cells in the composition disclosed herein. Such a kit may optionally include an identification statement or label or instructions relating to its use in the method described herein.

[0079]

[0100] The kit typically includes a label listing the contents and / or instructions for use. This also includes accompanying documentation, such as instructions for use. Typically, a set of instruction manuals would also be included.

[0080]

[0101] In some cases, the label is on the container or attached to the container. In some examples, the label is on the container if the letters, numbers, or other symbols forming the label are attached to the container itself, molded, or etched; the label is attached to the container if it is located within a receptacle or carrier that also holds the container, for example, as an accompanying document. In some examples, the label is used to indicate that the contents are to be used for a particular therapeutic application. The label also indicates instructions for the use of the contents, such as in the methods described herein.

[0081] Drugs, compositions, and their use

[0102] A composition comprising cells produced by the method described herein (e.g., in v itro compositions, pharmaceutical compositions, etc., and pharmaceuticals are disclosed herein. A pharmaceutically acceptable carrier, excipient, or stabilizer as needed (Remington, The Science and Practice of Pharmacy, 20th edition, Mac Together with k Publishing (2000), compositions or pharmaceuticals of desired purity can be prepared in the form of lyophilized formulations or aqueous solutions. As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any material that, when combined with the active ingredient, preserves the biological activity of the ingredient and is non-reactive with the target immune system. Examples, but not limited to, include any standard pharmaceutically acceptable carrier, e.g., phosphate-buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate-buffered saline solution (PBS) or ordinary (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro (ed.), Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0082]

[0103] Permitted carriers, excipients, or stabilizers are used in the dosage and concentration. Non-toxic to cypients; buffering agents, e.g., phosphates, citrates, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., serum albumin, gelatin, or The following may be included: immunoglobulins, etc.; 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 nonionic surfactants, such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0083]

[0104] The products described herein for in vitro culture or assay The use of cells is described herein. For example, the cells may be for use in immunofluorescence or fluorescence-activated cell sorting (FACS) assays. In some cases, the cells produced may be neural stem cells (NCS), pancreatic progenitor cells (PPC), ectoderm cells, mesoderm cells, endoderm cells, hepatocytes, or hepatic progenitor cells.

[0084]

[0105] Methods used herein to test novel drugs for safety and efficacy The use of neural stem cells (NSCs) produced by the method herein is described herein. For example, the effect can be determined by bringing the test agent into contact with a culture containing the cells to be produced. If the test agent is toxic to the cells, the growth of the culture may decrease and / or the cells may die. If the test agent is effective, the growth of the culture may increase. In the case of neural stem cells, the test agent may induce the production of motor neurons. The use of neural stem cells (NSCs) produced by the method herein for producing motor neurons in vitro or in vivo is described herein. The use of neural stem cells (NSCs) produced by the method herein in the manufacture of agents for the treatment of motor neuron diseases is described herein. The use of neural stem cells (NSCs) produced by the method herein in the manufacture of agents for the treatment of spinal cord injury is described herein. The use of neural stem cells (NSCs) is described herein.

[0085]

[0106] Methods used herein to test novel drugs for safety and efficacy The use of pancreatic progenitor cells (PPCs) produced by the method described herein is described herein. For example, a test agent can be brought into contact with a culture containing the cells to be produced to determine its effect. If the test agent is toxic to the cells, the growth of the culture may decrease and / or the cells may 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 cells and / or exocrine cells. The use of PPCs produced by the method described herein for producing endocrine cells and / or exocrine cells in vitro or in vivo is described herein. The use of PPCs produced by the method described herein in the manufacture of agents for the treatment of diseases or disorders caused by pancreatic trauma is described herein. The use of PPCs produced by the method described herein in the manufacture of agents for the treatment of pancreatic trauma is described herein. The use of PPCs produced by the method described herein for producing artificial tissue or organs (e.g., pancreas) in vitro is described herein. [Examples]

[0086]

[0107] This application refers to the following non-limiting examples provided as exemplary embodiments of this application. This can be better understood. The following embodiments are provided to illustrate the embodiments more completely, but should not be construed as limiting the broad scope of this application in any way.

[0087] Example 1: MPSC reached a population doubling of 89.

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

[0088] [Table 1]

[0089]

[0110] Seeding density of 10,000 cells / cm² 2 From 2,000 to 4,000 cells / cm² 2 When the concentration was reduced, the population doubling rate of MPSCs improved. 3000-5000 cells / cm² 2 A 3-day subculture of cells seeded at a density of 4000 / 3000 cells / cm² will produce a similar number of PDs. 2 Alternating 3-day / 4-day subculturing of seeded cells yielded 4000 cells / cm² for faster subculturing. 2 This produces a number of PDs similar to those produced by a 3-day subculturing. The culture environment may be 21%O2 / %CO2, or 2%O2 / 5%CO2 / 93%N2.

[0090]

[0111] A portion of the growth results from 90 days, i.e., more than 30 passages of a 3-day subculture. As shown in Figure 1, this is a line graph showing the growth curve of MPSCs over 3 days in subculture, measured by population doubling (PD) over a 90-day timeframe. MPSCs reached a maximum of 25 PD by approximately 12 days, a maximum of 50 PD by approximately 30 days, a maximum of 75 PD by approximately 63 days, and a maximum of 89 PD by approximately 90 days.

[0091]

[0112] MPSCs, when cultured in xenofree medium, take approximately 7 hours to double in age. It possesses an extended population doubling capacity of 0 to 80 times. On average, it takes about 27 hours for cells to double in a 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 the number of cells to double, and y is the percentage of cells in the G0 phase).

[0092]

[0113] This extended doubling capacity makes MPSC ideal for scaling up to industrial scale. By creating cell populations, the need for repeated isolation from donors or biological resources is eliminated. The large batch size capability of MPSCs will streamline therapeutic development and manufacturing, facilitating the realization of stem cell-based therapeutics in clinical settings by generating sufficient MPSCs from a single origin, thereby reducing the variability of products associated with multiple cell banks and the costs of producing and launching equivalent products from different donors.

[0093] Example 2: MPSC does not have chromosomal abnormalities.

[0114] Figures 2A-2D show the KAR of four different MPSC samples derived from different population doubling processes. The whole-genome view using YOSTAT® is shown. The KARYOSTAT® assay can enable digital imaging of chromosomal abnormalities. The size of structural abnormalities that can be detected exceeds approximately 2 Mb (megabases) for chromosome expansion and approximately 1 Mb for chromosome loss. Genomic DNA was purified from cells and added to GENECHIP® for KARYOTATE®. GENECCHIP® can determine chromosome copy number variants. Figures 2A–2D show whole-genome views showing all somatic cell chromosomes and sex chromosomes in a single frame. Figure 2A is an MPSC sample from a population doubling of 16.5, Figure 2B is an MPSC sample from a population doubling of 44.5, Figure 2C is an MPSC sample from a population doubling of 62.6, and Figure 2D is an MPSC from a population doubling of 71.5. The smoothed signal plot (y-axis on the right) is a log2 ratio smoothing depicting the signal intensity of the probe on the microarray. A value of 2 may represent a normal copy number state (CN=2). A value of 3 may represent chromosome expansion (CN=3). A value of 1 may represent chromosome loss (CN=1). Gray signals represent the raw signals for each individual chromosome probe, while black signals represent normalized probe signals used to identify copy number and abnormalities (if any). No observable chromosomal abnormalities were observed in Figures 2A–D. MPSC cells can undergo multiple population duplications without showing chromosomal abnormalities. For example, a monoclonal population can be expanded and then frozen for future use. Once cells are grown from a monoclonal cryopreserved culture, 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 immortality-associated genetic abnormalities or mutations.

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

[0115] MPSCs express HLA-G, an immune-privileged marker. (In adults or after birth) Unlike human mesenchymal stromal cells, MPSCs as used herein express human leukocyte antigen-G (HLA-G), a major histocompatibility complex class I antigen found only in the placenta, which binds to the HLA-G receptor on leukocytes and suppresses immune function through several mechanisms, including induction of apoptosis in activated T cells, modulation of natural killer (NK) and dendritic cell activity, and inhibition of T cell proliferation. Referring to Figure 3, this figure shows MPSCs stained with the primary antibody 4H84. For Figure 3, MPSCs were collected from cultures in MESENCULT® ACF Plus Medium. The 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 approximately 0.25–0.5 x 10⁶ cells per sample. 6 Individual cells were aliquoted into flow cytometry tubes, and the cells were centrifuged. The 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 made it possible to stain intracellular materials. After incubation, the cells were washed with flow cytometry washing buffer, centrifuged, and then stained with flow cytometry staining buffer (R&D The HLA-G primary antibody (e.g., 4H84 antibody) was resuspended in the Systems. Primary antibody staining occurred when the solution was added to the cells and incubated at room temperature. The cells were washed several times with flow cytometry washing 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 to 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 with flow cytometry washing buffer. After washing the cells, they were resuspended in flow cytometry staining buffer and incubated for approximately 0.5 x 10⁻⁶ 6The cells were adjusted to a concentration of 100%. After resuspending the cells, they were sampled by flow cytometry. The primary antibody MEM-G / 11 can recognize the membrane-bound HLA-G1 isoform. The primary antibody 4H84 can recognize the alpha domains of the seven HLA-G isoforms. Figure 3 shows MPSCs that have been permeabilized and stained with the primary antibody HLA-G 4H84. The staining indicates the presence of HLA-G isoforms in or on the MPSCs. The staining shows that approximately 76% of the cells were positive compared to the isotype control at a primary antibody dilution of 1:50. Figure 4 shows cells stained with the primary antibody 4H84 (bottom panel) and the primary isotype control antibody mouse IgG1 (top panel). The cells showed limited staining for the IgG1 antibody, and 99.64% of events were stained with the 4H84 antibody, indicating that this antibody is specific to MPSCs. As shown in Figure 3, HLA-G expression in MPSCs allows cells to have access to immune-privileged sites, such as the fetus.

[0095]

[0116] In this specification, MPSC is measured by fluorescence-activated cell sorting (FACS). When this occurred, the phenotype and morphology of human MSCs were observed by expressing characteristic markers. See Table 2 below.

[0096] [Table 2]

[0097]

[0118] In this specification, MPSCs are used as an alternative to mesenchymal stromal cells (MSCs). Human MSCs can offer a solution. They exhibit immunosuppressive effects, show three lineages of differentiation in vitro, and are safely delivered to patients for a variety of indications, and are approved to treat niche indications such as autoimmune perianal fistula and graft-versus-host disease. However, the broad adoption of MSC-based therapies has been hindered by the inability to produce large batches of MSCs due to the population doubling limit of approximately 30-40 doublings before cellular senescence is reached.

[0098]

[0119] In this specification, MPSC refers to natural saturation when measured by FACS. The killer cell phenotype was observed. Please refer to Table 3 below.

[0099] [Table 3]

[0100]

[0121] MPSCs are β-hCG, HLA-G, and heat shock protein 90 (HSP9). 0), and various cellular biomarkers, including CDX2, were expressed immunocytochemically (Figure 5A). However, MPSCs did not express the proliferation marker Ki-67, HSP70, the tumor suppressor p53, and the intercellular fusion protein syncytin (Figure 5B), which supports the concept that MPSCs occupy the primary position of the TE differentiation trophoblast. Specifically, MPSCs expressed HLA-A, B, 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 MPSC. Yes. Using Ab 4H84, all seven isoforms were barely detectable on the cell surface (top left column), but 68.7% of all seven HLA-G isoforms were detected in permeabilized MPSCs (bottom left column). With Ab MEM-G / 11, HLA-G G1 was only slightly detected on the cell surface (top center column), with 8.1% of HLA-G G1 being detected (top center column). Similarly, with Ab MEM-G9, HLA-G G1, G3, and G5 were detected on the cell surface (top right column), but none of the HLA-G were detected.

[0102]

[0123] Human MPSCs exhibit immune cell binding biomarkers. They bind cells to immunocytochemistry. The cells were characterized using analysis and FACS analysis. As a result, the MPSCs were found to be NK cell differentiation antigens (CD) 56, CD16 dim It was shown that various biomarkers bound to immune cells were expressed, including the inhibitory receptor KIR2DL4, CD11b, the activating receptor NKp46, and CD10 (Figure 6A); the 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 on hematopoietic stem cells (Figure 6F) and CD34 (Figure 6G); and CD38 on lymphocytes (Figure 6G). Subsequently, the expression of these biomarkers in MPSCs was analyzed using eight independent cell lines that showed similar patterns of NK and T cell biomarkers, and (CD16+CD56) + NK cells and CD107(+) cells showed the highest expression in the MPSC. FACS analysis revealed that while NK cell and T cell biomarkers comprised many immune cells in the MPSC, CD107(+)CD(16+56)(+) cells and CD8(+)CD(16+56)(+) cells constituted the majority of the cell populations in the MPSC.

[0103] Example 4: The significant portion of MPSC is monoclonal.

[0124] MPSC monoclonals were obtained from MPSC cultures. MPSCs were examined using an inverted microscope. Cells were placed on top and grown, and the cell type was recorded. Old culture medium was removed, and the cells were washed with sterile PBS. TRYPLE® solution was added to the MPSC culture. The cells were incubated at 37°C and 5% CO2 for approximately 6 minutes. After incubation, the cells were isolated. The TRYPLE® reaction was stopped by adding culture medium to the cells. The cells were collected and the number of cells was counted using a cell counter. Approximately 200 cells were taken out and placed in a centrifuge tube. The culture medium was replenished and the cells were divided 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 culture medium was changed every 2-3 days. The old medium was washed and the TRYPLE® solution was added to the cells. After a short incubation, the TRYPLE® reaction was stopped by adding culture medium and the cells were transferred to 6-well plates and grown at 37°C and 5% CO2. The cells were subcultured in 100 mm dishes and grown at 37°C and 5% CO2, with the culture medium changed every 2-3 days. The culture medium was changed every 2-3 days. When the monoclonal cells reached 80-95% thickness, they were frozen. Cells were expanded from donor ectopic tissue as wild-type passages mixed with other cells, or expanded into monoclones. Monoclones were expanded to provide multiple doses. For example, approximately 125,000 monoclones can be cultured for every 1 million cells derived from donor ectopic tissue. Each monoclone was 7 x 10⁶. 28 It may contain 100 million cells per dose, with each monoclonal being 7 x 10⁶ 20 This allows for the creation of a dosage. Total possibilities derived from each ectopic tissue collected: 125,000 x 7 x 10 20 Dose=8.8x10 25 Dosage. Each MPSC monoclone can support a complete product cycle.

[0104]

[0125] All vials of 1M cells potentially contain approximately 130,000 monoclonal cells. This can result in the following. Please refer to Table 4 below.

[0105] [Table 4]

[0106] Example 5: MPCS is pathogen-free

[0127] Regardless of whether the source cells were infected with the pathogen or did not contain the pathogen Furthermore, the human MPCS obtained herein is pathogen-free. Nine cell lines of MPSC were tested. PCR evaluation detected Corynebacterium bovis, Corynebacterium species (HAC2), EBV, HAdV, 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) in nutrient medium + cell adhesion substrate The cells were cultured and passaged 12 times at 37°C and 5% CO2. Fixed and permeabilized cells were prepared for surface and intracellular staining. The staining conditions are as shown in Table 5 below.

[0108] [Table 5]

[0109]

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

[0110] [Table 6]

[0111]

[0132] The results of the primary antibody dot plot (A-FL1 vs. SSC) were shown in 1:25 and 1:5 The dilution ratios of 0, 1:100, and 1:200 are shown in Table 7 below.

[0112] [Table 7]

[0113]

[0134] The results of the primary antibody dot plot (FL1 vs FL2) were shown at 1:25 and 1:50. The dilution ratios of 1:100 and 1:200 are shown in Table 8 below.

[0114] [Table 8]

[0115]

[0136] MPSC1 was cultured in nutrient medium + cell adhesion substrate at 37°C and 5% CO2 for 7 The cells were passaged several times (19.8 population doubling). Fixed and permeabilized cells were prepared for surface and intracellular staining using HLA-G 4H84 antibody and HLA-G MEM-G / 11 antibody. 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] Results of FLA-G 4H86 dot plot compared to control (FL1 vs SS) Table 10 shows the HLA-G staining of MPSC1, along with C).

[0118] [Table 10]

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

[0140] Four types of extraembryonic stem cell lines (e.g., human trophoblast stem cells) are used as source cells. Lethal pluripotent stem cell lines 1 (MPSC1), MPSC2, MPSC3, and MPSC4. A cell bank was developed by culturing the cell lines separately in nutrient media (e.g., MESENCULT® + cell adhesion substrate). Cell subcultures were grown at a rate of 3000-4000 cells / cm³. 2 Seeds were seeded at a density and cultured for 3 or 4 days. The endpoints of the study were 10PD, 35PD, 55PD, and 70PD. Subsequently, FACS characterization was performed. Phenotyping was evaluated using MPSC / NK markers and HLA-G. Furthermore, the function of the produced cells was assessed.

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

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

[0121]

[0142] The characteristics of C1 were as follows: MPSC1 was evaluated for its functionality. (3-series differentiation and secretome analysis).

[0122]

[0143] The characteristics of C2 were as follows: MPSC1 P13, 40.3PD The phenotypic determination and functional aspects (three-series differentiation and secretome analysis) were evaluated using FACS for MSC / NK markers and HLA-G.

[0123] Lethal pluripotent stem cell line 2

[0144] Lethal pluripotent stem cell lineage 2 (MPSC2) was used as the source cell. 3000 / 4000 cells / cm2 Cells were seeded, cultured in nutrient medium (e.g., MESENCULT® + cell adhesion substrate), and expanded. Three cell banks (CBs) were frozen: CB1: 8.1 PD; CB2: 29.3 PD; and CB3: 47.4 PD. Phenotypic and functional assays were performed. The mean doubling time (P4-P6) was 26.9 hours.

[0124]

[0145] The characteristics of C1 were as follows: MPSC2 passage 5 (P5), 11. The passage doubling (PD) of strain 2 was evaluated for phenotypic determination and function (three-series differentiation and secretome analysis) using FACS for MSC / NK markers and HLA-G.

[0125]

[0146] The characteristics of C2 were as follows: MPSC2 P16, 36.9PD The phenotypic determination and functional aspects (three-series differentiation and secretome analysis) were evaluated using FACS for MSC / NK markers and HLA-G.

[0126] Lethal pluripotent stem cell line 3

[0147] Lethal pluripotent stem cell lineage 3 (MPSC3) was used as the source cell. 3000 / 4000 cells / cm 2 Cells were seeded, cultured in nutrient medium (e.g., MESENCULT® + cell adhesion substrate), and expanded. Three cell banks (CBs) were frozen: CB1: 8.1 PD; CB2: 25.5 PD; and CB3: 37.3 PD. Phenotypic and functional assays were performed. The mean doubling time (P4-P6) was 32.7 hours.

[0127]

[0148] The characteristics of C1 were as follows: MPSC3 passage 5 (P5), 10. The passage doubling (PD) of strain 2 was evaluated for phenotypic determination and function (three-series differentiation and secretome analysis) using FACS for MSC / NK markers and HLA-G.

[0128]

[0149] The characteristics of C2 were as follows: MPSC3 P18, 33.3PD The phenotypic determination and functional aspects (three-series differentiation and secretome analysis) were evaluated using FACS for MPSC / NK markers and HLA-G.

[0129] Lethal pluripotent stem cell line 4

[0150] Lethal pluripotent stem cell lineage (MPSC4) was used as the source cell. 3000 / 4000 cells / cm 2 The cells were seeded and cultured in nutrient medium (e.g., MESENCULT® + cell adhesion substrate) and expanded over a total of approximately 6.5 PD. The cells were either frozen or characterized by FACS analysis for mesenchymal stem cells (MSCs) / natural killer NK markers and HLA-G. The mean doubling time from P4 to P5 was approximately 43.7 hours.

[0130] Growth curve

[0151] The proliferation curves of the four MPSC cell lines are shown in Figure 7. MPSC1 for this experiment It underwent two freeze / thaw cycles, which likely reduced the maximum number of PDs in the culture.

[0131]

[0152] Cell banks for MPSC1, MPSC2, MPSC3, and MPSC4 Growth curves and results from the second experiment of collection were obtained for (CB)1, 2, 3, and 4. Doubling of MPSC4 was slow, and experiments for that cell line were discontinued.

[0132] [Table 11]

[0133]

[0153] The phenotypic characteristics were evaluated for each cell line at the following passage doubling (PD) stage. It was done in between.

[0134] [Table 12]

[0135]

[0154] Cell surface antigen expression of MPSC / NK markers and HLA-G is measured in FACS. Therefore, we evaluated the cells. We also evaluated their adhesion to plastic under standard culture conditions, as well as their ability to differentiate into osteoblasts, adipocytes, and chondrocytes.

[0136] [Table 13]

[0137]

[0155] FACS characterization of MPSC markers and HLA-G for C1 and C2 The following applies:

[0138] [Table 14]

[0139] [Table 15]

[0140] [Table 16]

[0141] [Table 17]

[0142]

[0156] Function: Three-series differentiation was evaluated using the following differentiation protocol:

[0143] [Table 18]

[0144]

[0157] MPSC3 did not show differentiation 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 are not shown.

[0145]

[0158] In the third week, MPSC1, MPSC2, and MPSC3 C1 cells were compared to the control. In comparison, alizarin red staining showed bone formation. Data are not shown.

[0146] Example 9: Differentiation induced by hypoxia

[0159] Extraembryonic stem cell lines (e.g., human trophoblast stem cells) are grown until they reach a dense concentration (e.g., approximately 3000 cells / cm 2 ~about 9000 cells / cm 2 , or approximately 6000 cells / cm² 2 The cells were grown in nutrient medium (e.g., MESENCULT® + cell adhesion substrate). The cells were washed and the medium was replaced without supplementation. Hypoxia was induced in the chamber (e.g., cultured in a 2% O2 gas mixture for approximately 24 hours). The medium was collected and frozen until use. The medium from all three cell lines was tested using QUANTIBODY® Human Kiloplex Array (RAYBIOTECH® Life, Inc.). Then, 1000 types of proteins were quantitatively analyzed. Experiments were repeated for MPSC1 and MPSC2. Briefly, samples were processed, analyte concentrations (pg / mL) were determined, and compared with standard curves. Data were determined as the percentage of samples below the limit of detection (LOD), the percentage of samples above the LOD but less than 3 times the LOD, the percentage of samples within the best confidence interval, and the percentage of samples above the maximum value.

[0147]

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

[0148] [Table 19]

[0149]

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

[0150] [Table 20]

[0151]

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

[0152] [Table 21]

[0153]

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

[0154] [Table 22]

[0155]

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

[0156] [Table 23]

[0157] Example 10: Evaluation of immunosuppressive cell generation and IDO secretion

[0165] Three types of extraembryonic stem cell lines (e.g., human trophoblast stem cells) are used as source cells. Lethal pluripotent stem cell lineage 1 (MPSC1) P5 cells, MPSC2 P8 cells, and MPSC3 P8 cells were used. The cells were cultured separately in nutrient medium (e.g., MESENCULT® + cell adhesion substrate). Cell subcultures were passed at approximately 5,000 cells / cm³. 2 The cells were seeded at a density of [density], cultured for approximately 3 days, and treated with interferon-gamma (IFN-γ) at concentrations of 0 ng / mL (control), approximately 20 ng / mL, approximately 50 ng / mL, or approximately 100 ng / mL for 24 hours. The cells and supernatant were then collected. The immunosuppressive capacity of the resulting MPSCs was evaluated by ELISA.

[0158]

[0166] Indoleamine 2,3-dioxygenase (IDO) content upon IFN-γ stimulation IDO secretion was evaluated. Figure 8A shows the standard curve for the assay. Figure 8B shows the results for three cell lines stimulated with various concentrations of IFN-γ on IDO secretion, compared to a control. IFN-γ-primed MPSCs were not found 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 are cultured in a nutrient medium (e.g., MESE). Cells were cultured in NCULT® (a cell adhesion substrate). Cell subcultures were passed at approximately 5,000 cells / cm³. 2 The cells were seeded at a density of [density], cultured for approximately 3 days, and treated with interferon-gamma (IFN-γ) at 0 ng / mL (control), approximately 20 ng / mL, approximately 50 ng / mL, or approximately 100 ng / mL for 24, 48, and 72 hours. The cells and supernatant were then collected.

[0160]

[0168] Kynurenine secretion in response to IFN-γ stimulation was evaluated. Figure 9A shows the assay standard curve. The lines are shown. Figure 9B shows the results at 24, 48, and 72 hours, compared to the control and culture medium alone. The results of the effects of IFN-γ stimulation on kynurenine secretion at different concentrations are shown. IFN-γ-primed MPSCs did not statistically increase kynurenine secretion.

[0161] Example 12: Evaluation of immunosuppressive cell generation and IL-2 secretion

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

[0162]

[0170] We established a co-culture of MPSC1 cells and Jurkat cells. The sample was ( The samples included (1) MPSCs only, (2) MPSC1+ resting Jurkat cells, (3) MPSC1+ activated Jurkat cells, and (4) activated Jurkat cells only. MPSCs were distributed at approximately 2000-3000 cells / cm². 2 Jurkat cells were seeded at a density of approximately 50,000 to 500,000 cells / well. The cells were co-cultured for 24 or 48 hours, and the supernatant was collected. IL-2 secretion was evaluated by ELISA. Figure 10A shows the standard curve of the assay. Figure 10B shows the results of the effect of IFN-γ stimulation on IL-2 secretion at 24 and 48 hours. Co-cultures of MPSC1 and activated Jurkat cells induced the highest IL-2 secretion. A dose-dependent increase was observed. Figure 10C shows the results of co-cultures of approximately 3000 cells / cm³ at 24 hours compared to the control. 2 The effect of MPSC seeding density is shown. A dose-dependent increase was observed. Figure 10D shows the effect of approximately 2000 cells / cm³ in 24-hour co-culture compared to the control. 2The effect of MPSC seeding density is shown. A dose-dependent increase was observed. Figure 10E shows the effect of approximately 3000 cells / cm³ in 48 hours of co-culture compared to the control. 2 The effect of MPSC seeding density is shown. A dose-dependent increase was observed. Figure 10F shows the effect of approximately 2000 cells / cm³ in 48 hours of co-culture compared to the control. 2 The effect of MPSC seeding density is shown. A dose-dependent increase was observed. MPSCs increased, rather than decreased, IL-2 secretion by activated Jurkat cells. FACS phenotyping of samples was determined, 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 Individual cell stem cells (e.g., MPSC1 P4) are placed in a nutrient medium (e.g., For example, cells were thawed in MESENCULT® + cell adhesion substrate, or MEM-Alpha + STEMULATE. Then, the cells were expanded in P5 at 4000 cells / cm². 2 MESENCULT® + cell adhesion substrate or 4000 cells / cm² 2 MEM-alpha + STEMULATE. PPC or NSC differentiation began at P6. Culture and differentiation conditions were as follows:

[0165] [Table 25]

[0166]

[0172] Cell adhesion to microcarriers and suspension expansion were evaluated. Expansion was performed at 100°C. The procedure was performed in a mL bioreactor.

[0167] MPSC2

[0173] Approximately 1x106 Stem cells of individual cells (e.g., MPSC2 P4) were thawed in a nutrient medium (e.g., MESENCULT (trademark) + cell attachment matrix, or MEM-alpha + STEMULATE). Then, the cells were expanded at P5: 5000 cells / cm 2 of MESENCULT (trademark) + cell attachment matrix or 5000 cells / cm 2 of MEM-alpha + STEMULATE. PPC or NSC differentiation started at P6. The culture and differentiation conditions are as follows:

[0168]

Table 26

[0169] Differentiation experiment 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] [[ID=五十二]] [[ID=五十三]]MPSC1 culture condition 2: Neural stem cell markers[[ID=五十四]] [[ID=五十五]]

[0175] [[ID=五十六]] [[ID=五十七]] [[ID=五十八]]

Table 30

[0176] [[ID=六十四]] [[ID=六十五]] [[ID=六十六]]

Table 31

[0177] It should be noted that there are some consecutive line numbers with the same content in the original text which seem a bit odd. I've translated them as accurately as possible while maintaining the format. If there are any specific issues or corrections needed regarding the original text structure, it might be beneficial to review and clarify the source material. MPSC2 Culture Condition 1: Neural Stem Cell Marker

[0178] [Table 32]

[0179] [Table 33]

[0180] MPSC2 Culture Condition 2: Neural Stem Cell Marker

[0181] [Table 34]

[0182] [Table 35]

[0183]

[0174] The NCS markers of the cells produced include one or more of NCAD, NESTIN, SOX2, P AX6, 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 Marker

[0185]

Table 36

[0186]

Table 37

[0187] MPSC1 Culture Condition 2: Pancreatic Progenitor Cell Marker

[0188]

Table 38

[0189]

[0175] Bold characters indicate markers that consistently increased or decreased after FBF treatment.

[0190]

Table 39

[0191] MPSC2 Culture Condition 1: Pancreatic Progenitor Cell Marker

[0192]

Table 40

[0193]

Table 41

[0194] MPSC2 Culture Condition 2: Pancreatic Progenitor Cell Marker

[0195]

Table 42

Table 43

[0196] , , seem to be in a special format (maybe some kind of highlighting or specific code). Since the instruction is to preserve them exactly, they are shown in red in the translation for better visibility. If there is a specific meaning or expected formatting for these tags, it might need further clarification based on the context.

[0197]

[0176] The PPC markers of the cells produced are PDX1, FOXA2, SOC9, and This includes one or more of any combination thereof. For example, a PPC cell includes one of PDX1, FOXA2, and SOC. In another example, a PPC cell includes two of PDX1, FOXA2, and SOC. For example, a PPC may include PDX1 and FOXA2, PDX1 and SOC, or FOXA2 and SOC. In yet another example, a PPC cell includes 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 ) on the shaker, together with the microcarrier The cells were cultured in (1) MESENCULT®, (2) MESENCULT® + BSA, (3) MESENCULT® + PLU, or (4) Rooster medium. After being expanded for three days, the cells were analyzed by trypan blue exclusion test and live / dead cell imaging.

[0201]

[0178] Figure 11A is a graph showing the number of cells after 72 hours. Each bar represents the number of cells. Dead cells are shown at the top, and living cells are shown at the bottom. Figure 11B is a graph showing population doubling for each type in the culture medium.

[0202] Example 15: Expansion of MPSCs in a bioreactor

[0179] MPSC (approximately 4,600 cells / cm 2 ) together with the microcarrier, at approximately 7 rpm at 25 rpm Cells were cultured in Rooster MXC XF medium in 100 mL of PBS bioreactor for several days. Cell count and viability were determined. FACS characterization of cell markers was performed comparing adherent vs. suspension culture.

[0203]

[0180] Figure 12A is a graph showing cell counts for different culture durations. Δ D2-D 6 = 44,000,000 cells. Figure 12B is a graph showing the percentage of viable cells in culture. Figure 12C is a graph showing population doubling comparing adherent vs. suspension culture. Δ D2-D6 = 4.9PD. Adherent culture initially doubled faster than suspension culture, but over time, suspension culture achieved a faster rate of population doubling.

[0204]

[0181] MPSC and NK markers for adhesion and suspension cell culture are shown below. That is correct.

[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 (trademark) 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 just an example. 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.

Claims

1. A population of lethal pluripotent stem cells (MPSCs) that express HLA-G and insulin and can achieve population doubling of at least 89 within approximately 90 days of starting MPSC culture.

2. The population of MPSCs according to claim 1, which can achieve population doubling of approximately 25 to approximately 30 within approximately 12 days from the start of MPSC culture, population doubling of approximately 50 to approximately 55 within approximately 30 days, and / or population doubling of approximately 75 to approximately 80 within approximately 63 days.

3. A group of MPSCs according to claim 1 or 2, which can be doubled in approximately 22 to 27 hours.

4. A group of MPSCs according to claim 3, which can be doubled in approximately 25 hours.

5. A population of lethal pluripotent stem cells (MPSCs) that express HLA-G and insulin, and are free of pathogens.

6. A group of MPSCs according to any one of claims 1 to 5, wherein the MPSCs are bacteria-free.

7. A group of MPSCs according to any one of claims 1 to 6, wherein the MPSCs are virus-free.

8. A group of MPSCs according to any one of claims 1 to 7, wherein the MPSCs do not contain cytomegalovirus.

9. A group of MPSCs according to any one of claims 1 to 8, wherein the MPSCs do not include pathogens such as 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.

10. The group of MPSCs according to claim 9, wherein the MPSCs do not contain hepatitis viruses, and the hepatitis viruses include hepatitis A, hepatitis B, hepatitis C, or a combination thereof.

11. The group of MPSCs according to claim 9, wherein the MPSCs do not contain herpes simplex virus (HSV), and the herpes simplex virus (HSV) includes human herpesvirus 6 (HHV6), human herpesvirus 8 (HHV8), or a combination thereof.

12. The group of MPSCs according to claim 9, wherein the MPSCs do not contain human immunodeficiency virus, and the human immunodeficiency virus includes human immunodeficiency virus 1 (HIV1), human immunodeficiency virus 2 (HIV2), or a combination thereof.

13. The group of MPSCs according to claim 9, wherein the MPSC does not contain human papillomavirus, and the human papillomavirus includes HPV16, HPV18, or a combination thereof.

14. The group of MPSCs according to claim 9, wherein the MPSC does not contain herpes simplex virus, and the herpes simplex virus includes herpes simplex virus (HSV1), herpes simplex virus (HSV2), or a combination thereof.

15. The group of MPSCs according to claim 9, wherein the MPSCs do not include human T lymphotropic virus, and the human T lymphotropic virus includes human T lymphotropic virus (HTLV-1), human T lymphotropic virus (HTLV-2), or a combination thereof.

16. The group of MPSCs according to claim 9, wherein the MPSCs do not contain Corynebacterium, and Corynebacterium includes Corynebacterium bovis, Corynebacterium species (HAC2), or a combination thereof.

17. The group of MPSCs according to claim 9, wherein the MPSC does not contain hantavirus, and the hantavirus includes Hantan hantavirus, Seoul hantavirus, Syn Nombre hantavirus, or a combination thereof.

18. A population of MPSCs according to any one of claims 1 to 17, further expressing one or more proteins from among β-HCG, HSP90, CDX2, FGFR1, pAKT, pCREB1, HLA-A, HLA-B, HLA-C, or any combination thereof.

19. A population of MPSCs according to any one of claims 1 to 18, further expressing one or more proteins from among 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.

20. A population of MPSCs according to any one of claims 1 to 19, further expressing one or more proteins from among IL-6, IL-8, MCP-1, CLXL2, PDGF-AA, VEGF, PAI-1, IL-10, or any combination thereof.

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 proteins from among Ki-67, HSP70, p53, syncytin, or a combination thereof.

22. A population of MPSCs according to any one of claims 1 to 21, expressing one or more proteins from among CD44, CD90, CD105, CD146, CD166, HLA-A, HLA-B, HLA-C, or a combination thereof.

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 proteins from among CD19, CD45, HLA-DR, or a combination thereof.

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 proteins from among CD19, CD45, HLA-DR, or a combination thereof.

25. A group of MPSCs according to any one of claims 1 to 24, expressing CD16, CD56, or a combination thereof.

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.

27. A population of MPSCs according to any one of claims 1 to 26, wherein more than 96% of the MPSCs do not express CD3.

28. A population of MPSCs according to any one of claims 1 to 27, wherein at least 65% of the population of MPSCs express HLA-G.

29. The group of MPSCs according to claim 28, wherein HLA-G includes HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, HLA-G7, or any combination thereof.

30. The group of MPSCs according to claim 28 or 29, wherein HLA-G includes HLA-G2, HLA-G4, HLAG-6, HLA-G7, or any combination thereof.

31. A group of MPSCs according to any one of claims 28 to 30, wherein HLA-G includes HLAG-6, HLA-G7, or a combination thereof.

32. A population of MPSCs according to any one of claims 1 to 31, wherein less than 15% of the MPSC population expresses HLA-G1.

33. A population of MPSCs according to any one of claims 1 to 32, wherein at least 10% are monoclonal.

34. A population of MPSCs according to claim 25, wherein approximately 13% to approximately 15% are monoclonal.

35. at least 1 x 10 ６ A group of MPSCs according to any one of claims 1 to 34, comprising an MPSC.

36. A population of MPSCs according to any one of claims 1 to 35, wherein the MPSCs have a stable karyotype when measured by an array-based whole-genome assay.

37. A population of MPSCs according to any one of claims 1 to 36, wherein the MPSCs do not exhibit chromosomal abnormalities derived from population doubling when measured by an array-based whole-genome assay.

38. A population of MPSCs according to any one of claims 1 to 37, wherein the MPSCs do not show substantial chromosomal abnormalities originating from freezing and thawing when measured by an array-based whole-genome assay.

39. A method for growing a population of lethal pluripotent stem cells (MPSCs), comprising approximately 1,000 to 5,000 cells / cm³ in the culture medium. ２ A method comprising the steps of seeding a subculture of MPSCs at a density, and culturing the cells.

40. A method for growing a population of lethal pluripotent stem cells (MPSCs), comprising approximately 1,000 to 5,000 cells / cm³ in the culture medium. ２ A method comprising the steps of seeding a subculture of MPSCs at a density and culturing the cells, wherein the population of MPSCs expresses HLA-G and insulin.

41. The method according to claim 39 or 40, wherein the culture medium does not contain animal components.

42. The method according to any one of claims 39 to 41, wherein the culture medium does not contain serum.

43. The method according to claim 38, wherein the culture medium does not contain fetal bovine serum.

44. The method according to any one of claims 39 to 43, wherein the MPSC is cultured for approximately 3 days.

45. The method according to any one of claims 39 to 44, wherein the MPSC is cultured for about four days.

46. The subculture of MPSCs contains approximately 2,000 to 4,000 cells / cm². ２ The method according to any one of claims 39 to 45, wherein the seeds are sown at a density of .

47. A population of cells produced by the method described in any one of claims 39 to 46.

48. A method for producing cells, comprising the step of contacting a population of MPSCs according to any one of claims 1 to 38 with one or more inducers.

49. The method according to claim 48, wherein the cells produced are ectoderm cells.

50. The method according to claim 48, wherein the cells produced are mesoderm cells.

51. The method according to claim 48, wherein the cells produced are endoderm cells.

52. The method according to claim 48, wherein the cells produced are pancreatic cells or pancreatic progenitor cells (PPCs).

53. The method according to claim 52, wherein one or more inducers include bFGF (basic fibroblast growth factor).

54. The method according to claim 53, wherein one or more inducers further comprise 2-mercaptoethanol and nicotinamide.

55. The method according to any one of claims 52 to 54, wherein the PPC comprises β-HCG, CDX2, HLA-G, or any combination thereof.

56. The method according to 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.

57. The method according to claim 55 or 56, wherein the PPC further comprises PDX1, FOXA2, SOX9, or any combination thereof.

58. The method according to claim 48, wherein the cells produced are nerve cells (NCS) or neural progenitor cells.

59. The method according to claim 58, wherein one or more inducers include retinoic acid.

60. The method according to claim 58 or 59, wherein the NCS cells include RAR-β, CDX2, HLA-G, or any combination thereof.

61. The method according to 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.

62. The method according to claim 60 or 61, wherein the NCS cells further comprise N-CAD, NESTIN, SOX2, PAX6, or any combination thereof.

63. The method according to claim 48, wherein the cells produced are hepatocytes or hepatic progenitor cells.

64. The method according to claim 63, wherein one or more inducers include fibroblast growth factor (FGF), steroids, and cytokines.

65. The method according to claim 48, wherein the cells produced are natural killer cells and the inducer is FGF.

66. The method according to claim 65, wherein the natural killer cells are CD16+, CD56+, and CD3-.

67. The method according to claim 66, wherein the natural killer cells are further HLA-G+ and CDX2+.

68. The method according to claim 48, wherein the cells produced include adipocytes, chondrocytes, osteocytes, or any combination thereof.

69. The method according to claim 68, wherein the cells produced include adipocytes and chondrocytes.

70. The method according to claim 68, wherein the cells produced include adipocytes and osteocytes.

71. The method according to claim 68, wherein the cells produced include chondrocytes and osteocytes.

72. The method according to claim 68, wherein the cells produced include adipocytes, chondrocytes, and osteocytes.

73. The method according to claim 68, wherein the cells produced include adipocytes.

74. The method according to claim 68, wherein the adipocytes contain leptin, HOXC8, HOXC9, Ucp1, CIDEA, PRDM16, Zic1, Lhx8, Eva1, Epstiti1, 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.

75. The method according to claim 73 or 74, wherein the adipocytes include white adipocytes.

76. The method according to claim 75, wherein the white adipocytes contain leptin, HOXC8, HOXC9, or a combination thereof.

77. The method according to claim 73 or 74, wherein the adipocytes include brown adipocytes.

78. The method according to claim 77, wherein the brown adipose tissue comprises Ucp1, CIDEA, PRDM16, Zic1, Lhx8, Eva1, Epst1, or a combination thereof.

79. The method according to claim 73 or 74, wherein the adipocytes include beige adipocytes.

80. The method according to claim 79, wherein the beige adipocytes include Cd137, Tmem26, Tbx1, Cited1, Shox2, or a combination thereof.

81. The method according to claim 73 or 74, wherein the adipocytes include beige adipocyte precursors.

82. The method according to claim 81, wherein the beige adipocyte precursor comprises CD137, TMEM26, or a combination thereof.

83. The method according to claim 68, wherein the cells produced include chondrocytes.

84. The method according to claim 83, wherein the chondrocytes contain 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, otraprine / OTOR, URB, or a combination thereof.

85. The method according to claim 68, wherein the cells produced include osteocytes.

86. The method according to claim 85, wherein the osteocytes include preosteoblasts, osteoblasts, embedded osteoblasts, osteoid osteocytes, bone-calcifying osteocytes, or mature osteocytes.

87. The method according to claim 85 or 86, wherein the osteocytes contain RUNX2, OCN, E11, DMP1, PHEX, MEPE, sclerostin, CapG, ORP150, or a combination thereof.

88. The method according to claim 85 or 86, wherein the osteocytes include preosteoblasts, and the preosteoblasts include RUNX2.

89. The method according to claim 85 or 86, wherein the osteocytes include preosteoblasts, and the preosteoblasts include RUNX2.

90. The method according to claim 85 or 86, wherein the osteocytes include osteoblasts, and the osteoblasts include RUNX2 and OCN.

91. The method according to claim v, wherein the osteocytes include embedded osteoblasts, and the embedded osteoblasts include OCN, E11, DMP1, PHEX, and CapG.

92. The method according to claim 85 or 86, wherein the osteocytes include osteoid cells or calcified osteocytes, and the osteoid cells or calcified osteocytes include OCN, E11, DMP1, PHEX, MEPE, and CapG.

93. The method according to claim 85 or 86, wherein the osteocytes include mature osteocytes, and the mature osteocytes include DMP1, PHEX, MPEP, sclerostin, CapG, and ORP150.

94. A population of lethal pluripotent stem cells (MPSCs) expressing HLA-G and including a phenotype that includes one or more of the following: negative for indoleamine 2-3 deoxygenase (IDO) secretion, negative for kynurenine secretion, and positive for interleukin 2 (IL-2) secretion.

95. A population of MPSCs according to 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.

Images