WO2017014513A1 - Storage method and banking system of nt cell - Google Patents

Abstract

The present invention relates to a storage method and a banking system of cells prepared using the nuclear transfer (NT) technology of somatic cells with homozygous genotypes in genes of human leukocyte antigen (HLA)-A, HLA-B, HLA-DR, etc. The banking of NT cell-derived stem cells according to the present invention can be applied to allogeneic or heterogeneous patients and can provide transplantable cells and tissue materials for treatment of various diseases such as diabetes, osteoarthritis, Parkinson's disease, etc.

Description

NT cell storage method and banking system

The present invention is a cell prepared by using somatic cell nuclear transfer (NT) technology of somatic cells having a homozygous genotype in genes such as HLA (human leukocyte antigen) -A, HLA-B and HLA-DR It relates to the storage method and banking system.

Cell therapy is a field that is emerging as a new paradigm of the pharmaceutical industry by enabling 'fundamental treatment' through cells, especially stem cells, which were considered to be limitedly treated through drugs or surgical procedures. In particular, regenerative medicine is a new technology field that fights diseases by restoring or replacing tissues and organs damaged or degraded by aging, disease, accident, etc. It is a field that is emerging as a.

In order for cell therapy to be used more widely, the challenge of immune rejection must be solved. The cause of the immunorejection is caused by six HLA (HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR) cell surface proteins, also called Major Histocompatibility Antigen Complex (MHC). In humans, six species derived from the paternal gene and six species derived from the maternal gene, that is, a total of six pairs are expressed. Normal somatic cells express only three pairs of HLA-A, HLA-B, HLA-C belonging to MHC class I, and immune cells express a total of six pairs in total, both MHC class I and MHC class II. The role of HLA surface antigens is to display fragments of the proteins present in the cell on the surface of the cell so that infections or mutations that may have occurred in vivo are detected by the immune cells, which is why antigen-providing proteins ( Also known as antigen presenting protein.

Meanwhile, the Somatic Cell Nuclear Transfer (SCNT) technology refers to a technique of removing a nucleus of a somatic cell and transplanting the nucleus of an egg to replicate. By using somatic cells to separate the nucleus of the somatic cells and injecting the isolated nucleus into the nucleus from which the nucleus is removed to prepare a stem cell line that maintains the genetic characteristics of the somatic cells. This method has the advantage of eliminating the immune rejection reaction in that it uses the patient's own somatic cells, and enables the patient-specific treatment. Although NT's technology is advancing, the rate of formation from reconstructed oocytes to blastocysts is still very low. Therefore, various attempts have been made and demanded to increase this formation rate. In particular, the largest disorder of NT embryos is zygotic gene activation (ZGA), which occurs in 4- to 8-cell phases in large mammals, including humans. In particular, it is necessary to remove existing epigenetic barriers to increase success rate regardless of donor variability. Specifically, for successful production from the 2-, 4-, and 8-cell stages to blastocysts, it is necessary to dramatically increase the blastocyst success rate by changing the donor nucleus state without manufacturing defects or losses.

As a cell therapy for the treatment of regenerative medicine and intractable diseases as described above, there is a need for a method of producing a cell and a banking system capable of solving an immune rejection reaction and providing a patient-specific cell.

The present inventors have led to the present invention by developing a cell therapeutic agent or a cell for transplantation that does not cause immunorejection from homozygous cells.

Accordingly, an object of the present invention is to provide a method for storing NT cell-derived stem cells applicable to various diseases of the same kind or different kinds.

It is also an object of the present invention to provide a method for producing NT cell-derived stem cells and a banking system for the prepared cells.

The present invention comprises the steps of: a) screening for homozygous (Homozygous) from a plurality of donation tissue (donation);

b) isolating nuclei from Homozygous cells to produce NT cells;

c) generating stem cells from the prepared NT cells; And

d) provides a method for storing stem cells derived from an immunocompatible NT cell comprising cryopreserving a plurality of stem cells.

In addition, the present invention comprises the steps of a) screening homozygous (Homozygous) from a plurality of donation tissue (donation) tissue;

b) isolating nuclei from Homozygous cells to produce NT cells; And

c) generating a stem cell from the prepared NT cell; and provides a method for producing an immunocompatible NT cell-derived stem cell comprising the.

In addition, the present invention comprises the steps of a) screening homozygous (Homozygous) from a plurality of donation tissue (donation) tissue;

b) isolating nuclei from Homozygous cells to produce NT cells;

c) generating stem cells from the prepared NT cells; And

It provides a method for producing a cell differentiated from the stem cells, which are derived from an immunocompatible NT comprising: d) preparing the differentiated cells for cell transplantation from the prepared stem cells.

The present invention also includes a) means for collecting a plurality of donor tissues, b) means for screening collected tissues, c) means for preparing stem cells from tissues, and d) means for cryopreserving stem cells, It provides a banking system of immunocompatible NT-derived stem cells.

Banking of stem cells derived from NT cells according to the present invention enables the treatment of various diseases or disorders. Implantable cell and tissue materials can be provided for the treatment of various diseases such as diabetes, osteoarthritis and Parkinson's disease, and in particular offer the possibility of fundamental treatment of cell type specific defects. In particular, the homozygous cells can provide a therapeutic approach that can reduce the risk of immune rejection and immune tolerance.

1 is a diagram showing that when the number of cells less than 700 million according to the law on cord blood management and research is classified for disposal.

Figure 2 shows the chromosome test results of the donor cells of Example 1.

Figure 3 shows the chromosome test results of NT cells prepared in Example 3.

Figure 4 shows (a) genomic DNA test results and (b) mitochondrial DNA test results of NT cells prepared in Example 3 compared with donor somatic cells and donor oocytes.

Figure 5 shows the results of immunochemistry analysis of stem cell markers of NT cells prepared in Example 3.

Figure 6 shows the results of RT-PCR analysis of stem cell markers of NT cells prepared in Example 3.

7 is an experimental result for confirming the pluripotent ability to differentiate into trioderm-derived cells of NT cells prepared in Example 3, forming an embryoid body and confirmed after 14 days culture (a) immunohistochemistry of the trigeminal differentiation marker Results of analysis and (b) RT-PCR of markers and (c) histologic analysis of teratoma formed by injection into immunodeficient mice are shown.

Figure 8 shows that there is no difference as a result of comparing the markers of (a) morphology of embryonic stem cell-derived RPE cells and NT-derived RPE cells prepared in Example 3 and (b) RPE cells.

Hereinafter, the present invention will be described in detail.

In the present invention, "immunocompatibility homozygous" means that each of the HLA-A, HLA-B, and HLA-DR genes inherited from the donor's paternal and maternal genomes is completely identical and has three HLA genotypes instead of six. Means that.

In the present invention, "somatic cell" refers to any tissue cell in the body except sex cells or precursors thereof.

In the present invention, "stem cells" are capable of self-renewal (having the ability to pass multiple cell division cycles while maintaining an undifferentiated state) and exhibit at least one multidifferentiation capacity (the ability to differentiate into one or more specialized cells). It means a cell that can.

As used herein, "long term culture" means proliferation of cells under controlled conditions for at least two months or longer than at least 10 passages. Preferably, the long term culture is cultured for at least 4 months, at least 6 months or at least 1 year. Preferably, the long term culture is passaged for at least 15 passages, at least 18 passages or at least 20 passages. The duration of long term culture depends mainly on individual cells and can vary from cell line to cell line.

As used herein, "mature" refers to a process consisting of coordinated biochemical steps leading towards finally differentiated cell types.

In the present invention, "differentiation" refers to the adaptation of the cell to a particular form or function.

In the present invention, "differentiated cell" includes any somatic cell that is not pluripotent in its original form, as the term is defined herein. Thus, the term “differentiated cells” also refers to partially differentiated cells, such as multipotent cells, or cells that are stable, non-potentially partially reprogrammed or partially differentiated cells produced using any of the compositions and methods described herein. It includes. In some embodiments, the differentiated cells are cells that are stable intermediate cells, such as non-pluripotent, partially reprogrammed cells. It should be noted that placing a large number of primary cells in the culture may result in some loss of fully differentiated properties. Thus, simply culturing such differentiated or somatic cells does not cause them to become undifferentiated cells (eg undifferentiated cells) or pluripotent cells. Transfer of differentiated cells (including stable, non-pluripotent partially reprogrammed cell intermediates) to pluripotency requires reprogramming stimuli beyond stimuli that partially lose differentiated properties when placed in culture. In some embodiments, reprogrammed, partially reprogrammed cells are also extended passaged without loss of growth potential as compared to parent cells with lower likelihood of having generally only a limited number of divisions in culture. Has the ability to suffer. In some embodiments, the term “differentiated cells” also refers to cells of less specialized cell types (ie, increased likelihood) (eg, from undifferentiated cells or reprogrammed cells), where the cells are an intracellular differentiation process. Refers to cells of more specialized cell types (ie, reduced likelihood of occurrence) derived from).

In the present invention, hematopoietic stem cells, muscle cells, cardiomyocytes, liver cells, chondrocytes, epithelial cells, urinary organ cells, adipocytes, kidney cells, vascular cells, retinal cells, mesenchymal stem cells (MSC) and neuronal cells It is selected from the group consisting of but not limited to.

In the present invention, the "immunocompetent cell" refers to a cell in which the HLA-A, HLA-B and HLA-DR genes are homozygous-like, without being particularly limited thereto, and all HLA genotypes in which only three of the six pairs are identical. It may have the advantage of being transplantable to the beneficiaries of the combination.

In the present invention, "bank" means a storage location of stem cells, and may be used as it is or may be differentiated from the stored cells to the individual or another individual for therapeutic, clinical or research purposes as needed.

In the present invention, "administration" means introducing a certain substance into a patient in any suitable way and the route of administration of the substance can be administered via any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto. In addition, administration can be performed by any device capable of moving to a target cell.

The present invention comprises the steps of: a) screening for homozygous (Homozygous) from a plurality of donation tissue (donation); b) isolating nuclei from Homozygous cells to produce NT cells; c) generating stem cells from the prepared NT cells; And d) cryopreserving a plurality of stem cells; provides a method for storage of stem cells derived from immunocompatible NT cells.

Cells made via NT may carry the genetic material of the patient's nucleus and are in this respect individual patient specific. Thus, cell transplantation into patients with a significantly reduced risk for autologous transplantation, i.e. allogeneic rejection, is possible. Furthermore, the homozygous cells of the present invention are cells that match the HLA antigen type, and can be transplanted to a heterogeneous person without anti-HLA antibodies. That is, the NT-derived stem cells have a homozygous type and can be transplanted into immunocompatible cells.

Therefore, the screening in step a) is preferably selected that the genes of human leukocyte antigen (HLA) -A, HLA-B and HLA-DR is homozygous (homozygous). When HLA genotyping screens find 140 donors with different immunocompatible homozygotes, it has been announced that it is possible to secure immune-compatible cell lines that can be transplanted to over 90% of Japan's population. (A more efficient method to generate integration-free human iPS cells, Nature Methods 8, 409-412 (2011))

In the present invention, homozygous (homozygous) cells were screened based on data from a primary hospital donor umbilical cord blood bank. According to the current umbilical cord blood management and research law, if the number of cells is less than 700 million cells, the Ministry of Health and Welfare Umbilical Cord Blood Committee is authorized to discard them so that they can be used for research (see FIG. 1).

Table 1 below (International Journal of Immunogenetics (2013) 40: 515-523) examined the frequency of HLA A-B-DRB1 haplotype for a total of 4,128 cord blood (only 0.1% or more).

Therefore, the cells to be used in this study can be used primarily for cryopreservation blood (with less than 700 million cells), and in addition to all donor blood for research, registered with the Center for Organ Transplantation Management (KONOS). It may include. In addition, samples obtained by the hematopoietic stem cell donor network and medical institutions affiliated with the Cha Hospital can be used.

In addition, the NT cell production method in step b) is the step of denuclearizing oocytes; Fusing the nucleus of the somatic cell to denucleated oocytes; And culturing the fused oocytes in post activation medium.

The method for producing stem cells derived from NT comprises the steps of removing the nuclei of oocytes, adding one or more nuclei of one or more donor cells to generate nuclear transplanted (NT) oocytes, and incubating the NT eggs by incubation in an activation medium. Activating blast cells, and generating blastocysts from the activated NT oocytes.

In one embodiment, removing the nucleus of the oocyte includes removing the mid-phase II (MII) stage egg spindle. In various embodiments, the first pole body 1PBE is removed. In another embodiment, the method includes the step of stripping the cumulus cells before completion of maturation. In one embodiment, oocytes are observed by non-UV light based observation on 1PBE in real time. In another embodiment, the observation takes place in the absence of a staining or labeling agent such as Hoechst staining. In one embodiment, this involves the use of a polscope, eg, Research Instruments (CRi) Oosight ^™ image system. For example, this involves visualizing zona pellucid and spindle complexes in MII oocytes collected at 545 nm light. In another embodiment, removing the nuclei of oocytes comprises the use of contoured micropipettes that allow the removal of 1 PBE from the oocytes and the pores of the oocyte membranes. In another embodiment, removing the nuclei of oocytes comprises the use of a piezoelectric drill. In another embodiment, the step of removing nuclei of oocytes is carried out in a denucleation medium containing cytocalin B and optionally, a protein phosphatase inhibitor such as caffeine. Caffeine is a protein phosphatase inhibitor that inhibits premature activation and improves the growth of cloned embryos, thereby increasing the rate of blastocyst formation. Therefore, preferably, the denucleation of the oocytes is made in a medium containing a protein phosphatase inhibitor, and the protein phosphatase inhibitor may be caffeine.

In another embodiment, generating the nuclearly transplanted (NT) oocytes by adding one or more nuclei of one or more donor cells may comprise implanting the donor nuclei.

Implanting the donor nucleus involves the use of agents that modify the oocyte membrane structure. In one embodiment, removing the nucleus of the oocyte through the use of an agent to modify the oocyte membrane structure comprises fusion with somatic cells. For example, implanting a donor nucleus may comprise providing 3 to 4 donor cells with an injection pipette (eg, 12 μm diameter), paramyxovirus or paramyxovirus proteins such as Sendai virus, Releasing the donor cells in a predetermined amount of a solution containing the envelope protein or extract thereof. Thereafter, recovering the cells using an injection pipette at a distance (4 to 5 cells long) away from the linearly arranged donor cells, holding the oocytes with the holding pipette, and injecting the injection pipette with the donor cells into oocytes. This is followed by a step forward. Advancing the injection pipette involves non-destruction of the yolk plasma membrane, and in the perivitelline space, which is the space between the zona pellucida and the cell membrane to contact the nuclear donor cells with the yolk plasma membrane located below the zona pellucida. The insertion of nuclear donor cells. In various embodiments, the recovery of the pipette does not interfere with the contact between the yolk sac and the donor cell. In various embodiments, the cells are fused after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more minutes of donor cell insertion. In various embodiments, the cells are fused 10 minutes after donor cell insertion. Optionally, the process is repeated for cells that have not been successfully fused. In various embodiments, polyscopes, such as the Oosight ^™ image system, are used throughout the process.

For example, the method of implanting a donor nucleus may be performed by a direct injection method. In one embodiment, the step of implanting a donor nucleus may comprise electrical cell manipulation, eg, electrofusion. In another embodiment, the method may comprise separating the nucleus of the somatic cell nuclear donor, the stem cell nuclear donor, and the sperm cell nuclear donor. In another embodiment, the method comprises inserting one or more donor nuclei via a pipette or piezoelectric injection after the step of separating the somatic nuclei for NT. In various embodiments, the donor nucleus is derived from a cell, such as dermal fibroblasts, white blood cells, hair follicles, or other somatic cell nuclear donors. In another embodiment, the present invention discloses a method comprising isolation and preparation of a nucleus from sperm cell donation. In different embodiments, the separation of the nucleus may be tissue biopsy, transfusion, or other method for obtaining a tissue sample, mechanical separation, collagenase digestion, washing, centrifugation based density gradient separation, and / or standard culture medium. Processing of tissues through culture.

Preferably, the step of fusing the nucleus of the somatic cells is made in a medium containing Sendai virus or Sendai virus extract.

After fusing the nucleus of the somatic cell to the denucleated oocytes, the fused oocytes are transferred to a post activation medium and undergo an activation process.

C) generating stem cells from the prepared NT cells, specifically, activating NT oocytes by incubating nuclear transplanted (NT) oocytes in an activation medium, and generating blastocysts from the activated NT oocytes. And isolating endothelial cell (ICM) cells from the blastocyst, wherein the ICM cells can be further cultured as NT-hPSC cell lines.

Artificial oocyte activation relies on the imitation of calcium signal changes that occur during spontaneous sperm fertilization. Normal oocyte development relies on high levels of Metaphase Promoting Factor (MFP) activity to arrest oocytes in the mid-II (MII) phase. MII I block of cells is hampered by a cell change in calcium ions (Ca ^{2 +)} level due to the entry of sperm. Thereafter, targeted degradation of cyclin B (MPF regulatory subunit) occurs, which releases oocytes from cell cycle blocking, pronuclear formation, and meiosis and mitosis processes.

Oocyte activation relies on an artificial calcium-change strategy to release cultured oocytes from cell cycle blockade. Examples include the addition of calcium ion carriers, fat soluble molecules that transfer ions through the lipid bilayer, such as ionomycin and A23817. Alternative strategies rely on electrical activity or direct implantation of ions.

As with NT, after reconstitution of nuclear transplanted (ie reconstructed) oocytes, oocyte activation using calcium alteration techniques also occurs.

However, in addition to such calcium ion implantation, for example, the addition of protein phosphatase inhibitor caffeine to sheep oocytes during the preparation of NT may result in maturation-promoting factor (MPF) and mitogen-activated protein. It has been reported to increase the activity of kinases (MAPKs) and similar benefits have been reported in monkey oocytes, while the frequency of blastocyst formation did not increase. In addition, calcium activation via calcium ion carriers, electrical activation, or direct implantation is a naturally occurring modification that does not cause the same timing, spatial control, or calcium oscillation. Adding additional complexity, the effect on calcium has also been shown to be species specific. In some examples, the use of additional treatments with kinase inhibitors, such as 6-dimethylaminopurine (6-DMAP), and protein synthesis inhibitors, such as ethanol and cycloheximide (CHX), to increase MPF inactivation Used. Histone deacetylase inhibitors such as TSA have been associated with improved NT reprogramming. Treatment of TSA can promote the formation of blastocysts.

In one example, an electrical pulse may be applied during fusion and activation of somatic cells. Electrical activation includes electrical pulses in electrical cell fusion media. In various embodiments, the electrical cell fusion the medium is from 0.1 to 0.5 M mannitol, 0.01 to _{1 mM MgSO 4 .7H 2 O,} 0.01 to 1 mg / ml of polyvinyl alcohol, 1 to 10 mg / ml human serum albumin, 0.005? 0.5 mM CaCl ₂ .2H ₂ O. In one embodiment, an electrical cell fusion medium 0.3 M including _{mannitol, 0.1 mM MgSO 4 .7H 2 O} , 0.1 mg / ml polyvinyl alcohol, 3 mg / ml human serum albumin, 0.05 mM CaCl ₂ .2H ₂ O do.

In various embodiments, nuclear transplanted oocytes are treated in a post-activation medium for complete activation. In different embodiments, the activated reconstructed nuclear transplanted oocytes are then incubated in post-activation medium. In different embodiments, the post-activation medium is HEPES-removing medium, protein-removing medium, G1 or G2 medium, eggplant medium, eggplant aid medium, IVF medium, blastocyst forming medium, or global human embryo culture medium. In different embodiments, the post-activation medium is 6-DMAP, puromycin, ethanol, cycloheximide (CHX), trichostatin A (TSA), and cytochalasin B It may include any one or more selected from (CB). In different embodiments, the activated oocytes are 30 to 45, 45 to 60, 60 to 90, 90 to 120, 120 to 150, 150 to 180, 180 to 210, 210 to 240, 240 to 240 in post-activation medium. Incubate for less than 270, 300 to 330, 330 to 360, 360 to 390 minutes, or more than 390 minutes. In certain embodiments, the activated oocytes are incubated for 240, 300, or 360 minutes. In various embodiments, the activation and post-activation steps are performed under hypoxic conditions. In certain embodiments, hypoxic conditions are about 80-85%, 85-90%. 90 to 95%, 95% or more N ₂ , about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more O ₂ , and About 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more CO ₂ . In certain embodiments, low oxygen conditions include about 90% N ₂ , about 5% O ₂ , and about 5% CO ₂ . In various embodiments, the post-activation medium is 1, 2, 3, 4, 5, 5, or in a gas mixture, such as about 90% N ₂ , about 5% O ₂ , and about 5% CO ₂ . 1, 2, 3, 4, 5, 5, or more mM 6-DMAP in eggplant media, incubated at 37 ° C. for longer time.

After incubation in post-activation medium, post-activated oocytes are incubated in wash medium. In a different embodiment, the wash medium is HEPES-removing medium, protein-removing medium, G1 or G2 medium, eggplant medium, eggplant aid medium, IVF medium, blastocyst forming medium. In another embodiment, the culture medium does not require continuous medium exchange as global human embryo culture medium. In certain embodiments, the wash medium comprises TSA. In certain embodiments, post-activated oocytes are incubated for 240, 300, or 360 minutes in wash media comprising TSA. In one embodiment, the post-activated reconstructed nuclear transplanted oocytes are washed and further cultured. In one embodiment, the post-activated reconstituted nuclear transplanted oocytes are washed in 6-DMAP removal medium. In other embodiments, various disclosed media, such as HEPES-removing media, protein-removing media, G1 or G2 media, eggplant media, eggplant aid media, IVF media, blastocyst forming media, or global human embryo culture media, Optionally growth factors such as GM-CSF or IGF1. In various embodiments, the growth factor may be added at 1, 2, 3, 4, 5, 6, 7, or later days after nuclear transfer.

In another embodiment, the activating and / or post-activating step comprises adding a factor, derivatives and extracts isolated from sperm. In one embodiment, the human sperm factor is injected into the reconstructed ovum activated using any of the described infusion methods. In one embodiment, the human sperm factor is injected into the post-activated reconstructed egg using any of the described infusion methods. In various embodiments, after about 1, 2, 3, or 4 days, post-activated reconstituted nuclear transplanted oocytes are transferred to eggplant medium. In certain embodiments, after about 1 day, the post-activated reconstructed nuclear transplanted oocytes are transferred to eggplant medium. In various embodiments, the sperm factor includes a factor that is isolated from, for example, a cellular protein present inside or outside the sperm cell. In one embodiment, the total sperm extract is obtained using mechanical mixing of the surfactant and ejaculated sperm. In another embodiment, the whole cell extract is treated with DNAase I and RNAase. In another embodiment, the crude extract is washed with buffer and centrifuged (20,000 g for 2 hours). In another embodiment, fresh ejaculated human sperm is imported and centrifuged at 900 g for 10 minutes to remove semen plasma, followed by Sperm-TALP containing 5 mg / mL bovine serum albumin. After centrifugation concurrently with resuspension and setup of pellets in supernatant, removal of supernatant and nuclear separation media (NIM: 125 mM KCl, 2.6 mM NaCl, 7.8 mM Na ₂ HPO ₄ , 1.4 mM KH ₂ PO ₄ , 3.0 mM EDTA D. Sodium salt; pH 7.45), resuspend the pellet to a final concentration of 20 × 10 ⁸ sperm / mL, and centrifuge to remove Spurm-TALP. After removal of the perm-TALP, it contains 1 mM dithiothreitol, 100 mM leupeptin, 100 mM antipain, and 100 mg = mL soybean trypsin inhibitor 4 cycles of freezing (5 minutes for each cycle in liquid N ₂ ) with thaw (5 minutes for each cycle at 15 ° C.) with dense sperm pellet formation at 20,000 × for 50 minutes at 2 ° C. after resuspension of the pellets in the same volume to NIM. 5 minutes per cycle). Finally the resulting supernatant is carefully removed, titrated and maintained at -80 ° C until used.

In various embodiments, the post-activated reconstructed nuclear transplanted oocytes are further cultured into blastocysts. In one embodiment, the post-activated reconstituted nuclear transplanted oocytes are further cultured in SAGE eggplant medium, eg, Quinn's medium. In another embodiment, medium, for example 3i medium (Neuro basal medium 50%, DMEM / F-12 50%, N2 additive 1/200 v / v, B27 additive 1/100 v / v, 100 mM L-glutamine 1/100 v / v, 0.1M β-ME 1/1000 v / v, SU5402 (FGFR inhibitor) 2 μM, PD184352 (ERK cascade inhibitor) 0.8 μM, CHIR99021 (GSK3 inhibitor) 3 μM) or modified 3i medium (including 0.4 μM PD0325901 (MAPK inhibitor)) promotes pluripotency. In one embodiment, the additional culture is for at least 1, 2, 3, 4, 5, or 5 days. In one embodiment, additional cultures are provided in a culture medium having a reprogramming factor and / or methylation-modifying agent. In various embodiments, the additional culture is for 3 days in G2 medium to which any of CARM1 CARM1, Esrrb, Kdm4a, Kdm4b and Kdm4d Esrrb is added. For example, CARM1 and / or Esrrb may be provided at concentrations in medium of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 or more μg / ml, respectively. In some embodiments, CARM1 and / or Esrrb are each provided at a concentration in medium of 2 μg / ml.

In various embodiments, further incubation into blastocysts and induction of pluripotent stem cells (pSCs) from blastocysts comprises treating the cultured blastocysts with acidic Tyrode's solution to remove the zona pellucida (ZP). Include. In various embodiments, the treatment is for a few seconds (eg, 1 to 5). In various embodiments the removal of ZP is followed by a wash in HEPES-HTF medium. In various embodiments, isolation of the internal cell mass (ICM) comprises discarding the trophoblast of the blastocyst. In various embodiments, ICM cells are plated in a mouse embryonic feeder (MEF) prepared one day prior to plating. In some embodiments, the whole blastocyst is plated on MEFs. For example, the method includes stripping the zona pellucida of the blastocyst. In various embodiments, the method removes the zona pellucida of the blastocyst with 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1% pronase in Hepes-HTF medium. Steps. In one embodiment, the method comprises removing the zona pellucida of the blastocyst with 0.5% pronase in Hepes-HTF medium. In another embodiment, the method comprises pronase in TH3 medium (SAGE blastocyst medium) for 1-10, 10-20, 20-30, 30-60, 60-120, 120-180, or> 180 seconds. It includes the step of applying. In yet another embodiment, applying 0.5% pronase in HTF medium for 30-60 seconds. In one embodiment, the blastocyst is derived from reconstructed nuclear transplanted oocytes obtained from somatic cell nuclear transfer (NT) of donor cell nuclei into recipient oocytes. In one embodiment, the stem cell line is a somatic cell nuclear transfer human pluripotent stem cell (NT-hPSC) cell line.

In another embodiment, the invention includes a method for immunosurgery comprising mechanical dispersion of an internal cell mass (ICM) from trophectodermal cells. In various embodiments, the exfoliated 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, the method comprises washing the exfoliated blastocyst with TH3 (SAGE blastocyst medium) and incubating in guinea pig complement reconstituted with HECM-9 (SAGE blastocyst medium) at 37 ° C. for 30 minutes. do. In a different embodiment, the zona pellucida of the expanded blastocyst is removed with slight exposure (45-60 seconds) to 0.5% pronase or acidic tirod solution in TH3 (hepes-HTF) medium. In one embodiment, the method is laser assisted incubation using small bore pipetting, Ehsms zilos-tk unit (Hamilton Thorne) to selectively separate endocytosis from trophectoderm cells. mechanically spreading the cells using a hatching method.

In the present invention, preferably, the post activation medium is made from a medium containing TSA, and the post activation medium is 6-DMAP. More preferably, the cells are cultured in a medium containing 6-DMAP at the time of post activation, and then further cultured in a medium containing TSA.

In various embodiments, the nuclei of at least one donor cell can be altered by focusing epigenetic modifying agents to increase successful NT production. Epigenetic regulators specifically alter the state of methylation or acetylation of specific proteins or DNA to increase transcriptional efficiency and consequently to increase NT efficiency. Targets of these epigenetic regulators include histone acetyl transferase (HAT) proteins, histone deacetylase (HDAC) proteins, lysine dimethylase (KDM) domain proteins, and protein methyl transfer. Enzyme (PMT, protein methyl transferase) domain protein and the like, and includes at least one or more. These agents include small interfering RNA (siRNA), small molecules, proteins, peptides, antibodies, and the like. These agents act on epigenetic targets associated with reprogramming resistant regions. NT oocytes are cultured in the presence of agents that alter epigenetic status. Specific examples of these formulations are described in Tables 2-5 below. Histone acetyl transferase (HAT) protein, histone deacetylase (HDAC) protein, lysine dimethylase (KDM) domain protein, protein methyl transferase (PMT) protein It is not limited to the examples below.

Histone acetyl transferase (HAT) protein Target_ID (| domain #) Full name Uniprot _ID NCBI geneid ATAT1 alpha tubulin acetyltransferase 1 Q5SQI0-1 79969 CLOCK clock homolog (mouse) O15516-1 9575 CREBBP CREB binding protein Q92793-1 1387 ELP3 elongation protein 3 homolog (S. cerevisiae) Q9H9T3-1 55140 EP300 E1A binding protein p300 Q09472-1 2033 GTF3C4 general transcription factor IIIC, polypeptide 4, 90kDa Q9UKN8-1 9329 HAT1 histone acetyltransferase 1 O14929-1 8520 KAT2A / GCN5L2 K (lysine) acetyltransferase 2A Q92830-1 2648 KAT2B / PCAF K (lysine) acetyltransferase 2B Q92831-1 8850 KAT5 / TIP60 K (lysine) acetyltransferase 5 Q92993-1 10524 MYST1 K (lysine) acetyltransferase 8 Q9H7Z6-1 84148 MYST2 K (lysine) acetyltransferase 7 O95251-1 11143 MYST3 K (lysine) acetyltransferase 6A Q92794-1 7994 MYST4 K (lysine) acetyltransferase 6B Q8WYB5-1 23522 NCOA1 nuclear receptor coactivator 1 Q15788-1 8648 NCOA3 nuclear receptor coactivator 3 Q9Y6Q9-1 8202 TAF1 TAF1 RNA polymerase II, TATA box binding protein (TBP) -associated factor, 250 kDa P21675-1 6872 TAF1L TAF1 RNA polymerase II, TATA box binding protein (TBP) -associated factor, 210kDa-like Q8IZX4-1 138474

Histone deacetylase (HDAC) protein Target_ID (| domain #) Full name Uniprot_ID NCBI geneid HDAC1 histone deacetylase 1 Q13547_1 3065 HDAC10 | 1 histone deacetylase 10 Q969S8_1 83933 HDAC10 | 2 histone deacetylase 10 Q969S8_1 83933 HDAC10 | 1 histone deacetylase 10 Q969S8_2 83933 HDAC10 histone deacetylase 10 Q969S8_5 83933 HDAC11 histone deacetylase 11 Q96DB2_1 79885 HDAC2 histone deacetylase 2 Q92769_1 3066 HDAC3 histone deacetylase 3 O15379_1 8841 HDAC4 histone deacetylase 4 P56524_1 9759 HDAC5 histone deacetylase 5 Q9UQL6_1 10014 HDAC6 | 1 histone deacetylase 6 Q9UBN7_1 10013 HDAC6 | 2 histone deacetylase 6 Q9UBN7_1 10013 HDAC7 histone deacetylase 7 Q8WUI4_1 51564 HDAC8 histone deacetylase 8 Q9BY41_1 55869 HDAC9 histone deacetylase 9 Q9UKV0_1 9734 SIRT1 sirtuin 1 Q96EB6_1 23411 SIRT2 sirtuin 2 Q8IXJ6_1 22933 SIRT3 sirtuin 3 Q9NTG7_1 23410 SIRT4 sirtuin 4 Q9Y6E7_1 23409 SIRT5 sirtuin 5 Q9NXA8_1 23408 SIRT6 sirtuin 6 Q8N6T7_1 51548 SIRT6 sirtuin 6 Q8N6T7_2 51548 SIRT6 sirtuin 6 Q8N6T7_4 51548 SIRT7 sirtuin 7 Q9NRC8_1 51547

Lysine Dimethylase (KDM) Domain Protein Target_ID (| domain #) Full name Uniprot_ID NCBI geneid JARID2 jumonji, AT rich interactive domain 2 Q92833_1 3720 JHDM1D jumonji C domain containing histone demethylase 1 homolog D (S. cerevisiae) Q6ZMT4_1 80853 JMJD1C jumonji domain containing 1C Q15652_1 221037 JMJD5 jumonji domain containing 5 Q8N371_1 79831 KDM1A lysine (K) -specific demethylase 1A O60341_1 23028 KDM1B lysine (K) -specific demethylase 1B Q8NB78_1 221656 KDM1B lysine (K) -specific demethylase 1B Q8NB78_2 221656 KDM2A lysine (K) -specific demethylase 2A Q9Y2K7_1 22992 KDM2B lysine (K) -specific demethylase 2B Q8NHM5_1 84678 KDM3A lysine (K) -specific demethylase 3A Q9Y4C1_1 55818 KDM3B lysine (K) -specific demethylase 3B Q7LBC6_1 51780 KDM4A lysine (K) -specific demethylase 4A O75164_1 9682 KDM4B lysine (K) -specific demethylase 4B O94953_1 23030 KDM4C lysine (K) -specific demethylase 4C Q9H3R0_1 23081 KDM4D lysine (K) -specific demethylase 4D Q6B0I6_1 55693 KDM4DL lysine (K) -specific demethylase 4D-like B2RXH2_1 390245 KDM5A lysine (K) -specific demethylase 5A P29375_1 5927 KDM5B lysine (K) -specific demethylase 5B Q9UGL1_1 10765 KDM5C lysine (K) -specific demethylase 5C P41229_1 8242 KDM5D lysine (K) -specific demethylase 5D Q9BY66_1 8284 KDM6A lysine (K) -specific demethylase 6A O15550_1 7403 KDM6B lysine (K) -specific demethylase 6B O15054_1 23135 MINA MYC induced nuclear antigen Q8IUF8_1 84864 NO66 chromosome 14 open reading frame 169 Q9H6W3_1 79697

Protein methyl transferase (PMT) domain protein Target_ID (| domain #) Full name Uniprot_ID NCBI geneid ASH1L ash1 (absent, small, or homeotic) -like (Drosophila) Q9NR48_1 55870 CARM1 coactivator-associated arginine methyltransferase 1 Q86X55_1 10498 DOT1L DOT1-like, histone H3 methyltransferase (S. cerevisiae) Q8TEK3_1 84444 EHMT1 euchromatic histone-lysine N-methyltransferase 1 Q9H9B1_1 79813 EHMT2 euchromatic histone-lysine N-methyltransferase 2 Q96KQ7_1 10919 EZH1 enhancer of zeste homolog 1 (Drosophila) Q92800_1 2145 EZH2 enhancer of zeste homolog 2 (Drosophila) Q15910_1 2146 EZH2 enhancer of zeste homolog 2 (Drosophila) Q15910_5 2146 MDS1 MDS1 and EVI1 complex locus Q03112_3 2122 MLL myeloid / lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila) Q03164_1 4297 MLL2 myeloid / lymphoid or mixed-lineage leukemia 2 O14686_1 8085 MLL3 myeloid / lymphoid or mixed-lineage leukemia 3 Q8NEZ4_1 58508 MLL4 - Q9UMN6_1 9757 MLL5 myeloid / lymphoid or mixed-lineage leukemia 5 (trithorax homolog, Drosophila) Q8IZD2_1 55904 NSD1 nuclear receptor binding SET domain protein 1 Q96L73_1 64324 PRDM1 PR domain containing 1, with ZNF domain O75626_1 639 PRDM10 PR domain containing 10 Q9NQV6_1 56980 PRDM11 PR domain containing 11 Q9NQV5_1 56981 PRDM12 PR domain containing 12 Q9H4Q4_1 59335 PRDM13 PR domain containing 13 Q9H4Q3_1 59336 PRDM14 PR domain containing 14 Q9GZV8_1 63978 PRDM15 PR domain containing 15 P57071_1 63977 PRDM16 PR domain containing 16 Q9HAZ2_1 63976 PRDM2 PR domain containing 2, with ZNF domain Q13029_1 7799 PRDM4 PR domain containing 4 Q9UKN5_1 11108 PRDM5 PR domain containing 5 Q9NQX1_1 11107 PRDM6 PR domain containing 6 Q9NQX0_1 93166 PRDM7 PR domain containing 7 Q9NQW5_1 11105 PRDM8 PR domain containing 8 Q9NQV8_1 56978 PRDM9 PR domain containing 9 Q9NQV7_1 56979 PRMT1 protein arginine methyltransferase 1 Q99873_1 3276 PRMT2 protein arginine methyltransferase 2 P55345_1 3275 PRMT3 protein arginine methyltransferase 3 O60678_1 10196 PRMT5 protein arginine methyltransferase 5 O14744_1 10419 PRMT6 protein arginine methyltransferase 6 Q96LA8_1 55170 PRMT7 | 1 protein arginine methyltransferase 7 Q9NVM4_1 54496 PRMT7 | 2 protein arginine methyltransferase 7 Q9NVM4_1 54496 PRMT8 protein arginine methyltransferase 8 Q9NR22_1 56341 SETD1A SET domain containing 1A O15047_1 9739 SETD1B SET domain containing 1B Q9UPS6_1 23067 SETD2 SET domain containing 2 Q9BYW2_1 29072 SETD3 SET domain containing 3 Q86TU7_1 84193 SETD4 SET domain containing 4 Q9NVD3_1 54093 SETD5 SET domain containing 5 Q9C0A6_1 55209 SETD6 SET domain containing 6 Q8TBK2_1 79918 SETD6 SET domain containing 6 Q8TBK2_2 79918 SETD7 SET domain containing (lysine methyltransferase) 7 Q8WTS6_1 80854 SETD8 SET domain containing (lysine methyltransferase) 8 Q9NQR1_1 387893 SETDB1 SET domain, bifurcated 1 Q15047_1 9869 SETDB2 SET domain, bifurcated 2 Q96T68_1 83852 SETMAR SET domain and mariner transposase fusion gene Q53H47_1 6419 SMYD1 SET and MYND domain containing 1 Q8NB12_1 150572 SMYD2 SET and MYND domain containing 2 Q9NRG4_1 56950 SMYD3 SET and MYND domain containing 3 Q9H7B4_1 64754 SMYD4 SET and MYND domain containing 4 Q8IYR2_1 114826 SMYD5 SMYD family member 5 Q6GMV2_1 10322 SUV39H1 suppressor of variegation 3-9 homolog 1 (Drosophila) O43463_1 6839 SUV39H2 suppressor of variegation 3-9 homolog 2 (Drosophila) Q9H5I1_1 79723 SUV420H1 suppressor of variegation 4-20 homolog 1 (Drosophila) Q4FZB7_1 51111 SUV420H2 suppressor of variegation 4-20 homolog 2 (Drosophila) Q86Y97_1 84787 WHSC1 Wolf-Hirschhorn syndrome candidate 1 O96028_1 7468 WHSC1L1 Wolf-Hirschhorn syndrome candidate 1-like 1 Q9BZ95_1 54904

In general, methyl transferases can be inhibited by co-substrate analogues. Three kinds of co-substrate analogues that inhibit various kinds of methyltransferases are known. Sinefugin, a structurally similar antibiotic compound to S-adenosylmethionone (SAM), dimethylated co-substrate SAH and methylthioadenosine as feedback inhibitors. Lysine methyl group inhibitors include the first identified chaetocin and G9a (KMT1C) inhibitor Bix-01294. These are optional for SUV39H1 and PRM1. Bix-01338 is a rather nonselective inhibitor with no selectivity between lysine and arginine methyltransferase, showing an IC50 of 5 mM for G9a and an IC50 of 6 mM for PRMT1. UNC0224 has been proposed as a new inhibitor with IC50 15 mM against lysine methyltransferase G9a. Inhibitors of histone methyltransferases such as EPZ5676, EPZ005687 and GSK126 also show anticancer activity in various cancer animal models.

Protein arginine methylation is accomplished by PRMTs and is divided into two groups. Type I methyl transferases form asymmetrically substituted arginine residues, and type II methyl transferases form symmetrically substituted arginine residues. CARM1 shows affinity with proline-glycine-methionine-arginine (the so-called PGM motif). PRMT5 is also known to methylate the PGM motif. Cosubstrate analogs such as sinefungin can also be used as inhibitors of arginine methyltransferases (also known as AMIs as arginine methyltransferase inhibitors). AMI-1 is the most active inhibitor of PRMT1 with IC50 9 mM. Inhibitors of allatodapsone and stilbamidine induce hypomethylation in H4R3.

Another type of epigenetic target could be a way to increase NT efficiency. DNA methyltransferases (DNMTs) preferentially methylate the CpGnucleotide sequence of DNA. In mammals, three DNA methyltransferases have been identified: DNMT1, DNMT3A, and DNMT3. In general, methylation of these promoter sites inhibits the expression of genes by preventing transcription factors from binding to DNA. In addition, methylated DNA is bound by a methyl-CpG binding domain protein, which attracts histone modeling enzymes, consequently condensing chromatin structure to express genes. May induce a mechanism to suppress DNMT inhibitors can increase the efficiency of NT by preventing gene expression from being inhibited. Some examples include chlorogenic acid, mithramycin, azacytide, bisdemethoxycurcumim, decitabine, lomegutatrib, benzylguanine, sorafenib and sorafenib tosylate.

In addition, histone deacetylase (HDAC) functions to remove acetyl groups from histone N-acetyl lysine amino acids, resulting in more positively charged and strongly bound to negatively charged DNA. Condensation of DNA structures and genetic transcription are further inhibited. HDACs can be divided into four subgroups, depending on their location and function. Class I HDACs (subtypes 1, 2, 3, and 8) are found primarily in the nucleus and class II HDACs (4, 5, 6, 7, 9). 10) can travel through the nuclear membrane and are found in both the nucleus and the cytoplasm. Type III HDACs are called silent information regulator 2 (Sir2). Type IV HDACs (11 subtypes) are found in both the nucleus and cytoplasm and are primarily located in the brain, heart, and muscle cells. HDACs inhibitors have anticancer activity when administered in combination with other chemotherapy drugs. HDAC inhibitors can promote DN transcription, resulting in increased NT efficiency.

Epigenetic modifying agents, such as epigenetic chromatin and β histone modification agents, and / or DNA modifiers may be included in the post activation medium. In certain embodiments, it is a protein arginine methyl-transferase (PRMT1) and a coactivator-associated arginine methyltransferase 1 (CARM1 / PRMT4), a nuclear orphan receptor estrogen. receptor estrogen) may be selected from related receptor β (Esrrb) proteins and may also be selected from Lysine (K) -Specific Demethylase 4A (Kdm4a), Lysine-specific dimethylase 4B (Lysine (K)). -Specific Demethylase 4B, Kdm4b) or lysine specific dimethylase 4D (Lysine (K) -Specific Demethylase 4D, Kdm4d), respectively, can be selected from RNA or protein. In certain embodiments, methylation-modifying agents and / or DNA modifiers are expressed as modified recombinant proteins. For example, CARM1 and Esrrb may be a 7X arginine (7R) -cell-penetrating peptides (CPPs), or one of skill in the art, to enhance the penetration of proteins and peptides through cell and nuclear membranes, and to bind and / or transactivate DNA. May be modified with other proteins known to increase. In another embodiment, the method uses transcript talent-based reprogramming to allow octamer binding transcription factor-4 (Oct-4), sex determination site Y-box-2 (sex determining). region Y-box-2) (Sox-2), nanog, Kruppel-like factor-4 (Klk-4), MyoD, c-Myc, zinc finder protein-42 protein-42) (Rex-1 / Zfp-42), lefty A, teratocarcinoma-derived growth factor (Tdgf), and / or telomeric repeating binding epigenetic reprogramming of nuclear donor cells with factor) (Terf-1). In various embodiments, the method comprises direct piezoelectric injection, viral injection, liposome injection, or other intracellular injection. In various embodiments, the transcription factor can be delivered in the form of mRNA, protein, and / or cell extracts that can be applied prior to nuclear transfer into nucleated oocytes. In another embodiment, the method may comprise the use of HDAC inhibitors (Class I, II, and III), or DNMT3a and DNMT3b inhibitors.

Thus, the present invention preferably post activation medium comprises epigenetic modifying agents. More preferably, epigenetic modifying agents are histone acetyl transferase (HAT) proteins, histone deacetylase (HDAC) proteins, lysine dimethylase (KDM) domain proteins, Protein methyl transferase (PMT) domain proteins and DNA methyl transferases (DNA) are involved in any one or more selected from the group comprising DNA transferases (DNMTs). Preferably in the present invention, the method for producing NT cell-derived stem cells, the production of stem cells in step c) is the step of activating NT cells to generate blastocysts; Isolating endocytosis (ICM) cells from the resulting blastocysts; And further culturing the isolated inner cell population cells with stem cells.

Stem cells of the present invention can be cryopreserved for future use. In one embodiment the cryopreservative comprises one or more cryoprotectants including but not limited to dimethyl sulfoxide (DMSO), ethylene glycol, glycerol and propanediol; One or more culture media, including but not limited to DMEM, MEM and the patented media disclosed above; The stem cells are cryopreserved in a solution comprising one or more additional substances, including but not limited to sucrose, dextran, serum substitutes and HEPES buffer. In one embodiment, the solution comprises CryoStor ™ CS-10 media (BioLife Solutions Inc., Botel, Washington). In another embodiment, the serum replacement is Knockout serum replacement (Invitrogen 10828-028).

Cryopreservation of the prepared stem cells freezes the cells at a controlled rate or in a "manual" process. The controlled rate freezing procedure begins by turning on a controlled rate freezer and setting up a freezing program for tissue or cell freezing. The controlled rate freezer will use liquid nitrogen to reduce the temperature in the inner chamber (which will reduce the temperature of any contents of the chamber). The freezing program for cells begins by cooling the inner chamber to 4 ° C. and maintaining the temperature until it is stimulated to continue the procedure. While the controlled rate freezer cools, the cells are suspended in cryopreservation medium cooled to 4 ° C. The cell suspension is aliquoted into frozen vials in an amount of 1 ml per cryovial. The frozen vials are then labeled and placed in a controlled rate freezer chamber to stimulate the program to continue. First, the temperature of the chamber is maintained at 4 ° C. for an additional 10 minutes. Next, the chamber is cooled at a rate of -1 ° C / min until the temperature reaches -80 ° C. The chamber is then cooled at a rate of -50 ° C / min until the chamber reaches a temperature of -120 ° C. After 5 minutes at -120 ° C, the temperature of the frozen cells will equilibrate to -120 ° C. The frozen vials of the frozen cells are then transferred to liquid nitrogen Dewar for long term storage.

In addition, the present invention comprises the steps of a) screening homozygous (Homozygous) from a plurality of donation tissue (donation) tissue;

b) isolating nuclei from Homozygous cells to produce NT cells; And

c) generating a stem cell from the prepared NT cell; and provides a method for producing an immunocompatible NT cell-derived stem cell comprising the.

Preferably, the screening in step a) is that the genes of human leukocyte antigen (HLA) -A, HLA-B, and HLA-DR are homozygous, and in step b), NT production may include denuclearizing oocytes; Fusing the nucleus of the somatic cell to denucleated oocytes; Comprising a step of culturing the fused oocytes in a post activation medium, more preferably, the denucleation of the oocytes is made in a medium containing a protein phosphatase inhibitor (protein phosphatase inhibitor). In addition, the step of fusing the nucleus of the somatic cells is made in a medium containing Sendai virus or Sendai virus extract, the post-activation medium preferably comprises a histone deacetylase inhibitor (distone deacetylase inhibitor), It is more desirable to include TSA. The post activation medium preferably contains epigenetic modifying agents, more preferably the epigenetic modifying agents include histone acetyl transferase (HAT, histone acetyl). at least one selected from the group consisting of transferase (HDC) protein, histone deacetylase (HDAC) protein, lysine dimethylase (KDM) domain protein, and protein methyl transferase (PMT) domain protein. To get involved.

In addition, the present invention comprises the steps of a) screening homozygous (Homozygous) from a plurality of donation tissue (donation) tissue;

b) isolating nuclei from Homozygous cells to produce NT cells;

c) generating stem cells from the prepared NT cells; And

It provides a method for producing cells differentiated from stem cells derived from immunocompatible NT cells, comprising the steps of: preparing the differentiated cells for cell transplantation from the prepared stem cells.

After step c), the stem cells may be optionally added to cryopreservation, in which case, the step of thawing the cryopreserved cells prior to step d) may be selectively added.

Differentiated cells are derived from stem cells, hematopoietic stem cells, muscle cells, cardiomyocytes, liver cells, chondrocytes, epithelial cells, urinary organ cells, adipocytes, kidney cells, vascular cells, retinal cells, mesenchymal stem cells (MSCs) and neurons. There is no limitation, such as cells, it means any cell that has been differentiated into any one or more cells from the group consisting of them and can be used as a therapeutic agent through cell transplantation without limitation. Selecting immunoconjugated cells via HLA screening of xenogeneic patients from cryopreserved cell banking may be added. Immunoconjugation is to make banking possible by increasing the optimal availability of regenerative therapies as implants. Such a banking system can reduce the continuous supply of new oocytes by replacing NT production with autologous cells and enable a wide range of autologous or heterologous cell transplants. In particular, the screening step for xenotransplantation from these banking systems and the preparation of specific differentiated cells from the selected stem cells expand the range of use as more and more various cell therapies. It is possible to apply variously to the field of cell transplantation treatment that is used in the past. Specifically, the differentiation into vascular endothelial cells can lead to vascular related diseases, retinal pigment epithelial cells, and retinal related diseases through differentiation into neurons. The field of treatment such as neurodegenerative diseases through differentiation will not be limited.

In addition, the present invention is to provide a cell population comprising an immune-compatible stem cells prepared by the method for producing an immunocompatible NT cell-derived stem cells. The present invention also provides a composition comprising a cell population of NT cell-derived stem cells for the treatment of various diseases.

Cell composition of the present invention with respect to the total weight of the composition of the present invention contains 0.1 to 99.9% by weight as an active ingredient, may comprise a pharmaceutically acceptable carrier, excipient or diluent.

The compositions of the present invention may be in various oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutically effective amount is 0.0001 to 100 mg / kg, 0.001 to 10 mg / kg, but is not limited thereto. The dosage may vary depending on the weight, age, sex, health condition, diet, duration of administration, method of administration, elimination rate, severity of disease, and the like of the particular patient.

The composition can be administered orally or parenterally during clinical administration and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or intrathoracic injection during parenteral administration. And can be used in the form of general pharmaceutical formulations.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.

In addition, the present invention provides a means for collecting a plurality of donor tissue,

Means for screening immunocompatibility from the collected tissue,

Means for preparing stem cells from immunocompatible tissue, and

Means for cryopreserving stem cells; provides an immunocompatible NT cell-derived stem cell banking system comprising a.

The present invention provides a stem cell bank for storing NT cell derived stem cells obtained from multiple donors of an individual. Stored stem cells can be used as a source of cells for restoring a particular cell population of an individual's body for health reasons or for the treatment or clinical use of another individual. Stored stem cells can also be used for research applications. Stored stem cells may be thawed and used as stem cells after long-term preservation, or may be used or administered to a patient after differentiation into specific cells.

Hereinafter, the operation and effects of the invention will be described in more detail with reference to specific examples of the invention. However, these embodiments are only presented as an example of the invention, whereby the scope of the invention is not determined.

Example 1

Screening of Homozygous Cells and Donor Cell Selection from Donor Cells

Homozygous cells were screened for less than 700 million disposable cord blood at the Cha Hospital donated cord blood bank. Genomic DNA was extracted using Gentra Puregene ^™ Blood Kits (QIAGEN, Hilden, Germany) for HLA-A, B and DRB1 genotyping, and then SeCore A, B and DRB1 Locus Sequencing Kit (Invitrogen, Brown Deer, WI, USA) Sequence-based typing was used. In particular, exon 2-4 for HLA-A and B and exon 2 for HLA-DRB1 were amplified using a locus specific primer included in the kit, and ABI3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA) , USA) was used to sequence the PCR product formed, and the data using HLA SBT u-type software v3.0 (Invitrogen) and Sequencher (Gene Codes Corp., Ann Arbor, MI, USA) The analysis was carried out. In the end, HLA homozygous donor cells (A * 33: 03-B * 44: 03-DRB1 * 13: 02) (haplotype frequency: 4.6%) were most frequently used in donor blood If found, NT-cells could cover about 9% of the total population.

From the screening results, hematopoietic cells of HLA AB-DRB1 haplotype were selected as donor cells and cultured in a cell culture flask under 5% CO ₂ at 37 ° C. DMEM culture medium containing 10% DMSO and 30% FBS is used as a freezing solution, frozen in cryo vials and stored in a liquid nitrogen tank until use. These cells undergo a chromosome test (FIG. 2). Cells were thawed prior to NT and incubated confluency in 4 well dishes and these cells were synchronized to G0 / G1 phase with 2 days in 0.5% FBS DMEM / F12 medium.

Example 2

2. Recovery and Preparation of 1 Oocyte

It was approved under the Stem Cell Research Oversight (SCRO) Committee and the Essex Institutional Review Board (EIRB) of the CHA Regenerative Medicine Institute.

We recruited 20-32 year old women through web-based advertising and examined the reproductive, medical and psychological health status of the women according to the American Society for Reproductive Medicine (ASRM) guidelines. According to the test, women with BMI <28 Kg / m ² who passed both medical and psychological tests were conducted.

Ovarian stimulation was performed according to established clinical IVF guidelines (Tachibana et al., 2013). The women were sedated with Midazolam 5-7.5 mg (Versed, Roche, and Nutley. NJ, USA) and Fentanyl 50-75 ug (Abbott Pharmaceutical, Abbott Park, Ill. USA) 36 hours after Lupron or hCG injection. And oocytes were then recovered using the previously described ultrasound map. Fresh-isolated cumulus-oocyte cell complexes (COCs) in IVF medium (Quinn's IVF medium, SAGE Biopharma, Bedminster, NJ) were collected and 10% serum substitutes (SSS; Quinns Advantage Serum, Cooper Surgical ) Supplemented with HTF-Hepes medium (Global Medium) under 37 ° C conditions. COCs were treated with hyaluroniase (100 IU / ml, Sigma, St. Louis, Mo. USA), and then the oocytes were sorted according to maturity, and oocytes in the middle II (MII) phase were used for NT.

2.2 Denuclearization of oocytes, nuclear transfer and activation of somatic cells

The denuclearization of oocytes can be carried out by a previously known method (Tachibana et al., 2013), the denuclearization of oocytes and nuclear replacement of somatic cells can be performed by stage warmers, narishige micromanipulators. ), An Oosight ^™ imaging system (poloscopic microscopy), and an inverted microscope equipped with a laser. An inverted microscope with piezo may optionally be used instead of an inverted microscope with a laser.

The oocytes were placed in a droplet of HTF-Hepes medium (Global Medium) containing cytocarcin B (5 μg / ml) and caffeine (1.25 mM), and then the droplet was placed into tissue culture oil. Covered with, and placed for about 10 to 15 minutes under 37 ℃ conditions. Caffeine is a protein phosphatase inhibitor that inhibits premature activation and improves the growth of cloned embryos, thereby increasing the rate of blastocyst formation. After fixing the oocytes with the holding pipette so that the spindle is located near the 2-4 o'clock direction, the zona pellucida next to the spindle was drilled with a laser pulse, and the injection pipette was inserted through the drilled portion. The injection pipette was aspirated with spindles in contact with a small amount of cytoplasm surrounded by a plasma membrane. The transparent band may optionally use piezo pulses instead of laser pulses.

The donor cells were then aspirated into micropipettes and transferred to small drops containing Sendai virus envelope protein (HJV-E extract, Isihara Sangyo Kaisha). Then, the nuclear donor cells of Example 1 were inserted into a perivitelline space located opposite the first polar body.

When fusion is confirmed, the prepared eggs were further incubated for 30 minutes or 2 hours in Global 10% SPS medium.

Activation was performed under 0.25 mM d-sorbitol buffer containing 0.1 mM potassium acetate, 0.5 mM magnesium acetate, 0.5 mM HEPES, 1 mg / ml fatty acid-free BSA (fatty-acid-free BSA). Electrical pulses (2 × 50 μs DC pulses, 2.7 kV / cm) were added to the test. Activated cells were incubated for 4 hours in Global Medium (excluding serum) containing 2 mM DMAP under 5% CO ₂ , 37 ° C., and under 5% CO ₂ , 5% O ₂ , 90% N ₂ , 37 ° C. Incubated for 12 hours in Global Medium supplemented with 10% FBS, 12 μM BME (β-mercaptoethanol), CARM (2 μg / ml) and 10 nM TSA (Trichostatin A). Pronuclear formation was then confirmed and, under conditions of 5% CO ₂ , 5% O ₂ , 90% N ₂ and 37 ° C, up to 7 days in Global Medium supplemented with 10% FBS and 12 μM BME without TSA Incubated. Thereafter, CARM mRNA is injected into the blast furnace using a micro injection system at the 4-cell stage. HDAC1, SIRT2 or KDM4D can optionally be added in place of CARM.

Example 3

Preparation and Characterization of Stem Cell Line from NT Cells

The blastocyst cultured in Example 2 was treated with acidic Tyrode solution (pH 2.0) for several seconds to remove the zona pellucida (ZP). After removal of ZP, embryos were vigorously washed in Hepes-HTF medium to remove even trace amounts of Tyrode solution. Intracellular cell mass (ICM) was isolated using a laser-assisted blastocyst ablation system (Hamilton-Thorne Inc.) and the remaining portion of the blastocyst (nutrient membrane) was discarded to confirm that the blastocyst was no longer intact. ICMs were plated on MEFs prepared one day prior to plating, but whole embryos were plated if replicated blastocysts had indistinguishable ICMs. hPSC induction medium was serum replacement (5% SR, Invitrogen), FBS (10%, Hyclone), plasmamate (5%), bFGF (32 ng / ml), and human LIF (2000 units / mI, Sigma) Knockout-DMEM supplemented with -Aldrich) was included. After incubation of ICM for 3 days in the same medium without change, on day 4 about 1/3 of the medium was replaced. ½ of the medium was changed every other day from day 6. Initial outgrowth was observed within 7 days after plating.

Colonies with ESC-like shape were selected, characterized and cytogenetic analysis for further propagation. And colonies were expanded and cryopreserved before day 12. Cell characterization was performed by karyotype (G-banding), DNA fingerprinting and mitochondrial DNA genotyping. The results are shown in FIGS. 3 and 4, respectively. In order to confirm the expression of pluripotent stem cell markers of cells, alkaline phosphatase activity was confirmed by AP staining, Oct4, SSEA-4, TRA 1-60 and TRA 1-81 were analyzed by immunocytochemistry, and RT-PCR was analyzed. The expression of Oct4, Nanog, Sox-2 markers was analyzed. The results are shown in FIGS. 5 and 6. It was confirmed that the cells derived from these results were stem cells. In addition, in order to confirm the pluripotency capable of differentiating into 3 germ layers-derived cells, embryonic bodies (EBs) were formed in vitro and cultured to express the expression of trioderm-derived differentiation markers through Immunochemistry and RT-PCR. It confirmed (FIG. 7 (a) and (b)). In order to confirm the pluripotency in the body, stem cells were injected into the testis or subcutaneously of the immunodeficient mice to induce teratoma formation and histologically, the differentiation was confirmed through H-E and special staining. The results are shown in Figure 7 (c).

Example 4

NT Cell Cryopreservation and Banking

The cells prepared in Example 3 are sorted according to homozygous cells and stored and recorded in a document or program. In particular, the information of the cell donor is stored together. It can be used directly for future autologous or allogenic recipients (patients) or stored for use as differentiated cells.

[ Example  5]

Differentiation of NT-derived Stem Cells into Functional Retinal Pigment Epithelial Cells (RPE)

In order to induce differentiation into RPE, undifferentiated NT-derived stem cells were mechanically clump-form of NT-ES cells using a sterilized tip under an anatomical microscope (a fragment of approximately 300-600 undifferentiated embryos). Stem cell line). Clump-type NT-derived stem cells were supplemented with 15% (v / v) knockout ^TM DMEM (Thermo Scientific, CA, USA) supplemented in low attachment 6-well plates (Corning, CA, USA). ^TM serum replacement (Thermo), 1% (v / v) glutamax (Thermo), 1% (v / v) NEAA (Thermo), 1% (v / v) penicillin-streptomycin (Thermo) and 0.1 mM β-mercaptoethanol (Thermo)) incubated for 4 days in a suspended state. The cultured embryoid body is transferred to the culture dish and induces RPE differentiation while attached.

Isolation of Differentiated Retinal Pigment Epithelial Cells from NT-derived Stem Cells

Embryos were transferred to a 6-well culture dish coated with 0.1% gelatin coating and allowed to stand in the incubator for 3 days for attachment. Then, EBDM culture medium was replaced every 2-3 days, and retinal pigment epithelial cells were grown. Incubated for about 50-55 days until it appears. To separate the color-coded RPE cells by pigmentation, the cells were washed twice with physiological saline (DPBS containing Ca ^{2 +} Mg ^{2 +} (Thermo)) and physiological saline containing collagenase type 4 (Type IV collagenase). (Thermo) in DPBS with Ca ²⁺ Mg ²⁺ (Thermo)) and incubated in an incubator maintained at 37 ° C., 5% CO ₂ for 2 hours. To remove the enzyme, detached cell clusters separated from the culture dish were collected in a 50 ml tube and washed twice with DMEM-FBS culture using a centrifuge (1500 rpm, 5 minutes). The cell masses were transferred to a 60 mm Petri dish, and the pigmented cell clusters were collected from other non-pigmented cell masses using a glass pipette drawn under a dissecting microscope.

Maturation and Proliferation of Retinal Pigment Epithelial Cells Differentiated from NT-derived Stem Cells

Pigmented cell clusters are washed twice with physiological saline (DPBS without Ca ^{2 +} Mg ^{2 +} (Thermo)) without calcium and magnesium to isolate and culture single cells. : 1 mixture of 0.25% Trypsin-EDTA (Thermo) and Cell Dissociation Buffer (Thermo)) were isolated. Retinal pigment epithelial cells separated into single cells were washed with DMEM-FBS medium using a centrifuge (1500 rpm, 5 min), and then 200,000 cells / 4 well plate was prepared by releasing the cells with EGM2 medium (Lonza, PA, USA). In a well of 0.1% gelatin-coated 4well culture plate at a concentration of one well was incubated in EGM-2 culture until it is full. After about 3-4 days, the cells are filled with a culture plate with RPE differentiation medium (RGMM: 1: 1 mixture of EBDM and DMEM-FBS media) to show the shape and characteristics of retinal pigment epithelial cells. Was replaced and incubated for 7 days.

Retinal pigment epithelial cells differentiated from NT-derived stem cells were subcultured using the same separation enzyme solution as above, and functional retinal pigment epithelial cells were obtained through the proliferation process using EGM2 culture and the aging process using RGMM culture. Some retinal pigment epithelial cells from passages 2 and 3 were obtained using 2 million cells / mL / cryo using freezing solution (90% (v / v) FBS (Thermo) and 10% (v / v) DMSO (Sigma)). Freezing at the concentration of vial was stored until use, and some of the retinal pigment epithelial cells were characterized. Figure 8 shows that there is no difference in the morphology and differentiation markers of RPE cells of embryonic stem cell-derived RPE cells and NT-derived stem cells prepared in Example 3 (Fig. 8 (a), (b)).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Claims (22)

a) screening for homozygous from a plurality of donation tissues; b) isolating nuclei from Homozygous cells to produce NT cells; c) generating stem cells from the prepared NT cells; And d) A method for storing an immunocompatible NT cell-derived stem cell comprising cryopreserving a plurality of stem cells. The method of claim 1, wherein the screening in step a) is characterized in that the genes of human leukocyte antigen (HLA) -A, HLA-B and HLA-DR are homozygous. The method of claim 1, wherein the NT production in step b) comprises the steps of denuclearizing oocytes; Fusing the nucleus of the somatic cell to denucleated oocytes; And culturing the fused oocytes in a post activation medium. The method of claim 3, wherein the oocyte denuclearization is performed in a medium containing a protein phosphatase inhibitor. The method of claim 3, wherein the fusion of the nucleus of the somatic cells is performed in a medium containing Sendai virus or Sendai virus extract. The method of claim 3, wherein the post activation medium comprises a histone deacetylase inhibitor. The method of claim 3, wherein the post activation medium comprises epigenetic modifying agents. 8. The method of claim 7, wherein the epigenetic modifying agents are histone acetyl transferase (HAT) protein, histone deacetylase (HDAC) protein, lysine dimethylase (KDM) A method characterized in that it is involved in any one or more selected from the group comprising a domain protein and a protein methyl transferase (PMT) domain protein. According to claim 1, wherein the production of the stem cells in step c) is the step of activating NT cells to generate blastocysts; Isolating endocytosis (ICM) cells from the resulting blastocysts; And culturing the isolated inner cell population cells as stem cells. a) screening for homozygous from a plurality of donation tissues; b) isolating nuclei from Homozygous cells to produce NT cells; And c) generating a stem cell from the prepared NT cell; a method for producing an immunocompatible NT-derived stem cell comprising a. The method of claim 10, wherein the screening in step a) is characterized in that the genes of human leukocyte antigen (HLA) -A, HLA-B and HLA-DR are homozygous. The method of claim 10, wherein in step b) NT production comprises the steps of denuclearizing oocytes; Fusing the nucleus of the somatic cell to denucleated oocytes; And culturing the fused oocytes in a post activation medium. The method of claim 12, wherein the oocyte denuclearization is performed in a medium containing a protein phosphatase inhibitor. The method of claim 12, wherein the fusion of the nucleus of the somatic cells is characterized in that the medium comprising a Sendai virus or Sendai virus extract. The method of claim 12, wherein the post activation medium comprises TSA. The method of claim 12, wherein the post activation medium comprises epigenetic modifying agents. The method of claim 13, wherein the epigenetic modifying agents are histone acetyl transferase (HAT) protein, histone deacetylase (HDAC) protein, lysine dimethylase (KDM) And a domain protein and a protein methyl transferase (PMT) domain protein. a) screening for homozygous from a plurality of donation tissues; b) isolating nuclei from Homozygous cells to produce NT cells; c) generating stem cells from the prepared NT cells; And d) preparing a differentiated cell from an immunocompatible NT cell-derived stem cell comprising the step of preparing a differentiated cell for cell transplantation from the prepared stem cell. 19. The method of claim 18, wherein after step c), the stem cells are cryopreserved and thawing the cryopreserved cells is added. 19. The method of claim 18, wherein the differentiated cells of step d) are hematopoietic stem cells, muscle cells, cardiomyocytes, liver cells, chondrocytes, epithelial cells, urinary organ cells, adipocytes, kidney cells, vascular cells, retinal cells, mesenchymal cells Stem cells (MSC) and neuronal cells, characterized in that any one or more selected from the group consisting of. A cell population comprising an immunocompatible stem cell prepared by the method of claim 1. Means for collecting a plurality of donor tissues, Means for screening immunocompatibility from the collected tissue, Means for preparing stem cells from immunocompatible tissue, and Means for cryopreserving stem cells; an immunocompatible NT cell-derived stem cell banking system comprising a.

Images