Storage method and storage system for nt cells

Abstract

Provided are storage methods and storage systems for cells prepared using somatic cell nuclear transfer (NT) technology, the cells having homozygous genotypes for genes of human leukocyte antigen (HLA)-A, HLA-B, HLA-DR, etc. Storage of NT cell-derived stem cells can be applicable to autologous or allogeneic patients, and can provide transplantable cells and tissue materials for treatment of various diseases, such as diabetes, osteoarthritis, Parkinson's disease, etc.

Description

[0001] This application is a divisional application of the invention patent application filed on July 18, 2016, with application number 201680042142.1 and entitled "Storage Method and Storage System for NT Cells". Invention Field

[0002] This disclosure relates to a method and a cell storage system for storing cells prepared using somatic cell nuclear transfer (NT) technology, wherein the cells have a homozygous genotype of human leukocyte antigen (HLA)-A, HLA-B, HLA-DR, etc. Background of the Invention

[0003] Cell therapy is an emerging field in medicine that uses cells, particularly stem cells, to achieve a "fundamental cure" for diseases that are considered to be treatable only by drugs or surgery. Specifically, it is a new technological field for combating diseases by restoring the function of tissues and organs through regeneration or replacement, which are damaged or functionally degenerated due to aging, disease, accidents, etc., and are emerging as an alternative treatment for intractable diseases in the field of regenerative medicine.

[0004] However, for cell therapy to be more widely used, the complex problem of immune rejection must be addressed. Immune rejection is caused by six HLA (HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR) cell surface proteins called the major histocompatibility antigen complex (MHC). Humans express a total of six pairs of HLA genes, including six from the father and six from the mother. Normal somatic cells express only three pairs of HLA-A, HLA-B, and HLA-C, which belong to MHC class I, while immune cells express a total of six pairs, including MHC class I and MHC class II. The role of HLA surface antigens is to display fragments of proteins present in the cell on the cell surface, enabling immune cells to detect infections or mutations in vivo. For this reason, HLA surface antigens are also called antigen-presenting proteins.

[0005] Somatic cell nuclear transfer (SCNT) is a technique for replicating somatic cells by removing the nucleus from a somatic cell and transplanting it into an enucleated oocyte. In other words, it's a technique for generating stem cell lines using somatic cells, which retain the genetic characteristics of the somatic cells. This is achieved by isolating the nucleus of the somatic cell and injecting the isolated nucleus into an enucleated oocyte. The advantage of this method is that, because it uses the patient's own somatic cells, the immune rejection response is suppressed, thus enabling patient-specific treatment. Despite advancements in NT technology, the rate of blastocyst formation from reconstructed oocytes remains low. Therefore, various attempts have been made to improve blastocyst formation rates. In particular, the biggest obstacle in NT embryos is zygotic gene activation (ZGA), which occurs in the 4-8 cell stage in large mammals, including humans. To improve success rates regardless of donor variability, it is necessary to eliminate existing epigenetic barriers. Specifically, to successfully generate blastocysts from the 2, 4, and 8 cell stages, it is necessary to significantly increase blastocyst formation success rates by altering the epigenetic state of the donor nucleus during generation without any defects or loss.

[0006] Therefore, as described above, there is a need for methods for generating cells and cell storage systems for cell therapies in regenerative medicine and for the treatment of refractory diseases, wherein the cells eliminate immune rejection responses and enable patient-specific treatment. Invention Details

[0007] Technical issues

[0008] The inventors of this invention completed the invention by generating cell therapeutic agents or transplantable cells from homozygous cells that do not induce an immune rejection response.

[0009] A method is provided for storing autologous or allogeneic NT cell-derived stem cells suitable for the treatment of various diseases.

[0010] Methods for generating NT cell-derived stem cells and storage systems for NT cell-derived stem cells are provided.

[0011] Technical solution

[0012] According to one aspect of the present invention, a method for storing immunocompatible nuclear transfer (NT) cell-derived stem cells is provided, the method comprising:

[0013] a) Screening for homozygosity among multiple donor tissues;

[0014] b) Isolate nuclei from homozygous cells to generate NT cells;

[0015] c) Generate stem cells from the NT cells; and

[0016] d) Cryopreservation of the stem cells.

[0017] According to another aspect of the present invention, a method for generating immunocompatible nuclear transfer (NT) cell-derived stem cells is provided, the method comprising:

[0018] a) Screening for homozygosity among multiple donor tissues;

[0019] b) Isolate nuclei from homozygous cells to generate NT cells; and

[0020] c) Generate stem cells from the NT cells.

[0021] According to another aspect of the present invention, a method for generating differentiated cells from stem cells derived from immunocompatible NT cells is provided, the method comprising:

[0022] a) Screening for homozygosity among multiple donor tissues;

[0023] b) Isolate nuclei from homozygous cells to generate NT cells;

[0024] c) Generate stem cells from the NT cells; and

[0025] d) Generate differentiated cells from the stem cells for transplantation.

[0026] According to another aspect of the present invention, a storage system for stem cells derived from immunocompatible nuclear transfer (NT) cells is provided, the storage system comprising:

[0027] A device for collecting tissues from multiple donors;

[0028] Device for screening collected donor tissues;

[0029] Devices for generating stem cells from said tissue, and

[0030] A device for cryopreserving the stem cells.

[0031] Beneficial effects of the present invention

[0032] The storage of NT cell-derived stem cells according to one or more embodiments enables the treatment of various diseases or conditions, provides transplantable cell and tissue materials for the treatment of various diseases such as diabetes, osteoarthritis and Parkinson's disease, and particularly offers the possibility of fundamentally treating cell type-specific defects and therapeutic methods that reduce the risk of immune rejection response and immune tolerance with homozygous cells according to the embodiments. Brief description of the attached diagram

[0033] Figure 1The figure shows the classification of cord blood cells into transplantable cells or disposal cells under the current Korean Blood Cord Management and Research Act, which stipulates that cord blood with a cell count of less than 700 million or less should be discarded.

[0034] Figure 2 The results of chromosome testing of the donor cells in Example 1 are shown;

[0035] Figure 3 The results of chromosome testing of the nuclear transfer (NT) cells produced in Example 3 are shown;

[0036] Figure 4 The results of (a) genomic DNA testing and (b) mitochondrial DNA comparison of NT cells from Example 3, comparing donor somatic cells with donor oocytes, are shown.

[0037] Figure 5 Immunochemical results of stem cell markers in NT cells from Example 3 are shown;

[0038] Figure 6 The results of real-time polymerase chain reaction (RT-PCR) of stem cell markers in NT cells in Example 3 are shown.

[0039] Figure 7 The results show the pluripotency of NT cells in Example 3 that differentiated into 3 germ layer-derived cells, and specifically (a) the immunohistochemical results of 3 germ layer differentiation markers after the formation of embryo-like bodies and incubation for 14 days, (b) the RT-PCR results of the markers, and (c) the histological analysis results of teratomas injected into immunodeficient mice.

[0040] Figure 8 The results showed no differences in shape and differentiation markers between (a) embryonic stem cell-derived RPE cells and (b) retinal pigment epithelial (RPE) cells obtained from NT-derived stem cells in Example 3. Invention Details

[0041] The implementation plan will be described in more detail below.

[0042] As used in this article, the term "immunocompatibility antigen homozygosity" can refer to a situation where the genotypes of each HLA-A, HLA-B, and HLA-DR gene inherited from the donor's paternal and maternal lines are completely identical, and the donor has three HLA genotypes rather than six HLA genotypes.

[0043] As used in this article, the term "somatic cell" can refer to any tissue cell in the body other than reproductive (sexual) cells or their precursors.

[0044] As used herein, the term "stem cell" can refer to a self-renewing cell with at least one pluripotency (the ability to differentiate into at least one specific cell type) and the ability to undergo multiple cell division cycles while remaining undifferentiated.

[0045] As used herein, the term "long-term incubation" can refer to a long period of time during which cells proliferate under controlled conditions for two months or more, or for 10 passages (passage culture) or more. In some embodiments, long-term incubation can be performed for four months or more, six months or more, or one year or more. In some other embodiments, long-term incubation can refer to 15 passages or more, 18 passages or more, or 20 passages or more. The duration of long-term incubation depends primarily on the individual cells and can vary from cell line to cell line.

[0046] As used in this article, the term “maturation” can refer to a series of coordinated biochemical steps that lead to the eventual differentiation of cells.

[0047] As used in this article, the term “differentiation” can refer to the adaptation of cells to a particular type or function.

[0048] As used herein, the term "differentiated cell" can encompass any somatic cell that is not pluripotent in its initial form, as defined herein. Therefore, the term "differentiated cell" can also encompass partially differentiated cells, such as pluripotent cells, or stable non-pluripotent, partially reprogrammed, or partially differentiated cells using any of the compositions and methods described herein. In some embodiments, the differentiated cell can be a stable intermediate cell, such as a non-pluripotent, partially reprogrammed cell. It should be noted that adding a large number of primary cells to a culture product can result in a slight loss of fully differentiated characteristics. Therefore, simple incubation of differentiated cells or somatic cells does not guarantee that the cell will become an undifferentiated cell (e.g., undifferentiated cell) or a pluripotent cell. Transferring differentiated cells (including stable, non-pluripotent, partially reprogrammed cell intermediates) to pluripotent cells may require reprogramming stimuli exceeding levels that could cause partial loss of differentiated characteristics upon addition of the cells to the culture. In some embodiments, reprogrammed cells, such as partially reprogrammed cells, can generally be cultured in extended passage cultures without loss of growth potential compared to parental cells with a lower probability of generation and a limited capacity for division. In some implementations, the term "differentiated cell" may refer to a further specialized cell (i.e., with a reduced probability of generation) derived from less specialized types (i.e., cells with an increased probability of generation) (which undergo intracellular differentiation) (e.g., cells derived from undifferentiated cells or reprogrammed cells).

[0049] In some implementations, the differentiating cells may be selected from the group consisting of hematopoietic stem cells, cardiomyocytes, hepatocytes, chondrocytes, epithelial cells, urethral cells, adipocytes, kidney cells, vascular cells, retinal cells, mesenchymal stem cells (MSCs), and neurons. However, the implementation is not limited to this.

[0050] The term "immunocompatible cells" as used in this article is not specifically defined and can refer to cells that are homozygous for HLA-A, HLA-B, or HLA-DR genes, and can be implanted into recipients who have any combination of HLA genotypes, having 3 of the 6 pairs of identical genotypes.

[0051] As used herein, the term "bank" refers to a storage location for stem cells that can be used, either as is or differentiated, for therapeutic, clinical, or research purposes for the donor or other patients.

[0052] As used herein, the term "administration" can refer to the introduction of a particular substance into a patient in any appropriate manner, such as via any general route through which the substance can reach the target tissue. For example, administration can be intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, or rectal administration. However, the implementation method is not limited to these. Administration can also be performed using any device capable of reaching the target cells.

[0053] According to one aspect of this disclosure, a method for storing stem cells derived from immunocompatible nuclear transfer (NT) cells includes: a) screening for homozygosity in multiple donor tissues; b) isolating nuclei from homozygous cells to generate NT cells; c) generating stem cells from NT cells; and d) cryopreserving the stem cells.

[0054] Cells obtained through NT transplantation carry the patient's nuclear genetic material and are, in this respect, individual patient-specific. This form of autologous transplantation allows for cell transplantation into patients with a significantly reduced risk of autoimmune rejection. Furthermore, as cells with matching HLA antigen types, homozygous cells, as used in this article, can be transplanted into allogeneic patients without anti-HLA antibodies. In other words, NT-derived stem cells are homozygous and can be transplanted into immunocompatible cells.

[0055] Therefore, in step a), screening can be performed on cells homozygous for human leukocyte antigen (HLA)-A, HLA-B, and HLA-DR genes. It has been reported that if 140 individuals with different levels of immunocompatibility are identified through HLA genotyping, an immunocompatibility cell line suitable for transplantation into approximately 90% or more of the total Japanese population can be secured in advance (A more efficient method to generate integration-free human iPS cells, Nature Methods 8, 409-412 (2011)).

[0056] In one implementation, predominantly homozygous cells are screened based on data from the CHA Public Cord Blood Bank (iCord) in Korea. Under the current Cord Blood Management and Research Act, with the approval of the Cord Blood Committee of the Ministry of Health and Welfare, research can be conducted using cord blood units classified as disposal cells with a cell count of 700 million (see [link to relevant document]). Figure 1 ).

[0057] Table 1 represents the HLA AB-DRB1 haplotype frequency in 4,128 cord blood units (accounting for only 0.1% or more, International Journal of Immunogenetics (2013) 40: 515-523).

[0058] Table 1

[0059]

[0060] Therefore, suitable frozen umbilical cord blood (with a cell count of less than 700 million) can be used as the cells for this study. In addition to these cells, any donated umbilical cord blood registered with the Korean Network for Organ Sharing (KONOS) for research use can also be used. Furthermore, samples collected from hematopoietic stem cell donor networks and CHA hospital-affiliated medical facilities can also be used.

[0061] In some implementations, step b) of generating NT cells may include: enucleating an oocyte; fusing the nucleus of a somatic cell with the enucleated oocyte; and culturing the fused oocyte in an activated culture medium.

[0062] In some embodiments, a method for preparing NT-derived stem cells may include enucleating an oocyte; adding at least one nucleus of at least one donor cell to produce an NT oocyte; incubating the NT oocyte in an activation medium to activate the NT oocyte; and generating a blastocyst from the activated NT oocyte.

[0063] In other embodiments, oocyte enucleation includes removing the oocyte spindle during metaphase II (Mil) of cell division. In several embodiments, the first polar body (IPBE) is removed. In another embodiment, the method includes enucleating the cumulus cell before maturation is complete. In one embodiment, the IPBE of the oocyte is monitored using real-time, non-UV light-based monitoring. In another embodiment, monitoring occurs in the absence of staining agents or markers such as Hoechst staining. In one embodiment, this includes using a poloscope, such as the Research Instruments (CRi) Oosight™ imaging system. For example, this could include visualizing the zona pellucida and spindle complex in harvested '5 harvested Mil oocytes with 545 nm polarized light. In another embodiment, oocyte enucleation includes using a contoured micropipette with a wavy profile, allowing puncture of the oocyte membrane and removal of the IPBE from the oocyte. In another embodiment, oocyte enucleation includes using a piezoelectric drill. In other embodiments, enucleation is performed in an enucleation medium containing cytochalasin B and optionally a protein phosphatase inhibitor such as caffeine.

[0064] Caffeine, as a protein phosphatase inhibitor, can suppress premature activation, thereby improving the growth of cloned embryos and increasing the blastocyst formation rate. Therefore, enucleation of oocytes can be performed in a culture medium containing a protein phosphatase inhibitor. Caffeine can be a protein phosphatase inhibitor.

[0065] In some other embodiments, adding at least one nucleus of at least one donor cell to produce an NT oocyte may include transplanting at least one donor nucleus.

[0066] Donor nucleus transfer may include the use of agents that alter the oocyte membrane. In one embodiment, oocyte enucleation using an agent that alters the oocyte cell membrane structure includes fusion with a somatic cell. For example, donor nucleus transfer may include providing 3-4 donor cells and an injection pipette (e.g., 12 μm in diameter), discharging the donor cells in a solution containing paramyxovirus or paramyxovirus proteins, such as Sendai virus envelope proteins. This is followed by retrieval of the cells with the injection pipette at a distance of 4-5 cell lengths between the linearly arranged donor cells, holding the oocyte with a holding pipette, and advancing the injection pipette containing the donor cells into the oocyte. In various embodiments, advancing the injection pipette includes not damaging the oolema membrane and inserting a nuclear donor cell into the perivitelline space of the oocyte, i.e., the space between the zona pellucida and the cell membrane, so that the nuclear donor cell contacts the oolema membrane, which is located below the zona pellucida. In various embodiments, withdrawal of the pipette does not interfere with the contact between the oolema and the donor cell. In various embodiments, the oocytes are further incubated. In various embodiments, cell fusion is performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 minutes or longer after donor cell insertion. In several embodiments, cell fusion is performed 10 minutes after donor cell insertion. Optionally, the above procedure is repeated for cells that do not fuse successfully. In various embodiments, Oosight or similar technology is used throughout the process. TM Polar mirror of the imaging system.

[0067] In some embodiments, donor cell nucleus transfer is performed via direct injection. In one embodiment, donor nucleus transfer may include electrocytological manipulation, such as electrofusion. In other embodiments, the method may include isolating nuclei from somatic cell nuclei, stem cell nuclei, and germ cell nuclei. In other embodiments, the method may include isolating somatic cell nuclei for SCNT and then injecting one or more donor nuclei via pipette or piezoelectric injection. In several embodiments, the donor nuclei are derived from cells, such as skin fibroblasts, leukocytes, hair follicles, or any other somatic cell nucleus donor. In another embodiment, the invention describes a method comprising isolating and preparing nuclei from germ cell donors. In various embodiments, nucleus isolation includes tissue biopsy, blood sampling, or other means of obtaining tissue samples, processing the tissue by mechanical decomposition, collagenase digestion, washing, centrifuge-based density gradient separation, and / or culturing with standard culture media.

[0068] In some implementations, somatic cell nuclear fusion can be performed in a culture medium containing Sendai virus or Sendai virus extract.

[0069] After fusing the somatic cell nucleus with the enucleated oocyte, the fused oocyte can be transferred into an activated culture medium and then activated.

[0070] In some implementations, step c) of generating stem cells from NT cells may include: incubating NT oocytes in an activation medium to activate the NT oocytes; generating blastocysts from the activated NT oocytes; and isolating inner cell mass (ICM) cells from the blastocysts. The ICM cells may be further cultured as a nuclear transfer human pluripotent stem cell line (NT-hPSC).

[0071] Oocyte activation (artificial oocyte activation) depends on changes in calcium signaling that mimic natural sperm fertilization.

[0072] Normal oocyte development depends on high levels of metaphase-promoting factor (MPF) activity to arrest the oocyte at metaphase II (Mil). Mil oocyte arrest is influenced by the surge in intracellular calcium ions (Ca) induced by sperm entry. 2+ Changes in cyclin levels disrupt this process. This is followed by the targeted degradation of cyclin B (a regulatory subunit of MPF) that releases oocytes from arrest, pronuclear formation, and the initiation of meiosis and mitosis.

[0073] Oocyte activation relies on artificial calcium-modification strategies to release cultured oocytes from arrest. Examples include the addition of calcium ion carriers, i.e., lipid-soluble molecules that transport ions across the lipid bilayer, such as iomycin and A23817. Alternative strategies rely on electroactivation or direct injection of ions.

[0074] As is associated with NT, the reconstruction of transferred nucleated (i.e., reconstructed) oocytes is followed by oocyte activation using calcium alteration techniques.

[0075] For example, while the addition of the protein phosphatase inhibitor caffeine to sheep oocytes has been reported to increase the activity of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), and similar benefits have been reported in monkey oocytes, it does not enhance the frequency of blastocyst formation. Furthermore, calcium activation via calcium ionophores, electroactivation, or direct injection does not produce the same timing, spatial regulation, or duration of calcium oscillations as in natural fertilization. Adding further complexity is that the effects on calcium also appear to be species-specific. In some cases, additional treatments using kinase inhibitors such as 6-dimethylaminopurine (6-DMAP), ethanol, and protein synthesis inhibitors such as cycloheximide (CHX) have been employed to enhance MPF inactivation.

[0076] Histone deacetylase inhibitors, such as TAS, are associated with improved NT reprogramming. Treatment with TSA can promote blastocyst formation.

[0077] In some embodiments, an electrical pulse may be applied during the somatic cell nuclear fusion and activation process. Electroactivation may involve an electrical pulse in the electrofusion medium. In some embodiments, the electrofusion medium may contain 0.1 to 0.5 M mannitol, 0.01 to 1 mM MgSO4·7H2O, 0.01 to 1 mg / ml polyvinyl alcohol, 1 to 10 mg / ml human serum albumin, and 0.005 to 0.5 mM CaCl2·2H2O. In some other embodiments, the electrofusion medium may include 0.3 M mannitol, 0.1 mM MgSO4·7H2O, 0.1 mg / ml polyvinyl alcohol, 3 mg / ml human serum albumin, and 0.05 mM CaCl2·2H2O.

[0078] In several embodiments, nuclear transfer (NT) oocytes are treated in an activated culture medium to complete activation. In various embodiments, the activated, reconstituted NT oocytes are then incubated in the activated culture medium. In various embodiments, the activated culture medium is a HEPES-free medium, a protein-free medium, G1 or G2 medium, cleavage medium, cleavage-assisted medium, IVF medium, blastocyst formation medium, or global human embryo medium. In various embodiments, the activated culture medium includes 6-DMAP, puromycin, ethanol, cyclohexylimide (CHX), aspergillus sulfadiazine A (TSA), and / or cytochalasin B (CB). In various embodiments, activated oocytes are incubated in a post-activation culture medium for less than 30, 30-45, 45-60, 60-90, 90-120, 120-150, 150-180, 180-210, 210-240, 240-270, 300-330, 330-360, 360-390, or more than 390 minutes. In some embodiments, activated oocytes are incubated for 240, 300, or 360 minutes. In many embodiments, activation and post-activation steps are performed under reduced oxygen conditions. In some embodiments, reduced oxygen conditions include about 80-85%, 85-90%, 90-95%, 95% or more N2, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more O2, and about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more CO2. In some embodiments, the reduced oxygen conditions include approximately 90% N2, approximately 5% O2, and approximately 5% CO2. In multiple embodiments, the activated culture medium comprises 1, 2, 3, 4, 5, 5 mM or more of 6-DMAP in cleavage medium, said cleavage medium being incubated at a temperature such as 37°C in a gas mixture, such as approximately 90% N2, approximately 5% O2, and approximately 5% CO2, for 1, 2, 3, 4, 5, 5 hours or more.

[0079] After incubation in the activated medium, the activated oocytes are incubated in a washing medium. In various embodiments, the washing medium is a HEPES-free medium, a protein-free medium, G1 or G2 medium, cleavage medium, cleavage-assisted medium, IVF medium, or blastocyst formation medium. In another embodiment, the medium does not require continuous medium replacement, such as global human embryo culture medium. In some embodiments, the washing medium contains TSA. In some embodiments, the activated oocytes are incubated in a TSA-containing washing medium for 240, 300, or 360 minutes. In one embodiment, the activated reconstituted nuclear transfer oocytes are washed and further cultured. In one embodiment, the activated reconstituted nuclear transfer oocytes are washed in a 6-DMAP-free medium. In other embodiments, various of the aforementioned media, such as HEPES-free medium, protein-free medium, G1 or G2 medium, cleavage medium, cleavage-assisted medium, IVF medium, blastocyst formation medium, or global human embryo culture medium, optionally contain growth factors such as GM-CSF or IGF1. In several implementations, growth factors can be added 1, 2, 3, 4, 5, 6, 7 or more days after nuclear transfer.

[0080] In another embodiment, the activation and / or post-activation steps include the addition of factors isolated from sperm, their derivatives, and extracts. In one embodiment, human sperm factors are injected into activated reconstructed oocytes using any of the described injection methods. In one embodiment, human sperm factors are injected into activated reconstructed oocytes using any of the described injection methods. In several embodiments, activated reconstructed nuclear transfer oocytes are transferred to cleavage medium after approximately 1, 2, 3, or 4 days. In one embodiment, activated reconstructed nuclear transfer oocytes are transferred to cleavage medium after approximately 1 day. In several embodiments, the sperm factors include, for example, factors obtained from the isolation of cellular proteins present inside or outside sperm cells. In one embodiment, intact sperm extract is obtained by mechanical mixing with a detergent and ejaculated sperm. In another embodiment, intact sperm cell extract is treated with DNase I and RNase. In another embodiment, crude extract is washed in buffer and centrifuged (20,000 g, up to 2 hours). In other embodiments, fresh ejaculated human sperm is collected and centrifuged at 900g for 10 minutes to remove seminal plasma. The pellets are then resuspended in sperm-TALP containing 5 mg / mL bovine serum albumin and centrifuged under the same conditions. The supernatant is then removed, and the pellets are resuspended to a final concentration of 20 x 10⁻⁶ in nuclear separation medium (NIM: 125 mM KCl, 2.6 mM NaCl, 7.8 mM Na₂HPO₄, 1.4 mM KH₂PO₄, 3.0 mM EDTA disodium salt; pH 7.45).8 Sperm count / ml was measured and centrifuged to remove sperm-TALP. After removing sperm-TALP, the clumps were resuspended to the same volume with NIM containing 1 mM dithiothreitol, 100 mM leupeptin, 100 mM antiproteasome, and 100 mg / mL soybean trypsin inhibitor. This was followed by four cycles of freezing (5 minutes per cycle in liquid nitrogen) and thawing (5 minutes per cycle at 15°C) to form tight sperm clumps at 2°C for 50 minutes at 20,000X. Finally, the resulting supernatant was carefully collected, aliquoted, and kept at -80°C until use.

[0081] In several embodiments, the activated and reconstituted nuclear-transferred oocytes are further cultured into blastocysts. In one embodiment, the activated and reconstituted nuclear-transferred oocytes are further cultured in SAGE cleavage medium, such as Quinn medium. In another embodiment, the medium promotes pluripotency, such as 3i medium (Neuro basal medium 50%, DMEM / F-12 50%, N2 supplement 1 / 200 v / v, B27 supplement 1 / 100 v / v, 100mM L-glutamine 1 / 100 v / v, 0.1M B-ME 1 / 1000 v / v, SU5402 (FGFR inhibitor) 2μM, PD1 84352 (ERK cascade inhibitor) 0.8μM, CHIR99021 (GSK3 inhibitor) 3μM) or a modified 3i medium (containing PD0325901 (MAPK inhibitor) 0.4μM). In one embodiment, further culturing for 1, 2, 3, 4, 5, 5, or more days is performed. In one embodiment, additional culturing is provided in a medium containing a reprogramming factor and / or a methylation modifier. In several embodiments, further culturing for 3 days is performed in G2 medium supplemented with CARM1 and / or Esrrb. For example, CARM1 and / or Esrrb may be provided in the medium at concentrations of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 μg / ml or more. In some embodiments, CARM1 and / or Esrrb are provided in the medium at a concentration of 2 μg / ml.

[0082] In several embodiments, further culturing into blastocysts and deriving pluripotent stem cells (pSCs) from blastocysts includes treating the cultured blastocysts with acidic Tyrode solution to remove the zona pellucida (ZP). In several embodiments, the treatment is performed for several seconds (e.g., 1-5 seconds). In several embodiments, the blastocysts are washed in Hepes-HTF medium after ZP removal. In several embodiments, separation of the inner cell mass (ICM) includes discarding the feeder layer of the blastocyst. In several embodiments, ICM cells are plated onto mouse embryonic feeder cells (MEF) prepared one day prior to plating. In some embodiments, the entire blastocyst is plated onto MEF. For example, this method includes peeling off the zona pellucida of the blastocyst. In several embodiments, the method includes removing the zona pellucida of the blastocyst in Hepes-HTF medium with 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1% streptomycin. In one embodiment, the method includes removing the zona pellucida of the blastocyst with 0.5% streptomycin in Hepes-HTF medium. In another embodiment, the method includes applying streptomycin to TH3 (SAGE blastocyst medium) culture for 1-10, 10-20, 20-30, 30-60, 60-120, 120-180, or >180 seconds. In another embodiment, the method includes applying 0.5% streptomycin to HTF medium for 30-60 seconds. In one embodiment, the blastocyst is derived from a parthenogenetic organism (parthenote) obtained from oocytes through parthenogenesis. In one embodiment, the hPSC line is a parthenogenetic-derived human pluripotent hPSC (pn-hPSC) cell line. In another embodiment, the blastocyst is derived from a reconstructed nuclear transfer oocyte obtained from a donor cell nuclear docking recipient oocyte somatic cell nuclear transfer (SCNT). In one embodiment, the hPSC line is a somatic cell nuclear transfer human pluripotent hPSC (NT-hPSC) cell line.

[0083] In another embodiment, the present invention describes a method of immunosurgery, comprising mechanically dispersing the inner cell mass (ICM) from trophoblastic ectoderm cells. In several embodiments, enucleated blastocysts are treated with rabbit anti-human spleen serum at 37°C for about 10, 20, 25, 30, 35, 40, 45, or 60 minutes. In one embodiment, enucleated blastocysts are treated with rabbit anti-human spleen serum at 37°C for about 30 minutes. In one embodiment, the method comprises washing the enucleated blastocyst with TH3 (SAGE blastocyst medium) and incubating it at 37°C in guinea pig complement reconstituted with HECM-9 (SAGE blastocyst medium) for 30 minutes. In various embodiments, the zona pellucida of the dilated blastocyst is removed by brief exposure (45-60 seconds) in TH3 (hepes-HTF) medium to 0.5% streptomycin or acidic Tyrode solution. In one embodiment, the method optionally includes separating inner cell mass cells from trophectoderm cells using mechanical cell dispersion via micropipette or laser-assisted incubation using a Zilos-tk unit (Hamilton Thorne).

[0084] In some embodiments, the activated culture medium can be cultured in a medium containing TSA. The activated culture medium may contain 6-DMAP. In some other embodiments, after culturing in a medium containing 6-DMAP during the activation period, further culturing can be carried out in a medium containing TSA.

[0085] In several implementations, to increase nuclear NT success rates, the nucleus of at least one donor cell can be modified by contacting an epigenetic modifier. Epigenetic modifiers can enhance NT efficiency by altering the methylation or acetylation state of specific proteins or DNA. Targets of epigenetic modifiers may include at least one of histone acetyltransferase (HAT) proteins, histone deacetylase (HDAC) proteins, lysine dimethyltransferase (KDM) domain proteins, and protein methyltransferase (PMT) domain proteins. These agents may include small interfering RNA (siRNA), small molecules, proteins, peptides, antibodies, etc. These agents can act on epigenetic targets associated with reprogrammed resistance regions. NT oocytes can be cultured in the presence of such epigenetic modifiers. Examples of these agents are presented in Tables 2-5. Examples of histone acetyltransferase (HAT) proteins, histone deacetylase (HDAC) proteins, lysine dimethyltransferase (KDM) domain proteins, and protein methyltransferase (PMT) domain proteins are not limited to those shown in the table below.

[0086] Table 2. Histone acetyltransferase (HAT) protein

[0087]

[0088] Table 3. Histone deacetylase (HDAC) proteins

[0089]

[0090] Table 4. Lysine dimethyltransferase (KDM) domain proteins

[0091]

[0092] Table 5. Protein Methyltransferase (PMT) Domain Proteins

[0093]

[0094]

[0095]

[0096] Typically, methyltransferases can be inhibited by co-substrate analogs. Three types of co-substrate analogs are known to inhibit multiple types of methyltransferases: sinefugin, an antibiotic compound structurally similar to S-adenosylmethionine (SAM), and dimethylated co-substrate SAH and methylthioadenosine as feedback inhibitors. Lysine methyltransferase inhibitors may include the first identified inhibitor, chaetocin, and BIX-01294, a G9a (KMT1C) inhibitor selective for both SUV39H1 and PRM1. The compound BIX-01338 (a mildly non-selective inhibitor without selectivity between lysine and arginine methyltransferases) inhibits G9a with an IC50 of 5 mM and PRMT1 with an IC50 of 6 mM. UNC0224 is a novel inhibitor of the lysine methyltransferase G9a with an IC50 of 15 mM. Histone methyltransferase inhibitors such as EPZ5676, EPZ005687 and GSK126 have also shown anticancer activity in various animal cancer models.

[0097] Protein arginine methylation can be carried out by PRMTs, which are classified into two groups: type I methyltransferases and type II methyltransferases. Type I methyltransferases can form asymmetrically substituted arginine residues, while type II methyltransferases can form symmetrically substituted arginine residues. CARM1 shows an affinity for the proline-glycine-methionine-arginine motif (the so-called PGM motif). PRMT5 is also known to methylate the PGM motif. Co-substrate analogs such as sinefungin can also be used as arginine methyltransferase inhibitors (also known as AMI). AMI-1 is the most active inhibitor with an IC50 of 9 mM. Allatodapsone and stilbamidine inhibitors can induce hypomethylation of H4R3.

[0098] Another type of epigenetic target can enhance NT efficiency. DNA methyltransferases (DNMTs) preferentially methylate the CpG nucleotide sequence of DNA. Three mammalian DNA methyltransferases, DNMT1, DNMT3A, and DNMT3, have been identified. Typically, methylation of these promoter regions can inhibit gene expression by interfering with the binding of transcription factors to DNA. Additionally, methylated DNA is bound to proteins with methyl-CpG binding domains. These proteins can attract histone modeling enzymes, thus condensing chromatin structure and inducing gene expression repression mechanisms. DNMT inhibitors can suppress gene expression repression, thus enhancing NT efficiency. Examples of DNMT inhibitors include several compounds, including chlorogenic acid, styromycin, azacytide, bisdemethoxycurcumim, decitabine, lomegutatrib, benzylguanine, sorafenib, and sorafenib tosylate.

[0099] Furthermore, histone deacetylases (HDACs) remove acetyl groups from the N-acetylsine amino acids of histones, producing histones with a higher positive charge, which then bind strongly to negatively charged DNA. DNA condensation and genetic transcription can be further inhibited. HDACs can be divided into four subgroups based on their location and function. Type I HDACs (subtypes 1, 2, 3, and 8) are mainly located in the nucleus, while type II HDACs (subtypes 4, 5, 6, 7, 9, and 10) can cross the nuclear membrane and are present in both the nucleus and cytoplasm. Type III HDACs are called silencing signal regulator 2 (Sir2), and type IV HDACs (subtype 11) are present in both the nucleus and cytoplasm and are mainly located in brain, heart, and muscle cells. When administered in combination with other chemically synthesized drugs, HDAC inhibitors can exhibit anticancer activity. HDAC inhibitors can promote DNA transcription, thus increasing NT efficiency.

[0100] The activated culture medium may include epigenetic modifiers, such as epigenetic chromatin and β-histone modifiers, and / or DNA modifiers. In some embodiments, these epigenetic modifiers may be selected from the group consisting of: protein arginine methyltransferase (PRMT1) and co-activator-associated arginine methyltransferase 1 (CARM1 / PRMT4) or solitary nuclear receptor estrogen-associated receptor β (Esrrb) protein; or may be RNA or protein selected from lysine-specific demethylase 4A (lysine (K)-specific demethylase 4A, Kdm4a), lysine-specific demethylase 4B (lysine (K)-specific demethylase 4B, Kdm4b), or lysine-specific demethylase 4D (lysine (K)-specific demethylase 4D, Kdm4d). In some embodiments, the methylation modifier and / or DNA modification is expressed as a modified recombinant protein. For example, CARM1 and Esrrb can be modified with 7xarginine (7R) cell-penetrating peptide (CPP) or any other protein known to those skilled in the art to enhance protein and peptide penetration through cell and nuclear membranes, enhance DNA binding, and / or transactivation. In other embodiments, the method may include exogenously reprogramming nuclear donor cells using transcription factor-based reprogramming, wherein the reprogramming is performed with octamer-binding transcription factor-4 (Oct-4), sex-determining region Y-box-2 (Sox-2), nanog, Kruppel-like factor-4 (Klk-4), MyoD, c-Myc, zinc finder protein-42 (Rex-1 / Zfp-42), leftyA, teratoma-derived growth factor (Tdgf), and / or telomere repeat binding factor (Terf-1). In various embodiments, the method includes direct piezoelectric injection, viral injection, liposome injection, or other intracytoplasmic injection methods. In various embodiments, transcription factors can be delivered in the form of mRNA, protein, and / or cell extracts, which can be applied prior to nuclear transfer to enucleated oocytes. In other embodiments, the method may include the use of HDAC inhibitors (class I, II, and III) or DNMT3a and DNMT3b inhibitors.

[0101] In some embodiments, the activated culture medium may include an epigenetic modifier. In some embodiments, the epigenetic modifier may involve at least one selected from histone acetyltransferase (HAT) protein, histone deacetylase (HDAC) protein, lysine dimethyltransferase domain protein, protein methyltransferase (PMT) domain protein, and DNA methyltransferase (DNMT). In a method for generating NT cell-derived stem cells, according to one or more embodiments, step c) of generating stem cells may include: activating NT cells and generating blastocysts; isolating inner cell mass (ICM) cells from the generated blastocysts; and further culturing the isolated ICM cells into stem cells.

[0102] In some embodiments, stem cells can be cryopreserved for future use. In some embodiments, the cryopreservative may include at least one cell protectant, including, for example but not limited to, dimethyl sulfoxide (DMSO), ethylene glycol, glycerol, and propylene glycol; at least one culture medium, including but not limited to DMEM, MEM, and the patented culture media disclosed above; and a solution containing at least one additional material, said additional material including but not limited to sucrose, dextran, serum substitutes, and HEPES buffer. Stem cells can be cryopreserved in solution. In some embodiments, the solution may include CryoStor® CS-10 medium (BioLife Solutions Inc., Bothell, Washington, USA). In some other embodiments, the serum substitute may be KnockOut. TM Serum substitute (Invitrogen, catalog number 10828-028).

[0103] Cryopreservation of the resulting stem cells can include freezing cells at a controlled rate or in “manual” mode. Rate-controlled freezing can be initiated by turning on the rate-controlled cryostat and setting the tissue or cell freezing program. The rate-controlled cryostat can use liquid nitrogen to lower the temperature of the chamber (thus lowering the temperature of any contents within the chamber). The cell freezing program can begin by cooling the chamber to 4°C and holding that temperature until the process is facilitated to continue. While the rate-controlled cryostat is cooling, cells can be suspended in cryopreservation medium cooled to 4°C. 1 ml of cell suspension is dispensed into each cryotube. The cryotubes are then labeled and placed in the chamber of the rate-controlled cryostat, and the program is facilitated to continue. First, the chamber temperature is held at approximately 4°C for another 10 minutes. Next, the chamber is cooled at a rate of -1°C / min until the chamber temperature reaches -80°C. Then, the chamber is cooled at a rate of -50°C / min until the chamber temperature reaches -120°C. After holding the temperature at -120°C for 5 minutes, the frozen cells reach equilibrium at -120°C. Then, the cryotubes containing the frozen cells are transferred to liquid nitrogen (Dewar) for long-term storage.

[0104] According to another aspect of this disclosure, a method for generating immunocompatible NT cell-derived stem cells includes: a) screening for homozygosity in multiple donor tissues; b) isolating nuclei from homozygous cells to generate NT cells; and c) generating stem cells from NT cells.

[0105] In some embodiments, in step a), screening may involve screening for cells homozygous for human leukocyte antigen (HLA)-A, HLA-B, and HLA-DR genes. In some embodiments, step b) of generating NT cells may include: enucleating oocytes; fusing the nucleus of a somatic cell with the enucleated oocyte; and culturing the fused oocyte in an activated culture medium. In some other embodiments, enucleation of oocytes may be performed in a culture medium containing a protein phosphatase inhibitor. Fusion of the somatic cell nucleus may be performed in a culture medium containing Sendai virus or Sendai virus extract. The activated culture medium may include a histone deacetylase inhibitor. For example, the activated culture medium may include TSA. The activated culture medium may include an epigenetic modifier. For example, the epigenetic modifier may involve at least one selected from the group consisting of: histone acetyltransferase (HAT) protein, histone deacetylase (HDAC) protein, lysine dimethyltransferase (KDM) domain protein, and protein methyltransferase (PMT) domain protein.

[0106] In one or more embodiments, a method for generating differentiated cells from stem cells derived from immunocompatible NT cells includes: a) screening for homozygosity in multiple donor tissues; b) isolating nuclei from homozygous cells to generate NT cells; c) generating stem cells from NT cells; and d) generating differentiated cells for transplantation from stem cells.

[0107] Optionally, the method may further include cryopreserving the stem cells after step c). In this case, the method may optionally further include thawing the cryopreserved stem cells before step d).

[0108] Differentiated cells can refer to cells differentiated into at least one of the following groups, but not limited to: hematopoietic stem cells, cardiomyocytes, hepatocytes, chondrocytes, epithelial cells, urethral cells, adipocytes, kidney cells, vascular cells, retinal cells, mesenchymal stem cells (MSCs), and neurons, as well as any cells used as therapeutic agents through cell transplantation. Methods may also include screening for immunocompatible cells through HLA screening of allogeneic patients. Immunocompatibility can increase the optimal availability of transplantable materials for regenerative therapies, enabling cell storage. Such storage systems can reduce the continuous supply of new oocytes by using autologous cells to replace NT cells and also enable various autologous or allogeneic cell transplantations. Specifically, the steps of screening cells from the storage system for allogeneic cell transplantation and generating differentiated cells from the screened stem cells can expand the scope of use as a variety of cell therapeutic agents. This invention can be integrated into the conventional field of cell transplantation therapy in various ways, and can be applied to various therapeutic areas, such as treating vascular-related diseases by differentiating into vascular endothelial cells, treating retinal-related diseases by differentiating into retinal pigment epithelial cells, and treating degenerative neuronal diseases by differentiating into nerve cells. However, the embodiments are not limited thereto.

[0109] According to aspects of this disclosure, the present invention provides cell masses comprising immunocompatible nuclear transfer (NT) cell-derived stem cells generated by a method according to any embodiment, and compositions comprising cell masses comprising NT cell-derived stem cells for treating various diseases.

[0110] In some embodiments, the cell composition may contain about 0.1 to 99.9 wt% of cells as the active ingredient relative to the total weight of the composition, and may include pharmaceutically acceptable carriers, excipients, or diluents.

[0111] In some embodiments, the composition can be any of a variety of oral or parenteral formulations. Common diluents or excipients, such as fillers, expanders, binders, wetting agents, disintegrants, or surfactants, can be used to formulate the composition. Solid formulations for oral administration can include, for example, tablets, pills, powders, granules, or capsules. These solid formulations can be prepared by mixing at least one compound with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to common excipients, lubricants such as magnesium stearate, talc, etc., can also be used. Liquid formulations for oral administration can be suspensions, liquids, emulsions, syrups, etc., and can include a variety of excipients, such as wetting agents, sweeteners, flavoring agents, preservatives, etc., in addition to common diluents such as water or liquid paraffin. Formulations for parenteral administration can include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, or suppositories. For example, non-aqueous solvents and solvents used for suspension can be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, or injectable esters such as ethyl oleate. As suppository bases, Witepsol, polyethylene glycol (macrogol), Tween 61, cacao paper, glyceryl laurin, glycerin gelatin, etc., can be used.

[0112] The pharmaceutically effective dose can be from about 0.0001 to about 100 mg / kg, and in some embodiments it is from about 0.001 to about 10 mg / kg. However, the embodiments are not limited to this. The dosage can vary with body weight, age, sex, health status, diet, duration of administration, method of administration, clearance rate, severity of disease, etc.

[0113] The composition can be administered orally or parenterally. Parenteral administration can be via intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine injection, intracerebral injection, or intrathoracic injection. The composition can be used in the form of a general pharmaceutical formulation. The composition can also be administered by injection.

[0114] The composition according to one or more embodiments can be used alone or in combination with surgery, radiotherapy, hormone therapy, chemotherapy, and any method using biological response modifiers.

[0115] According to another aspect of this disclosure, a storage system for immunocompatible nuclear transfer (NT) cell-derived stem cells includes: means for collecting multiple donor tissues; means for screening for immunocompatibility in the collected donor tissues; means for generating stem cells from the immunocompatible tissues; and means for cryopreserving the stem cells.

[0116] This invention provides a stem cell bank for storing NT cell-derived stem cells obtained from multiple individual donors. The stored stem cells can be used as a cell source to restore specific cell populations from donors or for therapeutic or clinical use in other individuals. The stored stem cells can also be used for research applications. The stored stem cells can be stored long-term and then used or administered to patients either by thawing as is or by differentiating into specific cell types.

[0117] The present invention also relates to the following embodiments:

[0118] 1. A method for storing immunocompatible nuclear transfer (NT) cell-derived stem cells, the method comprising:

[0119] a) Screening for homozygosity among multiple donor tissues;

[0120] b) Isolate nuclei from homozygous cells to generate NT cells;

[0121] c) Generate stem cells from the NT cells; and

[0122] d) Cryopreservation of the stem cells.

[0123] 2. The method of implementation scheme 1, wherein in step a), cells homozygous for human leukocyte antigen (HLA)-A, HLA-B, and HLA-DR genes are screened.

[0124] 3. The method of implementation scheme 1, wherein step b) of generating NT cells includes:

[0125] Enucleate the oocyte;

[0126] The nucleus of a somatic cell is fused with that of an enucleated oocyte; and

[0127] Fusion oocytes were cultured in post-activation medium.

[0128] 4. The method of implementation scheme 3, wherein the oocyte is enucleated in a culture medium containing a protein phosphatase inhibitor.

[0129] 5. The method of implementation scheme 3, wherein the fusion of the nuclei of the somatic cells is carried out in a culture medium containing Sendai virus or Sendai virus extract.

[0130] 6. The method of embodiment 3, wherein the activated culture medium contains a histone deacetylase inhibitor.

[0131] 7. The method of embodiment 3, wherein the activated culture medium contains an epigenetic modifier.

[0132] 8. The method of embodiment 7, wherein the epigenetic modifier relates to at least one selected from the group consisting of: histone acetyltransferase (HAT) protein, histone deacetylase (HDAC) protein, lysine dimethyltransferase (KDM) domain protein, and protein methyltransferase (PMT) domain protein.

[0133] 9. The method of embodiment 1, wherein step c) of generating the stem cells includes:

[0134] The NT cells are activated and blastocysts are produced;

[0135] Inner cell mass (ICM) cells were isolated from the generated blastocyst; and

[0136] The isolated ICM cells were further cultured into the stem cells described above.

[0137] 10. A method for generating immunocompatible nuclear transfer (NT) cell-derived stem cells, the method comprising:

[0138] a) Screening for homozygosity among multiple donor tissues;

[0139] b) Isolate nuclei from homozygous cells to generate NT cells; and

[0140] c) Generate stem cells from the NT cells.

[0141] 11. The method of implementation scheme 10, wherein in step a), cells homozygous for human leukocyte antigen (HLA)-A, HLA-B, and HLA-DR genes are screened.

[0142] 12. The method of embodiment 10, wherein step b) of generating the NT cells includes:

[0143] Enucleate the oocyte;

[0144] The nucleus of a somatic cell is fused with that of an enucleated oocyte; and

[0145] Fusion oocytes were cultured in activated culture medium.

[0146] 13. The method of embodiment 12, wherein the enucleation of the oocyte is performed in a culture medium containing a protein phosphatase inhibitor.

[0147] 14. The method of embodiment 12, wherein the fusion of the nuclei of the somatic cells is carried out in a culture medium containing Sendai virus or Sendai virus extract.

[0148] 15. The method of embodiment 12, wherein the activated culture medium comprises trichostatin A (TSA).

[0149] 16. The method of embodiment 12, wherein the activated culture medium contains a histone deacetylase inhibitor.

[0150] 17. The method of embodiment 13, wherein the epigenetic modifier relates to at least one selected from the group consisting of: histone acetyltransferase (HAT) protein, histone deacetylase (HDAC) protein, lysine dimethyltransferase (KDM) domain protein, and protein methyltransferase (PMT) domain protein.

[0151] 18. A method for generating differentiated cells from stem cells derived from immunocompatible NT cells, the method comprising:

[0152] a) Screening for homozygosity among multiple donor tissues;

[0153] b) Isolate nuclei from homozygous cells to generate NT cells;

[0154] c) Generate stem cells from the NT cells; and

[0155] d) Generate differentiated cells from the stem cells for transplantation.

[0156] 19. The method of embodiment 18, further comprising, after step c), cryopreserving the stem cells and thawing the cryopreserved stem cells.

[0157] 20. The method of embodiment 18, wherein in step d), the differentiated cell is at least one cell selected from the group consisting of: hematopoietic stem cells, cardiomyocytes, hepatocytes, chondrocytes, epithelial cells, urethral cells, adipocytes, renal cells, vascular cells, retinal cells, mesenchymal stem cells (MSCs), and neurons.

[0158] 21. A cell population comprising immunocompatible stem cells generated by the method described in embodiment 1.

[0159] 22. A storage system for immunocompatible nuclear transfer (NT) cell-derived stem cells, said storage system comprising:

[0160] A device (means) for collecting tissues from multiple donors;

[0161] Device used to screen for immunocompatibility in collected donor tissues;

[0162] Devices for generating stem cells from immunocompatible tissues, and

[0163] A device for cryopreserving the stem cells.

[0164] One or more embodiments of the present disclosure will now be described in detail with reference to the following examples. However, these examples are for illustrative purposes only and are not intended to limit the scope of one or more embodiments of the present disclosure.

[0165] [Example 1]

[0166] Screening for homozygous cells and selecting donor cells from donated cells.

[0167] Homozygous cells were screened from cord blood with fewer than 700 million cells discarded from the Korean CHA public cord blood bank (iCord). Gentra Puregene was used for HLA-A, B, and DRB1 genotyping. TM After genomic DNA extraction using a blood test kit (QIAGEN, Hilden, Germany), sequence-based genotyping was performed using the SeCore A, B, and DRB1 locus sequencing kit (Invitrogen, Brown Deer, WI, USA). Specifically, exons 2-4 of the HLA-A and HLA-B genes and exon 2 of the HLA-DRB1 gene were used as locus-specific primers for amplification using a kit. The resulting PCR products were then sequenced using an ABI 3130XL genetic analyzer (Applied Biosystems, Foster City, CA, USA), and data analysis was performed using HLA SBT u-type software v3.0 (Invitrogen) and Sequencher (Gene Codes Corp., Ann Arbor, MI, USA). These methods identified the most common (likely) HLA homozygous donor cells (A cells) in Koreans from donated umbilical cord blood. 33:03-B 44:03-DRB1 13:02) (Monotype frequency: 4.6%), ensuring that nuclear transfer (NT) cells (if generated therefrom) are used for approximately 9% of the total population of South Korea.

[0168] As a result of the screening, hematopoietic stem cells with the HLA AB-DRB1 haplotype were selected as donor cells and then cultured in cell culture flasks at 5% CO2 and 37°C. The cells were frozen in cryovials together with Dulbecco's modified Eagle's medium (DMEM) containing 10% dimethyl sulfoxide (DMSO) and 30% fetal bovine serum (FBS) (as the cryopreservation solution) and stored in liquid nitrogen until use. Chromosome analysis was performed on the cells (see [link to relevant documentation]). Figure 2Cells were thawed before nuclear transfer (NT), then cultured in confluence in 4-well dishes, and then synchronized in the G0 / G1 phase for 2 days in 0.5% FBS DMEM / F-12 medium.

[0169] [Example 2]

[0170] 2.1. Oocyte recovery and production

[0171] This experiment was conducted with the approval of the CHA Regenerative Medicine Institute (CHARMI) Stem Cell Research Oversight Committee (SCRO) and the Essex Institutional Review Board (EIRB).

[0172] Women aged 20-32 are recruited through online advertising, and their reproductive, medical, and mental health status is tested according to guidelines from the American Society for Reproductive Medicine (ASRM). Women with a BMI <28 kg / m² are also considered. 2 Furthermore, the experiment was conducted on women who passed medical and psychological tests.

[0173] Ovarian stimulation was performed according to established clinical IVF guidelines (Tachibana et al., 2013). Each woman was sedated for approximately 36 hours with 5–7.5 mg midazolam (Versed, Roche, and Nutley, NJ, USA) and 50–75 μg fentanyl (Abbott Pharmaceutical, Abbott Park, Ill. USA) following an injection of leuprorelin acetate or hCG, and oocytes were retrieved using a previously described ultrasound imaging technique. Freshly isolated cumulus-oocyte complexes (COCs) were collected from IVF medium (QuinnIVF medium, SAGE Biopharma, Bedminster, NJ) and then incubated at approximately 37°C in HTF-Hepes medium (Global Medium) with a 10% serum alternative supplement (SSS; Quinns Advantage Serum, Cooper Surgical). COCs were treated with hyaluronic acid (100 IU / ml, Sigma, St. Louis, Mo. USA) and oocytes were sorted according to maturity for NT using Midterm II (MII) oocytes.

[0174] 2.2. Enucleation of oocytes and replacement and activation of somatic cell nuclei

[0175] Enucleation of oocytes can be performed using publicly known methods (Tachibana et al., 2013). A stage heater, Narischige micromanipulator, and Oosight device can be used. TM An imaging system (polar microscopy) and a laser-equipped inverted microscope are used for oocyte enucleation and somatic cell nuclear replacement. Alternatively, a piezoelectric inverted microscope can be used instead of a laser-equipped inverted microscope.

[0176] Oocytes were placed in droplets of HTF-Hepes medium (global medium) containing cytochalasin B (5 μg / ml) and caffeine (1.25 mM), and the droplets were covered with oil for tissue culture at approximately 37°C for about 10 to 15 minutes. Caffeine, as a protein phosphatase inhibitor, inhibits premature activation to improve the growth of cloned embryos and thus increase the blastocyst formation rate. The oocytes were then fixed with a holding pipette to position the spindle approximately at the 2 to 4 o'clock position, and the zona pellucida adjacent to the spindle was punctured with a laser pulse. An injection pipette was inserted into the open portion to collect a small amount of cytoplasm surrounded by the plasma membrane and the contacting spindle. Optionally, a piezoelectric pulse could be used instead of a laser pulse to puncture the zona pellucida (ZP).

[0177] Next, nuclear donor cells were collected using a micropipette and transferred to a small droplet containing Sendai virus envelope protein (HJV-E extract, Isihara Sangyo Kaisha). The nuclear donor cells from Example 1 were then inserted into the periovarian space opposite the first polar body.

[0178] After confirming fusion, the resulting oocytes were cultured in 10% SPS medium for approximately 30 minutes or 2 hours.

[0179] Cells were activated by applying electrical pulses (2 x 50 μs DC pulses, 2.7 kV / cm) in 0.25 mM d-sorbitol buffer containing 0.1 mM potassium acetate, 0.5 mM magnesium acetate, 0.5 mM HEPES, and 1 mg / ml fatty acid-free BSA. Activated cells were then cultured in global medium (excluding serum) containing 2 mM DMAP at 5% CO2 and 37°C for approximately 4 hours, followed by further culture in global medium supplemented with 10% FBS, 12 μM BME (β-mercaptoethanol), CARM (2 μg / ml), and 10 nM TSA (tributamol A) at 5% CO2, 5% O2, and 90% N2 at 37°C for approximately 12 hours. Subsequently, after confirming pronuclear formation, cells were cultured for up to 7 days in a TSA-free global medium supplemented with 10% FBS and 12 μM BME at 37°C under conditions of 5% CO2, 5% O2, and 90% N2. Then, at the 4-cell stage, CARM mRNA was injected into blastomeres using a microinjection system. Optionally, HDAC1, SIRT2, or KDM4D can be added to replace CARM.

[0180] [Example 3]

[0181] Stem cell preparation and characterization analysis from NT cells

[0182] The blastocysts cultured in Example 2 were treated with acidic Tyrode solution (pH 2.0) for a few seconds to remove the zona pellucida (ZP). After ZP removal, the embryos were vigorously washed in Hepes-HTF medium to remove trace amounts of Tyrode solution. The inner cell mass (ICM) was separated using a laser-assisted blastocyst excision system (Hamilton-Thorne Inc.), and the remaining portion of the blastocyst (trophoblast) was discarded to confirm that the blastocyst was no longer intact. The ICM was plated onto prepared MEF the day before plating, where the entire embryo was plated when the cloned blastocyst had an indistinguishable ICM. The hPSC induction medium contained serum substitutes supplemented with Knockout-DMEM (5% SR, Invitrogen), FBS (10%, Hyclone), plasma (5%), bFGF (32 ng / ml), and human LIF (2000 units / ml, Sigma-Aldrich). Incubate the ICM in the same medium for 3 days without changing the culture medium, and replace about 1 / 3 of the medium on the 4th day. From the 6th day onwards, replace about 1 / 2 of the medium every other day. Confirm initial growth (growth) within 7 days after plating.

[0183] For further proliferation, colonies with ESC-shaped structures were selected, characterized, and subjected to cytogenetic analysis. Colonies were amplified and frozen before day 12. Cell characterization was performed using chromosome testing (G-banding), DNA fingerprinting, and mitochondrial DNA genotyping. Figure 3 and 4 The results are shown in the figure. To confirm the expression of pluripotent stem cell markers, alkaline phosphatase activity was confirmed by AP staining, Oct4, SSEA-4, TRA 1-60, and TRA 1-81 were analyzed by immunocytochemistry, and the expression of Oct4, Nanog, and Sox-2 markers was analyzed by RT-PCR. Figure 5 and 6 The results are shown in the figure. From these results, it was found that the derived cells were stem cells. Furthermore, to confirm the pluripotency of cells differentiating into 3 germ layer-derived cells, embryoids (EBs) were formed and cultured in vitro, followed by immunochemistry and RT-PCR to identify the expression patterns of 3 germ layer-derived differentiation markers (see [reference]). Figure 7 (A and 7B). To confirm in vivo pluripotency, stem cells were injected into the testes or subcutaneous tissue of immunodeficient mice to induce teratoma formation, and pluripotency was histologically determined by HE and special staining. Figure 7 The results are shown in C.

[0184] [Example 4]

[0185] Cryopreservation and storage of NT cells

[0186] The cells prepared according to the homozygous classification in Example 3 are stored and recorded in a document or program. Specifically, information about the cell donor is stored together so that they can be used directly or in the form of differentiated cells for future use in autologous or allogeneic recipients (patients).

[0187] [Example 5]

[0188] Functional retinal pigment epithelial (RPE) cells differentiated from NT-derived stem cells.

[0189] To induce differentiation into RPE, undifferentiated NT-derived stem cells were mechanically separated into several clumps of NT-ES cells (each containing approximately 300-600 undifferentiated embryonic stem cell lines) under a dissecting microscope using sterile tips. This was followed by supplementation with 15% (v / v) Knockout. TM Serum (EBDM; Knockout) TMNT-derived stem cells were seeded in clumps of DMEM (Thermo Scientific, 1% (v / v) Glutamax (Thermo), 1% (v / v) NEAA (Thermo), 1% (v / v) penicillin-streptomycin) in a low-attachment 6-well plate (Corning, CA, USA) and cultured in a floating state for 4 days. Cultured embryos were transferred and attached to culture dishes to induce RPE differentiation.

[0190] Isolation of retinal pigment epithelial cells differentiated from NT-derived stem cells

[0191] Embryoids were placed into 6-well culture dishes coated with 0.1% gelatin and incubated statically for 3 days. Cells were then cultured for approximately 50-55 days with EBDM medium changed every 2-3 days until retinal pigment epithelial cells emerged from the embryoids. To separate the RPE cells colored due to pigmentation, the cells were washed twice with physiological saline (DPBS (Thermo) containing Ca²⁺ and Mg²⁺) and then cultured for approximately 2 hours in physiological saline containing type IV collagenase (Type IV collagenase dissolved in DPBS (Thermo) containing Ca²⁺ and Mg²⁺) at approximately 37°C with 5% CO₂. To remove the enzyme, cell clusters separated from the culture dishes were collected in 50 ml tubes and washed twice with DMEM-FBS culture solution using a centrifuge (1500 rpm, 5 min). The cell clusters were transferred to 60 mm culture dishes, and then retinal pigment epithelial cells (pigment cell clusters) were collected from other cell clusters using thin glass pipettes under a dissecting microscope.

[0192] Maturation and proliferation of retinal pigment epithelial cells differentiated from NT-derived stem cells

[0193] Retinal pigment epithelial cell clusters (pigmented cell clusters) were washed twice with physiological saline solution (DPBS (Thermo) without Ca2+ and Mg2+), and then treated with a dissociation enzyme solution (a 1:1 mixture of 0.25% trypsin-EDTA (Thermo) and cell dissociation buffer (Thermo)) to isolate single cells. The isolated retinal pigment epithelial cells were washed with DMEM-FBS culture solution using a centrifuge (1500 rpm, 5 min), then resuspended in EGM2 culture solution (Lonza, PA, USA) using a centrifuge (1500 rpm, 5 min), transferred to 4-well culture dishes coated with 0.1% gelatin, and cultured in EGM-2 culture solution until each 4-well culture dish contained 200,000 cells. After about 3 to 4 days, when the culture dish is full of cells, the medium is replaced with RPE differentiation culture solution (a 1:1 mixture of RGMM, EBDM and DMEM-FBS medium) to reveal the shape and characteristics of the retinal pigment epithelial cells, and the cells are then incubated for another 7 days.

[0194] Retinal pigment epithelial cells (RPEs) were passaged using the same isocyanate solution as described above, and functional RPEs were obtained by proliferation using EGM2 medium and aging using RGMM medium. RPEs obtained from the second and third passages were frozen at a concentration of 2 million cells / mL in a cryoflask using freezing solutions (90% v / v FBS (Thermo) and 10% v / v DMSO (Sigma)) and stored until use. Some RPEs were used for characterization analysis. Figure 8 A and 8B show no difference in shape and differentiation markers between RPE cells derived from embryonic stem cells and RPE cells obtained from NT-derived stem cells in Example 3.

[0195] It should be understood that the embodiments described herein should be considered only in the descriptive sense and not for limiting purposes. The description of features or aspects within each embodiment should generally be regarded as other similar features or aspects that can be used in other embodiments.

[0196] Although one or more embodiments have been described with reference to the accompanying drawings, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the spirit and scope defined by the appended claims.

Claims

1. A method for storing immunocompatible nuclear transfer (NT) cell-derived stem cells, the method comprising: a) Screening for homozygosity of human leukocyte antigen (HLA)-A, HLA-B, and HLA-DRB1 in multiple donor tissues, including isolating HLA-A, HLA-B, and HLA-DRB1 haplotype homozygous cells from multiple donor tissues; The HLA-A, HLA-B, and HLA-DRB1 haplotype homozygous cells contained A 33:03-B 44:03-DRB1 13:02 HLA-A, HLA-B, and HLA-DRB1 haplotype homozygous cells; b) Isolate the nuclei from the homozygous cells to generate NT cells; c) Generate stem cells from the NT cells; and d) Cryopreservation of the stem cells, Step b) in generating NT cells includes: b1) Enucleate the oocyte to form an enucleated oocyte; Enucleation is performed in a culture medium containing protein phosphatase inhibitors; b2) The nucleus of the homozygous cell is fused with the enucleated oocyte to form a fused oocyte; The fusion is carried out in a culture medium containing Sendai virus or Sendai virus extract; b3) Activate the fused oocytes by electrical pulses; and b4) The fused oocytes were post-activated in a post-activation medium. The activated culture medium contains 6-dimethylaminopurine (6-DMAP), trichostatin A (TSA), and an epigenetic modifier involved in the modification of lysine demethylase (KDM) domain proteins. The epigenetic modifier is in contact with the nucleus of at least one homozygous cell, and Step c) in generating the stem cells includes: c1) Activate the NT cells and generate blastocysts from the activated NT cells; c2) Separate the inner cell mass (ICM) cells from the generated blastocyst; and c3) The isolated ICM cells are further cultured into the stem cells.

2. A method for generating immunocompatible nuclear transfer (NT) cell-derived stem cells, the method comprising: a) Screening for homozygosity of human leukocyte antigen (HLA)-A, HLA-B, and HLA-DRB1 in multiple donor tissues, including isolating HLA-A, HLA-B, and HLA-DRB1 haplotype homozygous cells from multiple donor tissues; The HLA-A, HLA-B, and HLA-DRB1 haplotype homozygous cells contained A 33:03-B 44:03-DRB1 13:02 HLA-A, HLA-B, and HLA-DRB1 haplotype homozygous cells; b) Isolate the nuclei from the homozygous cells to generate NT cells; and c) Generate stem cells from the NT cells. Step b) in generating NT cells includes: b1) Enucleate the oocyte to form an enucleated oocyte; Enucleation is performed in a culture medium containing protein phosphatase inhibitors; b2) The nucleus of the homozygous cell is fused with the enucleated oocyte to form a fused oocyte; The fusion is carried out in a culture medium containing Sendai virus or Sendai virus extract; b3) Activate the fused oocytes by electrical pulses; and b4) The fused oocytes were post-activated in an activated culture medium. The activated culture medium contains 6-dimethylaminopurine (6-DMAP), trichostatin A (TSA), and an epigenetic modifier involved in the modification of lysine demethylase (KDM) domain proteins. The epigenetic modifier is in contact with the nucleus of at least one homozygous cell, and Step c) in generating the stem cells includes: c1) Activate the NT cells and generate blastocysts from the activated NT cells; c2) Separate the inner cell mass (ICM) cells from the generated blastocyst; and c3) The isolated ICM cells are further cultured into the stem cells.

3. A method for generating differentiated cells from stem cells derived from immunocompatible NT cells, the method comprising: a) Screening for homozygosity of human leukocyte antigen (HLA)-A, HLA-B, and HLA-DRB1 in multiple donor tissues, including isolating HLA-A, HLA-B, and HLA-DRB1 haplotype homozygous cells from multiple donor tissues; The HLA-A, HLA-B, and HLA-DRB1 haplotype homozygous cells contained A 33:03-B 44:03-DRB1 13:02 HLA-A, HLA-B, and HLA-DRB1 haplotype homozygous cells; b) Isolate the nucleus from homozygous cells to generate NT cells; c) Generate stem cells from the NT cells; and d) Generate differentiated cells from the said stem cells for transplantation. Step b) in generating NT cells includes: b1) Enucleate the oocyte to form an enucleated oocyte; The denucleation is carried out in a culture medium containing a protein phosphatase inhibitor; b2) The nucleus of the homozygous cell is fused with the enucleated oocyte to form a fused oocyte; The fusion is carried out in a culture medium containing Sendai virus or Sendai virus extract; b3) Activate the fused oocytes by electrical pulses; and b4) The fused oocytes were post-activated in an activated culture medium. The activated culture medium contains 6-dimethylaminopurine (6-DMAP), trichostatin A (TSA), and an epigenetic modifier involved in the modification of lysine demethylase (KDM) domain proteins. The epigenetic modifier is in contact with the nucleus of at least one homozygous cell, and Step c) in generating the stem cells includes: c1) Activate the NT cells and generate blastocysts from the activated NT cells; c2) Separate the inner cell mass (ICM) cells from the generated blastocyst; and c3) The isolated ICM cells are further cultured into the stem cells.

4. The method of claim 3, further comprising, after step c), cryopreserving the stem cells and thawing the cryopreserved stem cells.

5. The method of claim 3, wherein in step d), the differentiated cell is at least one cell selected from the group consisting of: hematopoietic stem cells, cardiomyocytes, hepatocytes, chondrocytes, epithelial cells, urethral cells, adipocytes, renal cells, vascular cells, retinal cells, mesenchymal stem cells (MSCs), and neurons.

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