KR20180078160A - A method for manufacturing of mesenchymal stem cell - Google Patents

Abstract

The present invention relates to a method for manufacturing mesenchymal stem cells from human pluripotent stem cells and mesenchymal stem cells manufactured by the method. The mesenchymal stem cells manufactured according to the present invention enable treatment of various diseases or disorders. The present invention can provide cells and tissue materials which can be transplanted for various diseases such as diabetes, osteoarthritis, and Parkinson′s disease. The method comprises the steps of: a) generating human pluripotent stem cells; forming the human pluripotent stem cells into embryos; and manufacturing mesenchymal stem cells by culturing the embryos.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for producing mesenchymal stem cells,

The present invention relates to a method for producing mesenchymal stem cells from human pluripotent stem cells and mesenchymal stem cells prepared by the method.

Cell therapy is an emerging paradigm in the medical field by allowing 'fundamental treatment' through cells, especially stem cells, where it is thought that limited treatment is possible through drugs or surgical operations in the treatment of diseases. In particular, regenerative medicine is a new technology field against diseases by regenerating or replacing tissues and organs that have been damaged or deteriorated by aging, diseases, accidents, etc., Is a field that has been highlighted.

On the other hand, Mesenchymal Stem Cell (MSC) has been considered as one that can be usefully used for repairing damaged tissues in the field of regenerative medicine. However, in the process of obtaining mesenchymal stem cells, a surgical factor is required, and sometimes there is a disagreement between the characteristics of the obtained mesenchymal stem cells. Therefore, in order to obtain a large amount of mesenchymal stem cells, a method for producing mesenchymal stem cells from pluripotent stem cells such as embryonic stem cells and nuclear transfer pluripotent stem cells is required.

The inventors of the present invention have developed a method for producing mesenchymal stem cells by forming embryoid bodies from human pluripotent stem cells, leading to the present invention.

Accordingly, it is an object of the present invention to provide a method for producing mesenchymal stem cells from human pluripotent stem cells.

It is another object of the present invention to provide mesenchymal stem cells prepared by the above method.

The present invention provides a method of producing a human pluripotent stem cell (hPSC) comprising: a) producing a human pluripotent stem cell (hPSC); b) forming the human pluripotent stem cells into an embryoid body (EB); And c) culturing the embryoid body to prepare mesenchymal stem cells (MSCs).

For example, the human pluripotent stem cells can be selected from the group consisting of nuclear transfer pluripotent stem cells (NT-hPSC), hematopoietic-derived pluripotent stem cells (pn-hPSC), reprogrammed pluripotent stem cells (iPSC) , Embryonic Stem Cell).

For example, the step a) is a step of separating nuclei from homozygous cells to generate nuclear-engrafting human pluripotent stem cells (NT-hPSCs), which are homozygous from a plurality of donation tissues prior to the step a) Or < / RTI >

In another example, the screening may be a homozygote for the genes of human leukocyte antigen (HLA) -A, HLA-B, and HLA-DR.

For example, the step a) is a step of generating nuclear transfer human pluripotent stem cells (NT-hPSC), and the step a) includes the steps of removing nuclei of oocytes; Adding one or more nuclei of one or more donor cells to produce nuclear-engrafted (NT) oocytes; Incubating in a post-activated medium to activate said NT oocyte; Generating a blastocyst from the activated NT oocyte; And isolating the inner cell mass (ICM) cells from the blastocyst, wherein the ICM cells are further capable of incubating with NT-hPSC cells.

In another example, the step of removing the nuclei of the oocyte may be performed in a medium containing a protein phosphatase inhibitor.

As another example, the protein phosphatase inhibitor may be caffeine.

In yet another embodiment, the step of adding one or more nuclei of the one or more donor cells may involve direct injection.

In yet another embodiment, the step of adding one or more nuclei of the one or more donor cells may involve somatic cell fusion.

In yet another example, one or more nuclei of the one or more donor cells may be contacted with epigenetic modifying agents.

In another example, the eugenic regulator may be a histone acetyl transferase (HAT) protein, a histone deacetylase (HDAC) protein, a lysine dimethylazase (KDM) domain protein, (PMT) protein domain transferase protein.

In another example, the epigenetic regulator may be siRNA (small interfering RNA), a low molecular weight protein, a peptide, or an antibody.

In another example, the eugenic regulator may be at least one selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc, and Lin2.

In yet another embodiment, the step of adding nuclear of at least one of the donor cells to produce nuclear-engrafted (NT) oocytes may be in a medium comprising Sendai virus, a protein thereof or an extract thereof.

In yet another example, the one or more donor cells may be somatic cells or germ cells.

In another example, the post-activation medium in the step of activating NT oocytes is 6-DMAP, puromycin, ethanol, CHX (cycloheximide), TSA (trichostatin A), and CB (cytochalasin B ) May be included.

In another example, the NT oocyte may be cultured in the presence of an epigenetic regulator.

In another example, the step a) may be carried out in the absence of ultraviolet light.

As another example, the step a) may include injecting sperm factors into an oocyte.

For example, the step a) is a step of generating hemihydrate-derived human pluripotent stem cells (pn-hPSC), wherein the step a) comprises the steps of activating oocytes; Incubating the activated oocytes in a post-activated medium to produce a hemihydrate; Incubating the semi-adulterate in a culture medium to form a blastocyst; And separating the inner cell mass (ICM) cells from the blastocyst, wherein the inner cell mass is further cultured with pn-hPSC.

In another example, oocytes can be activated by applying electrical and / or chemical stimulation.

In another example, the chemical stimulation in the step of activating the oocyte may be incubation in an activated medium.

In another example, the activation medium may be one comprising a calcium ion carrier.

In another example, the calcium ion carrier may comprise at least one member selected from the group consisting of ionomycin, A23187, beauvericin, X-537A, and avenaciolide.

In yet another embodiment, the post-activation medium in the step of generating the semi-adulterated body is selected from the group consisting of 6-DMAP, puromycin, ethanol, CHX (cycloheximide) , TSA (trichostatin A), and CB (cytochalasin B).

As another example, the post-activation medium in the step of generating the hemihydrate may comprise a compound that does not block the second polar body emission.

In yet another example, the oocyte may comprise middle stage II (MII) stage oocytes.

In another example, the oocyte may comprise middle stage I (MI) stage oocytes.

In another example, the oocyte may comprise a single pronuclear oocyte.

In another example, the pn-hPSC may be heterozygous or homozygous.

In another example, the pn-hPSC may comprise multipotent cells having at least 1% of the population and an immunocompetent human leukocyte antigen (HLA) haplotype.

In another example, the HLA haplotype may be one comprising HLA-A, HLA-B, and HLA-DR.

As another example, the step a) may include injecting a sperm factor into an oocyte.

In another example, the oocyte may be incubated in the presence of CARM1 and / or Esrrb.

For example, step b) may be a suspension culture of hPSC.

In another example, the suspension culture period may be from 1 day to 14 days.

In another example, step b) comprises culturing hPSC in DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) supplemented with knockout-serum replacement (KSR) .

As another example, step b) may be performed by separating hPSC into two or more clumps and then culturing.

For example, the step c) comprises culturing the EB in a medium supplemented with a TGF-beta inhibitor and / or low glucose, or culturing the EB in a medium supplemented with a TGF-beta inhibitor; And culturing the EB in a medium with low glucose conditions.

In another example, the EB can be cultured under conditions free of bFGF (bFGF free).

As another example, it may be the EB adherent culture in the medium of the low glucose condition.

In another embodiment, the step c) comprises culturing the EB for 10 to 18 days in a medium supplemented with a TGF-beta inhibitor; And culturing the EB for 12 to 20 days in a medium with low glucose conditions.

In another embodiment, the step c) comprises culturing the EB in a DMEM / F12 medium supplemented with a TGF-beta inhibitor and KSR; And culturing the EB in DMEM / F12 medium with low glucose conditions supplemented with serum and antibiotics.

In another embodiment, the step c) comprises: culturing the EB in a medium supplemented with a TGF-beta inhibitor and / or a medium in a low glucose condition; And culturing the outgrowth of EB cultured in the medium in an MSC proliferation medium, or culturing the EB in a medium supplemented with a TGF-beta inhibitor; Culturing said EB in a medium with low glucose conditions; And culturing the resultant of EB cultured in the medium in an MSC proliferation medium.

As another example, the MSC growth medium may be DMEM medium supplemented with at least one member selected from the group consisting of serum, antibiotics, non-essential amino acids (NEAA) and? -Mercaptoethanol.

As another example, the result of EB may be separated into single cells, followed by adherent culture, followed by culturing in MSC proliferation medium.

For example, step c) may be a step of culturing under bFGF free conditions.

In another embodiment, the step c) comprises culturing EB in a medium in which bFGF is excluded, followed by culturing with addition of a TFG-? Inhibitor; And culturing the EB in low glucose conditions.

For example, more than 80% of the MSCs may be positive for the markers CD29, CD44 and CD90, and more than 20% may be positive for the marker CD105.

In another example, more than 80% of the MSCs may be positive for the markers CD29, CD44, CD90 and CD105.

In another example, less than 5% of the MSCs may be negative for the markers TRA1-60, SSEA4, CD34, and CD45.

In another example, less than 3% of the MSCs may be negative for the markers TRA1-60, SSEA4, CD34 and CD45.

In another example, the MSC may be capable of replicating at least 5 passages in cell culture.

In another example, the MSC may be capable of replicating at least 10 passages in cell culture.

In another example, the MSC may be capable of replicating at least 15 passages in cell culture.

As another example, the doubling time of the MSC may be 35 to 48 hours.

As another example, the MSC may have a doubling time of 41 to 45 hours.

In another example, the MSC may be one or more of the following types of selected from the group consisting of hematopoietic stem cells, muscle cells, myocardial cells, liver cells, chondrocytes, epithelial cells, urinary tract cells, kidney cells, blood vessel cells, retinal cells, Lt; / RTI >

In another embodiment, the method comprises: d) separating a single cell from the prepared mesenchymal stem cell (MSC); And e) culturing the isolated single cells to produce single cell-derived MSCs.

In another example, the mesenchymal stem cells of step d) may be cells of the early passage, for example P1 to P9.

As another example, the culture of step e) may be cultured in a medium containing bFGF.

The present invention also provides mesenchymal stem cells (MSCs) prepared by the above method.

For example, more than 80% of the MSCs may be positive for the markers CD29, CD44 and CD90, and more than 20% may be positive for the marker CD105.

In another example, more than 80% of the MSCs may be positive for the markers CD29, CD44, CD90 and CD105.

In another example, less than 5% of the MSCs may be negative for the markers TRA1-60, SSEA4, CD34, and CD45.

In another example, less than 3% of the MSCs may be negative for the markers TRA1-60, SSEA4, CD34 and CD45.

In another example, the MSC may be capable of replicating at least 5 passages in cell culture.

In another example, the MSC may be capable of replicating at least 10 passages in cell culture.

In another example, the MSC may be capable of replicating at least 15 passages in cell culture.

As another example, the doubling time of the MSC may be 35 to 48 hours.

As another example, the MSC may have a doubling time of 41 to 45 hours.

In another example, the MSC may be one or more of the following types of selected from the group consisting of hematopoietic stem cells, muscle cells, myocardial cells, liver cells, chondrocytes, epithelial cells, urinary tract cells, kidney cells, blood vessel cells, retinal cells, Lt; / RTI >

The mesenchymal stem cells prepared according to the present invention enable the treatment of various diseases or disorders. Diabetes, osteoarthritis, and Parkinson ' s disease.

FIG. 1 is a graph showing that when the number of cells is less than 700 million cells according to the current UCB management and research laws, it is classified as disposal.
Fig. 2 shows the result of chromosomal analysis of the donor cell of Example 1. Fig.
FIG. 3 shows the result of chromosome analysis of the NT-hPSC prepared in Example 1. FIG.
FIG. 4 shows the result of (a) genomic DNA test of NT-hPSC prepared in Example 1 and (b) mitochondrial DNA test as compared with donor somatic cell and donor oocyte.
Fig. 5 shows immunochemistry analysis results of the stem cell markers of NT-hPSC prepared in Example 1. Fig.
Fig. 6 shows RT-PCR analysis of the stem cell marker of NT-hPSC prepared in Example 1. Fig.
FIG. 7 is a graph showing the results of experiments for confirming the ability of the NT-hPSC to differentiate into triploid derived cells prepared in Example 1. As shown in FIG. 7, the embryoid bodies were formed and cultured for 14 days. immunohistochemistry analysis results, (b) RT-PCR results of marker, and (c) tissue analysis of teratoma formed by injection into immunodeficient mice.
FIG. 8 shows that there is no difference between (a) the morphology of RPE cells derived from embryonic stem cells and the type of RPE cells derived from NT-hPSC prepared in Example 1 and (b) the markers of RPE cells.
9 shows a process of manufacturing MSC from the hPSC according to the third embodiment.
10 is a microscopic photograph of the hPSC according to Example 3 during MSC production.
11A shows the doubling time of CHA-hES 15 derived MSC (CHA-hES 15 MPC) and CHA-NT4 derived MSC (CHA-NT4-MPC) (CHA-hES 15 MPC), CHA-NT4-derived MSC (CHA-NT4-MPC) and hBM-MSC, MSC (CHA-NT4-MPC) and hBM-MSC (Bar: mean ± SE, n = 3, triplicate).
Fig. 12 shows flow cytometric analysis results of CHA-hES 15-derived MSC (CHA-hES 15 MPC) and CHA-NT4 derived MSC (CHA-NT4-MPC).
Figure 13 shows the adipogenesis, osteogenesis and chondrogenesis of CHA-hES 15 derived MSC (CHA-hES 15 MPC), CHA-NT4 derived MSC (CHA-NT4-MPC) and hBM- ). The results are shown in FIG.
FIG. 14 shows the results of RT-PCR analysis of the expression of bone formation markers (COL1, Runx2), lipogenesis markers (PPARγ, C / EBPα) and cartilage development markers (COMP, Sox9).
Figure 15 shows (a) the cumulative number of cells of CHA-hES 15 single-derived MSC (CHA-hES15 (single-derived) -MPC), CHA-hES 15 derived MSC (CHA- hES 15 MPC) and hBM- cell numbers and (b) cumulative doubling numbers.
FIG. 16 shows flow cytometric analysis results of CHA-hES 15 single cell-derived MSC (CHA-hES15 (single-derived) -MPC).

Hereinafter, the present invention will be described in detail.

All technical terms used in the present invention are meant to be understood by those skilled in the art to which the present invention pertains in general, unless otherwise defined. In addition, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention.

In the present invention, " stem cells " are capable of self-renewing (having the ability to pass multiple cell division cycles while maintaining undifferentiated state) and exhibit one or more pluripotency (ability to differentiate into one or more specialized cells) Cells that can be treated. Mesenchymal Stem Cell (MSC) is a multipotential stem cell capable of differentiating into mesodermal cells such as bone, cartilage, fat, muscle cells and various mesodermal cells or nerve cells.

The term " somatic cell " in the present invention means any tissue cell in the body except for a sex cell or its precursor.

&Quot; Maturation " as used herein refers to a process consisting of harmonious biochemical steps leading to a finally differentiated cell type.

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

&Quot; Differentiated cells " in the present invention include any somatic cell that is not pluripotent in its original form as the term is defined herein. Thus, the term " differentiated cell " may also be a partially differentiated cell, such as a multipotent cell, or a cell that is a stable, non-functional partially reprogrammed or partially differentiated cell produced using any of the compositions and methods described herein . In some embodiments, the differentiated cells are stable intermediate cells, such as cells that are non-functional, partially reprogrammed cells. It should be noted that placing many of the primary cells in the culture can slightly erase the fully differentiated characteristics. Thus, simply culturing these differentiated or somatic cells does not make them become undifferentiated cells (e. G. Undifferentiated cells) or pluripotent cells. Transfer to differentiation potential of differentiated cells (including stable, non-functional partially reprogrammed cell intermediates) requires reprogramming stimulation beyond stimulation to partially attenuate the differentiated characteristics when placed in culture. In some reprogrammed, reprogrammed, partially reprogrammed cells can also be grown in extended subculture without loss of growth potential, as compared to parent cells that generally have a lower probability of development having only a limited number of cleavage in cultures To have the ability to suffer. In some embodiments, the term " differentiated cell " also encompasses a cell of a less specialized cell type (i. E., Increased likelihood of development) (e.g., from an undifferentiated or reprogrammed cell) (I. E., Reduced likelihood) stemming from < / RTI > Preferably, in the present invention, hematopoietic stem cells, muscle cells, cardiomyocytes, liver cells, chondrocytes, epithelial cells, urinary organs, adipocytes, kidney cells, vascular cells, retinal cells, mesenchymal stem cells (MSC) And the like.

In the present invention, " immunoconjugate homozygous junction " means that the genotype of each of the HLA-A, HLA-B and HLA-DR genes inherited from the donor's paternal and maternal systems is completely identical, .

In the present invention, the term " immunomodulatory cell " refers to a cell in which HLA-A, HLA-B and HLA-DR genes are homozygous and homologous, and all three HLA- The recipient can benefit from the transplantation.

The term " bank " in the present invention means a storage site of stem cells, and may be used as it is or in a different person, or in other individuals, for therapeutic, clinical or research purposes from stored cells as necessary.

In the present invention, " administering " means introducing a predetermined substance into a patient in any appropriate manner, and the administration route of the substance can be administered through any conventional route so long as it can reach the target tissue. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal. In addition, administration can be performed by any device capable of transferring to a target cell.

The present invention provides a method of producing a human pluripotent stem cell (hPSC) comprising: a) producing a human pluripotent stem cell (hPSC); b) forming the human pluripotent stem cells into an embryoid body (EB); And c) culturing the embryoid body to prepare mesenchymal stem cells (MSCs).

The pluripotent stem cells may be selected from the group consisting of nuclear transfer pluripotent stem cells (NT-hPSC), hematopoietic-derived pluripotent stem cells (pn-hPSC), reprogrammed pluripotent stem cells (iPSC), or embryonic stem cells Cell), but the present invention is not limited thereto.

Induced Pluripotent Stem Cell (iPSC) refers to pluripotency cells obtained by differentiation from differentiated cells (for example, somatic cells) and is capable of differentiating into various organ cells. iPSC can be obtained by reprogramming cells differentiated by their demetalization inducers.

Wherein the human embryonic stem cell comprises omnipotent cells derived from an inner cell mass of a human gland. Such human embryonic stem cells are, for example, CHA-hES3 (Jumi Kim, Sung-Hwan Moon, Soo-Hong Lee, Dong- Ryul Lee, Gou- Young Koh, and Hyung-Min Chung, Effective Isolation and Culture of Endothelial Cells in Embryoid Body Differentiated from Human Embryonic Stem cells (2007) STEM CELLS AND DEVELOPMENT 16: 269.280), but the present invention is not limited to these examples. In addition, human embryonic stem cells can be easily constructed by those skilled in the art.

The step a) is a step of separating nuclei from homozygous cells to generate nuclear-engrafting human pluripotent stem cells (NT-hPSC), wherein a homozygous or heterozygous state is formed from a plurality of donation tissues before step a) And screening.

Cells produced through NT may have genetic material in the nucleus of the patient and are individual patient specific in this respect. Thus, it allows for cell transplantation in patients with significantly reduced risk of autotransplantation, i.e., immune rejection in the same species. Further, the homozygous cells in the present invention are cells matched with the HLA antigen type, and are capable of being transplanted into a heterozygous person without an anti-HLA antibody. That is, the NT-derived stem cells have a homozygous type and are capable of being transplanted into an immunocompetent cell.

Therefore, it is preferable that the screening selects that the genes of human leukocyte antigen (HLA) -A, HLA-B and HLA-DR are homozygous. HLA genotypic screening has revealed that it is possible to obtain an immunocompetent cell line that can be transplanted to more than 90% of the total population of Japan once they find 140 donors with different immuno- A more efficient method to generate integration-free human iPS cells, Nature Methods 8, 409-412 (2011)).

In the present invention, homozygous cells were screened on the basis of data of Cha Hospital donated cord blood bank. According to the current umbilical cord blood management and research laws, when the number of cells is less than 700 million, it is approved for approval by the Ministry of Health and Welfare, the Ministry of Health and Welfare, and can be used for research as needed (see FIG. 1).

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

Therefore, the cells to be used may be frozen cord blood (suitable for research purposes) (with less than 700 million cells), and may include all of the donated cord blood for research registered with the Center for Organ Transplantation Management (KONOS) have. In addition, a sample obtained from a hematopoietic stem cell donor network or a medical institution under Chae Byung-won can be used.

The step of generating nuclear transfer human pluripotent stem cells (NT-hPSC) comprises the steps of removing nuclei of oocytes; Adding one or more nuclei of one or more donor cells to produce nuclear-engrafted (NT) oocytes; Incubating in a post-activated medium to activate said NT oocyte; Generating a blastocyst from the activated NT oocyte; And isolating the inner cell mass (ICM) cells from the blastocyst, wherein the ICM cells are further capable of culturing into NT-hPSC cells.

For example, the step of removing the nucleus of the oocyte includes the step of removing mid-stage II (MII) step oocyte spindle. In various embodiments, the first polar body (1PBE) is removed. In another embodiment, the method comprises peeling the cumulus cells before completion of maturation. In an embodiment, oocytes are observed in real-time with non-UV light based observations on 1PBE. In another embodiment, observations take place in the absence of dyeing or labeling agents, such as Hoechst staining. In an embodiment, this includes the use of a polscope, for example, a Research Instruments (CRi) Oosight ^TM imaging system. For example, this involves visualizing the zona pellucid and spindle complexes in MII oocytes harvested at 545 nm. In yet another embodiment, the step of removing the nuclei of the oocyte comprises the use of a contoured micropipette with a contour allowing the removal of 1PBE from the oocyte membrane and from the oocyte membrane. In yet another embodiment, the step of removing the nuclei of the oocyte comprises the use of a piezoelectric drill. In another embodiment, the step of removing the nuclei of the oocyte is performed in a denucleating medium containing cytochalasin B and, optionally, a protein phosphatase inhibitor, such as caffeine. Caffeine is a protein phosphatase inhibitor that inhibits premature activation to improve the growth of cloned embryos and consequently increases the rate of blastocyst formation. Therefore, preferably, the enucleation of the oocyte is performed in a medium containing a protein phosphatase inhibitor, and the protein phosphatase inhibitor may be caffeine.

In yet another embodiment, the step of generating nuclear-engrafted (NT) oocytes by adding one or more nuclei of one or more donor cells may comprise grafting the donor nucleus. The step of transplanting donor nuclei involves the use of agents that modify the oocyte membrane structure. In one embodiment, the step of removing the nucleus of the oocyte through the use of an agent that modifies the oocyte membrane structure comprises fusion with somatic cells. For example, the step of transplanting donor nuclei may include providing three to four donor cells with an injection pipette (e.g., 12 占 퐉 diameter), administering paramyxovirus or paramyxovirus proteins, such as Sendai virus, And then discharging the donor cells in a certain amount of the solution containing the coat protein or the extract thereof. Thereafter, cells were harvested using an injection pipette at a distance (4 to 5 cells long) from the linearly arranged donor cells, holding the oocytes with a holding pipette, the injection pipette with the donor cells harvested from the oocyte Lt; / RTI > The step of advancing the injection pipette comprises the nondestructive action of the yolk prostate membrane, and the step of contacting the nuclear donor cell with the yolk prostate membrane located below the zona pellucida, in the perivitelline space of the oocyte, Lt; RTI ID = 0.0 > donor < / RTI > In various embodiments, the recovery of the pipette does not interfere with the contact between the yolk membrane 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, cells are fused 10 minutes after donor cell insertion. Optionally, the process is repeated for cells that have not successfully fused. In various embodiments, Polyskop, e.g., the Oosight ^TM imaging system, is used throughout.

For example, donor nuclear transfer can be done by direct injection method. In one embodiment, the step of transplanting donor nuclei may comprise electrocellular manipulation, for example, electrofusion. In another embodiment, the method can comprise separating the somatic cell nuclear donor, the stem cell nuclear donor, and the nucleus of the sperm cell nuclear donor. In another embodiment, the method comprises inserting one or more donor nuclei via pipette or piezoelectric injection after separating somatic nuclei for NT. In various embodiments, the donor nucleus is derived from a cell, for example, a dermal fibroblast, a leukocyte, a hair follicle, or other somatic cell nuclear donor. In yet another embodiment, the present invention discloses a method comprising the isolation and preparation of nuclei from sperm cell donation. In a different embodiment, the separation of the nuclei may be accomplished by other methods for obtaining tissue biopsies, transfusions, or tissue samples, mechanical separation, collagenase digestion, washing, centrifugation based density gradient separation, and / Includes processing of tissue through culture.

Preferably, the step of fusing the nuclei of the somatic cells is carried out in a medium containing Sendai virus or Sendai virus extract.

Activation may be accomplished using an electrical and / or chemical method prior to incubation in the post-activation medium. The electrical and / or chemical method can be activated by activating calcium oscillation known in the art. For example, it can be activated by incubation in an activated medium as a chemical method.

Artificial oocyte activation depends on mimicry of changes in calcium signaling during spontaneous spermatogenesis. Normal oocyte development depends on a high level of Metaphase Promoting Factor (MFP) activity to arrest oocytes at mid-stage II (MII) stage. MII I block of cells is hampered by a cell change in calcium ions (Ca ^{2 +)} level due to the entry of sperm. Thereafter, target degradation of the cyclin B (MPF regulatory subunit) occurs, which releases the oocyte from cell cycle arrest, progenitor formation, and meiosis and mitosis.

Oocyte activation is dependent on an artificial calcium-changing strategy to release oocytes cultured from cell cycle arrest. Examples include the addition of calcium ion carriers, liposoluble molecules that transfer ions through lipid bilayers, such as ionomycin and A23817. Alternative strategies rely on electrical activity or direct injection of ions.

Following reconstruction of a nuclear-implanted (i.e., reconstructed) oocyte, as is associated with NT, oocyte activation using calcium-altering techniques also occurs.

However, in addition to this calcium ion implantation, the addition of protein phosphatase inhibitor caffeine to sheep oocytes, for example in the manufacture of NT, has been associated with the maturation-promoting factor (MPF) and mitogen-activated protein Increased activity of kinases (MAPKs) has been reported, and similar benefits have been reported in monkey oocytes, whereas the incidence of blastocyst formation has not increased. In addition, calcium activation through calcium ion transport, electrical activation, or direct injection does not cause the same timing, spatial conditioning, or calcium oscillation as a spontaneous correction. Adding additional complexity, the effect on calcium has also been shown to be species-specific. In some instances, the use of additional treatments with protein synthesis inhibitors such as kinase inhibitors, such as 6-dimethylaminopurine (6-DMAP), ethanol and cycloheximide (CHX) Is used. A histone deacetylase inhibitor such as TSA is associated with improved NT reprogramming. Treatment of TSA can promote the formation of blastocysts.

In one example, an electrical pulse can be applied during the fusion process and activation of somatic cells. Electrical activation involves electrical pulses in an electrical cell fusion medium. In various embodiments, the electrocellular fusogenic medium comprises 0.1 to 0.5 M mannitol, 0.01 to 1 mM MgSO ₄ .7H ₂ O, 0.01 to 1 mg / ml polyvinyl alcohol, 1 to 10 mg / ml human serum albumin, 0.5 mM CaCl ₂ .2H ₂ O and a. In one embodiment, the electrocellular fusogenic medium comprises 0.3 M 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 transfer oocytes are treated in a post-activation medium for complete activation. In a different embodiment, the activated reconstructed nuclear-engrafted oocytes are then incubated in a post-activation medium. In a different embodiment, the post-activation medium is a HEPES-depleted medium, a protein-depleted medium, a G1 or G2 medium, a compartment medium, a compartment co-medium, an IVF medium, a blastocyst formation medium, or a global human embryo culture medium. In a different embodiment, the post-activation medium is selected from the group consisting of 6-DMAP, puromycin, ethanol, cycloheximide (CHX), trichostatin A (TSA), and cytochalasin B ) CB. ≪ / RTI > In a different embodiment, the activated oocytes are cultured in post-activation medium at a concentration of 30-45, 45-60, 60-90, 90-120, 120-150, 150-180, 180-210, 210-240, 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, the hypoxic condition is about 80-85%, 85-90%. 90 to 95%, 95% or more of N _2, 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, the hypoxic condition comprises about 90% N ₂ , about 5% O ₂ , and about 5% CO ₂ . In various embodiments, the post-active medium a gas mixture, for example, about 90% N _2, and about 5% O _2, and about 5% CO 1 of _2, 2,3, 4,5, 5, or DMAP containing 1, 2, 3, 4, 5, 5 or more mM of the confluent medium incubated for a further period of time, for example at 37 ° C.

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

In yet another embodiment, the activation and / or post-activation may add factors separated from the sperm, its derivatives and extracts. In one embodiment, the human sperm factor is injected into the activated reconstructed oocyte using any of the injection methods described. In one embodiment, the human sperm factor is injected into the post-activated reconstructed oocyte using any of the injection methods described. In various embodiments, after about 1, 2, 3, or 4 days, the post-activated reconstructed nuclear-engrafted oocytes are transferred to the compartmental medium. In certain embodiments, after about one day, the post-activated reconstituted nuclear-engrafted oocytes are transferred to a compartmentalized medium. In various embodiments, the sperm factor comprises, for example, a factor separated from the cell protein that is present inside or outside the sperm cell. In one embodiment, the total sperm extract is obtained using mechanical mixing of a surfactant and an assessed 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, freshly sperm human sperm are imported and centrifuged at 900 g for 10 minutes to remove sperm plasma, followed by a sperm-TALP containing 5 mg / mL bovine serum albumin, after the centrifugation at the same time re-suspended and the setting of the pellets in the removal of the supernatant and nuclear separation medium (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) to a final concentration of 20 x 10 ⁸ sperm / mL, to remove the resuspension of the pellet, and the SPECT-TALP. After the SPECT-TALP was removed, the cells were treated with 1 mM dithiothreitol, 100 mM leupeptin, 100 mM antipain, and 100 mg = mL soybean trypsin inhibitor (5 min per cycle in liquid N ₂ ) and thawing (at 15 ° C) with 4 cycles of sperm pellet formation at 20,000 × for 50 min at 2 ° C after resuspension of the pellet in the same volume as the NIM 5 minutes per cycle). Finally, the resulting supernatant is carefully removed, titrated, and kept at -80 ° C until used.

In various embodiments, the post-activated reconstructed nuclear-engrafted oocytes are further cultured in blastocysts. In one embodiment, the post-activated reconstituted nuclear-engrafted oocytes are further cultured in a SAGE compartmental medium, for example, Quinn's medium. In yet another embodiment, the medium, e.g., 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, 0.1 M beta-ME 1/1000 v / v, SU5402 (FGFR inhibitor) 2 μM, PD184352 (ERK cascade inhibitor) 0.8 μM, CHIR99021 μM) or modified 3i medium (including 0.4 μM PD0325901 (MAPK inhibitor)) promote pluripotency. In one embodiment, the additional incubation is for 1, 2, 3, 4, 5, or more days. In one embodiment, additional incubation is provided in a culture medium having reprogramming factors and / or methylation-altering agents. In various embodiments, the additional incubation is for 3 days in G2 medium supplemented with either CARM1 CARM1, Esrrb, Kdm4a, Kdm4b and Kdm4d Esrrb. For example, CARM1 and / or Esrrb may be provided at a concentration in the 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 the medium of 2 [mu] g / ml.

In various embodiments, additional cultivation into blastocysts and induction of pluripotent stem cells (pSCs) from blastocysts can be accomplished by treating the blastocysts cultured to remove zipper cells with acidic Tyrode's solution . In various embodiments, the treatment is for a few seconds (e.g., 1 to 5). Following removal of ZP in various embodiments followed by washing in HEPES-HTF medium. In various embodiments, isolation of intracytoplasmic carbon (ICM) comprises discarding the trophoblast of the blastocyst. In various embodiments, ICM cells are plated in a mouse embryonic feeder (MEF) prepared prior to plating day. In some embodiments, whole blastocysts are plated onto MEFs. For example, the method includes the step of removing the zona pellucida. In various embodiments, the method comprises removing 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- . 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 administering to the mammal a therapeutically effective amount of a proanase in a TH3 medium (SAGE blastocyst medium) for 1 to 10, 10 to 20, 20 to 30, 30 to 60, 60 to 120, 120 to 180, . In another embodiment, the method comprises applying 0.5% proanthrocyte in HTF medium for 30 to 60 seconds. In one embodiment, the blastocysts are derived from reconstituted nuclear-engrafted oocytes obtained from somatic cell nuclear transfer (NT) of donor cell nuclei to committed oocytes. In one embodiment, the stem cell line is a somatic cell nuclear transfer human pluripotent stem cell (NT-hPSC) hPSC cell line.

In yet another embodiment, the invention includes a method for immunosurgery comprising mechanical dispersion of intracellular mass (ICM) from a trophectodermal cell. In various embodiments, the blasted blastocysts are treated with rabbit anti-human spleen serum for about 10, 20, 25, 30, 35, 40, 45, or 60 minutes at 37 占 폚. In one embodiment, the method includes the steps of washing the blasted blastocyst with TH3 (SAGE blastocyst medium), incubating the reconstituted Guinea pig complement with HECM-9 (SAGE blastocyst medium) at 37 DEG C for 30 minutes do. In a different embodiment, the zona pellucida of the expanded blastocyst is removed with a slight exposure (45-60 seconds) to a 0.5% fertilizer or acidic tiod solution in TH3 (hepes-HTF) medium. In one embodiment, the method further comprises the steps of small bore pipetting, laser assisted incubation using an Ehsms zilos-tk unit (Hamilton Thorne) to selectively isolate the inner cell mass from the malignant ectodermal cells, hatching method) to mechanically diffuse the cells.

In the present invention, preferably, the post-active medium is in a medium containing TSA, and the post-active medium is 6-DMAP. More preferably, post-activation is performed in a culture medium containing 6-DMAP, followed by culturing in a medium containing TSA.

In various embodiments, the nuclei of at least one donor cell may be altered by attracting it with epigenetic modifying agents to enhance the rate of successful NT-hPSC production. The eugenic regulator specifically increases the efficiency of transcription by changing the state of methylation or acetylation of a specific protein or DNA and consequently enhances NT efficiency. The targets of these eukaryotic regulators are histone acetyl transferase (HAT), histone deacetylase (HDAC) protein, lysine dimethylazase (KDM) domain protein, protein methyl transferase Enzyme protein (PMT) domain protein, and the like. These agents include small interfering RNA (siRNA), small molecules, proteins, peptides, antibodies, and the like. These agents act on the eugenic target associated with the reprogramming resistant region. NT oocytes are cultured in the presence of agents that alter the health status. Specific examples of these formulations are described in Tables 2 to 5 below. A histone acetyl transferase (HAT) protein, a histone deacetylase (HDAC) protein, a lysine dimethylazase (KDM) domain protein, and a protein methyl transferase (PMT) The present invention is not limited to the following examples.

The histone acetyl transferase (HAT) protein Target_ID (| domain #) Full name Uniprot _ID NCBI geneide 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, 90 kDa 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, 210 kDa-like Q8IZX4-1 138474

The histone deacetylase (HDAC) protein Target_ID (| domain #) Full name Uniprot _ID NCBI geneide 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 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 back six Q8N6T7_1 51548 SIRT6 back six Q8N6T7_2 51548 SIRT6 back six Q8N6T7_4 51548 SIRT7 sirtuin 7 Q9NRC8_1 51547

Lysine dimyrylase (KDM) domain protein Target_ID (| domain #) Full name Uniprot _ID NCBI geneide 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 geneide 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 variant 4-20 homolog 1 (Drosophila) Q4FZB7_1 51111 SUV420H2 suppressor of variant 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, the methyl transferase can be inhibited by co-substrate analogues. Three types of co-substrate analogs inhibiting various types of methyltransferases are known. Sinefugin, a structurally similar antibiotic compound to S-adenosylmethionone (SAM), dimerized co-matrix SAH, and methylthioadenosine as feedback inhibitors. Lysine methyl group inhibitors include chaetocin, the first identified inhibitor, and Bix-01294, a G9a (KMT1C) inhibitor. They are selective for SUV39H1 and PRM1. The compound Bix-01338 was a non-selective inhibitor of selectivity between lysine and arginine methyl group transferase, showing an IC50 of 5 mM for G9a, while an IC50 of 6 mM for PRMT1. UNC0224 is presented as a new inhibitor with an IC50 of 15 mM for lysine methyl transferase G9a. Inhibitors of histone methyltransferases such as EPZ5676, EPZ005687 and GSK126 also show antitumor activity in various animal models of cancer.

Protein arginine methylation is carried out by PRMTs and is divided into two groups. Type I methyl transport enzymes induce asymmetrically substituted arginine residues and type II methyl transport enzymes form symmetrically substituted arginine residues. CARM1 shows affinity with proline-glycine-methionine-arginine (so-called PGM motif). PRMT5 is also known to methylate PGM motifs. Common substrate analogs such as Sinefungin may also be used as inhibitors of arginine methyl group transferase (also known as AMIs as arginine methyl group transferase inhibitors). AMI-1 is the most active inhibitor with an IC50 of 9 mM for PRMT1. Allatodapsone and stilbamidine inhibitors induce hypomethylation in H4R3.

Another type of epigenetic target may be to increase NT efficiency. DNA methyltransferases (DNMTs) preferentially methylate the CpG nucleotide sequence of DNA. Three mammalian DNA methyltransferases have been identified for DNMT1, DNMT3A and DNMT3. In general, methylation of these promoter regions prevents gene expression by interfering with the binding of transcription factors to DNA. In addition, methylated DNA is bound by a methyl-CpG binding domain protein, which attracts histone modeling enzymes and consequently condenses the chromatin structure, Can be induced. DNMT inhibitors can consequently inhibit gene expression and thus increase NT efficiency. Some compounds are exemplified: chlorogenic acid, mithramycin, azacytide, bisdemethoxycurcumim, decitabine, lomegutatrib, benzylguanine, sorafenib and sorafenib tosylate.

In addition, histone deacetylase (HDAC) functions to remove acetyl groups from the N-acetyllysine amino acid of histone, resulting in more positively charged and strongly bound to negatively charged DNA. Condensation of the DNA structure and genetic transcription are further suppressed. HDACs can be divided into four subgroups according to their location and function. Class I HDACs (subtypes 1, 2, 3 and 8) are mainly found in the nucleus and class II HDACs (4, 5, 6, 7, 9 , 10) can migrate through the nuclear membrane and are found in both nuclear and cytoplasmic. Type III HDACs are called silent information regulator 2 (Sir2), and type IV HDACs (11 subtypes) are found in both nucleus and cytoplasm, mainly in the brain, heart, and muscle cells. HDACs inhibitors have anticancer activity when administered in combination with other chemical synthetic drugs. HDAC inhibitors can promote DN transcription and consequently increase NT efficiency.

The post-active medium can include epigenetic modifying agents, such as epigenetic chromatin and histone modification agents, and / or DNA modifiers. In a particular embodiment, it can be used in combination with the protein arginine methyl-transferase (PRMT1) and coactivator-associated arginine methyltransferase 1 (CARM1 / PRMT4), nuclear oropharyngeal nuclear (Lysine (K) -Specific Demethylase 4A, Kdm4a), lysine-specific dimethylazate 4B (Lysine (K) -Specific Demethylase 4B, Kdm4b) or lysine-specific dimethylazase 4D (Lysine (K) -Specific Demethylase 4D, Kdm4d). In certain embodiments, the methylation-altering agent and / or DNA modifier is expressed as a modified recombinant protein. For example, CARM1 and Esrrb can enhance the permeation of proteins and peptides through 7X arginine (7R) -cell-permeable peptides (CPPs), or to the artisan through cell membranes and nuclear membranes, Lt; RTI ID = 0.0 > protein. ≪ / RTI > In another embodiment, the method uses octamer binding transcription factor-4 (Oct-4), sex determining region Y-box-2 (sex determining (Y-box-2) (Sox-2), nanog, Kruppel-like factor-4 (Klk-4), MyoD, c-Myc, zinc finder (Rex-1 / Zfp-42), lefty A, teratocarcinoma-derived growth factor (Tdgf), and / or telomeric repeating binding factor (Terf-1) of the nuclear donor cell. In various embodiments, the methods include intracellular infusion of direct piezoelectric infusion, viral infusion, liposomal infusion, or other methods. In various embodiments, the transcription factor may be delivered in the form of an mRNA, protein, and / or cell extract that may be applied prior to nuclear transfer to the nucleated oocyte. In other embodiments, the method can include the use of HDAC inhibitors (Class I, II, and III), or DNMT3a and DNMT3b inhibitors.

Thus, the present invention preferably comprises a post activation medium comprising epigenetic modifying agents. More preferably, the epigenetic modifying agents are selected from the group consisting of histone acetyl transferase (HAT), histone deacetylase (HDAC) protein, lysine dimethylazase (KDM) domain protein, A protein methyl transferase (PMT) domain protein, and DNA methyltransferases (DNMTs). In the present invention, preferably, the method for producing stem cells derived from NT cells comprises the steps of: (a) culturing stem cells in the step (c); Isolating an inner cell population (ICM) cell from the resulting blastocyst; And further culturing the separated inner cell population cells as stem cells.

Wherein said step a) is a step of generating hemihydrate-derived human pluripotent stem cells (pn-hPSC), said step a) comprising the steps of: activating oocytes; Incubating the activated oocytes in a post-activated medium to produce a hemihydrate; Incubating the semi-adulterate in a culture medium to form a blastocyst; And separating the inner cell mass (ICM) cells from the blastocyst, wherein the inner cell mass is further cultured with pn-hPSC.

Activation of oocytes can activate oocytes by applying electrical and / or chemical stimuli.

In one embodiment, the method comprises incubating Medium II (MII) stage oocytes in an activated medium to initiate the formation of unit gonadal oocytes. In certain embodiments, the activation medium comprises a calcium ion carrier. In a different embodiment, the calcium ion carrier is ionomycin, A23187, vuverixin, X-537A, avenaciolide, a monomacrocyclic polyether, or a macrobiocyclic compound or cryptate. In a different embodiment, the activation medium comprises an alcohol, e. G. Ethanol. In a different embodiment, the activation medium comprises thimerosal. In different embodiments, the activation medium is a HEPES-depleted medium, a protein-depleted medium, a G1 or G2 medium, a compartment medium, a compartment co-medium, an IVF medium, a blastocyst formation medium, or a global human embryo culture medium. In another embodiment, the method comprises electrical activation of MII stage oocytes. In one embodiment, the electrical activation comprises an electrical pulse in an electrical cell fusion medium. In various embodiments, the electrocellular fusogenic medium comprises 0.1 to 0.5 M mannitol, 0.01 to 1 mM MgSO ₄ .7H ₂ O, 0.01 to 1 mg / ml polyvinyl alcohol, 1 to 10 mg / ml human serum albumin, 0.5 mM CaCl ₂ .2H ₂ O and a. In one embodiment, the electrocellular fusogenic medium comprises 0.3 M 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 other embodiments, one or more oocytes are injected first with a sperm factor and then activated electrically.

In certain embodiments, the MII stage oocyte is cultured at 1 < RTI ID = 0.0 > 1 < / RTI > at 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more minutes, and then washed in the wash medium. In certain embodiments, the MII stage oocytes are exposed to the calcium ion carrier for 5 minutes at a temperature of < RTI ID = 0.0 > 37 C < / RTI > In a different embodiment, the wash medium is a HEPES-depleted medium, a protein-depleted medium, a G1 or G2 medium, a compartment medium, a compartment co-medium, an IVF medium, a blastocyst formation medium.

In a different embodiment, the activated unit gonadotropic oocytes are then incubated in a post-activation medium. In a different embodiment, the post-activation medium is a HEPES-depleted medium, a protein-depleted medium, a G1 or G2 medium, a compartment medium, a compartment co-medium, an IVF medium, a blastocyst formation medium, or a global human embryo culture medium. In a different embodiment, the post-activation medium comprises 6-DMAP, puromycin, ethanol, cycloheximide (CHX), trichostatin A (TSA), and / or cytochalasin B (CB). In a different embodiment, activated unit gonadotropic oocytes are cultured in post-activation medium at 30, 30-45, 45-60, 60-90, 90-120, 120-150, 150-180, 180-210, 210 To 240, 240 to 270, 300 to 330, 330 to 360, 360 to 390 minutes, or more than 390 minutes. In certain embodiments, the activated unit gonadal 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, the hypoxic condition is about 80-85%, 85-90%. 90 to 95%, 95% or more of N _2, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more of 0 _2, and , About 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more CO ₂ . In certain embodiments, the hypoxic condition comprises about 90% N ₂ , about 5% O ₂ , and about 5% CO ₂ .

After incubation in the post-activated medium, the post-activated unit gonadotropic oocytes are incubated in the wash medium. In a different embodiment, the wash medium is a HEPES-depleted medium, a protein-depleted medium, a G1 or G2 medium, a compartment medium, a compartment co-medium, an IVF medium, a blastocyst formation medium. In yet another embodiment, the culture medium does not require continuous media exchange such as global human embryo culture medium. In certain embodiments, the wash medium comprises TSA. In certain embodiments, the post-activated oocytes are incubated for 240, 300, or 360 minutes in a wash medium comprising TSA.

After incubation in the wash medium, the post-activated unit gonadotropic oocytes are transferred to the culture medium. In a different embodiment, the culture medium is a HEPES-depleted medium, a protein-depleted medium, a G1 or G2 medium, a compartment medium, a coarse co-medium, an IVF medium, or a blastocyst formation medium. In yet another embodiment, the culture medium does not require continuous media exchange such as global human embryo culture medium. In a different embodiment, the post-activated unit gonadal oocyte can be cultured in confluent medium for 1, 2, 3, 4 or more days. In certain embodiments, incubation in refractory medium is for 2 days. After incubation in calf medium, oocytes are cultured in blastocyst formation medium for 1, 2, 3, 4 or more days. In certain embodiments, the incubation in the blastocyst formation medium is for 3 days.

In one embodiment, the step of activating the oocyte comprises washing in confluent medium followed by incubation in the activation medium for 5 minutes at 37 占 폚. In various embodiments, the activation medium comprises 5 [mu] M ionomycin. In various embodiments, oocytes are treated in a post-activation medium for complete activation. In various embodiments, the post-activation medium comprises 2 mM 6-DMAP in collagen medium for 4 hours at 37 ° C in 5% CO ₂ /5% O ₂ /90% N ₂ atmosphere. In one embodiment, the activated oocytes are washed, and further incubated. In one embodiment, the activated unit gonadal oocytes are washed in a 6-DMAp removal medium. In one embodiment, the activated unit gonadal oocytes are further cultured in a SAGE deficient medium, such as a queen medium. In another embodiment, the medium, e.g., 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, 0.1 M beta-ME 1/1000 v / v, SU5402 (FGFR inhibitor) 2 μM, PD184352 (ERK cascade inhibitor) 0.8 μM, CHIR99021 μM) or modified 3i medium (including 0.4 μM PD0325901 (MAPK inhibitor)) promote pluripotency. In one embodiment, the additional incubation is for 2 days. In one embodiment, additional incubation is provided in a culture medium having reprogramming factors and / or methylation-modifying agents. In various embodiments, the additional incubation is for 3 days in G2 medium supplemented with CARM1 and / or Esrrb. In various embodiments, activated unit gonadotropic oocytes are further cultured in blastocysts. In various embodiments, additional cultivation into blastocysts and induction of pluripotent stem cells (pSCs) from blastocysts can be accomplished by incubating the blastocysts cultured to remove zipper cells with acidic tie load solution (e.g., pH 2.0) . In various embodiments, the treatment is for a few seconds (e.g., 1 to 5). Following removal of ZP in various embodiments followed by washing in HEPES-HTF medium. In various embodiments, isolation of intracytoplasmic carbon (ICM) comprises discarding the trophoblast of the blastocyst. In various embodiments, ICM cells are plated into mouse embryo feeders (MEFs) that were prepared one day prior to plating. In some embodiments, the whole embryo is plated on MEFs. In various embodiments, the PSC induction medium contains a serum replacement (5% SR, Invitrogen), FBS (10%, Hyclone), plasminate (5%), bFGF (32 ng / ml), and human LIF , Sigma-Aldrich).

The pn-hPSCs are derived from heterozygous or homozygous oocytes. In certain embodiments, the heterozygous pn-hPSCs are more than 1%, 2%, 3%, 4%, 5%, 6% or more of the population of the ethnic group, as determined by the allelic variable frequency in the ehtnic population, Lt; RTI ID = 0.0 > HLA < / RTI > In certain embodiments, homozygous pn-hPSC is derived using the unit reproductive of one prokaryotic oocyte. In certain embodiments, homozygous pn-hPSCs are derived using the unit reproduction of one nucleated homozygous oocyte. In another embodiment, homozygous pn-hPSCs are obtained using unit generations involving a second polar extrusion. In certain embodiments, homozygous pn-hPSCs are present in 1%, 2%, 3%, 4%, 5%, 5% or more of a population of HLA serotypes that are immunoconjugated with 1%, 2%, 3%, 4%, 5% 3%, 4%, 5%, 5% or more HLA haplotype having HLA serotypes that are immunoconjugated. For example, the HLA haplotype A * 01, B * 08, and DRB1 * 03 are presented with a frequency of more than 5% in the Caucasian race. In another embodiment, the HLA-serotype includes allelic variants of HLA-A, HLA-B, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ and HLA-DR. In other embodiments, the HLA-serotype includes allelic variants of HLA-A, HLA-B, and HLA-DR. In another embodiment, pn-hPSC is derived from mid-stage I (MI) stage oocytes. In another embodiment, the pn-hPSCs are derived from midterm II (MII) stage oocytes. In another embodiment, the pn-hPSC comprises a mitochondrial genome that is identical or nearly identical to a donor oocyte.

The pn-hPSC includes the normal 46 chromosome, XX karyotype. In another embodiment, the pn-hPSC can form all three embryonic cell layers. In another embodiment, the pn-hPSC is selected from the group consisting of phase-specific embryonic antigen-4 (SSEA-4), SSEA-3, tumor rejection antigen 1-81 (Tra- 1-81), Tra- 1-60, (Rex-1 / Zfp-42), lefty A, teratocarcinoma-derived growth factor-4 (Oct-4), sex regulatory region Y-box-2 (Sox-2), nanog, zinc finger protein- Expressing one or more pluripotency markers comprising Tdgf, a telomeric repeating binding factor (Terf-1), and developmental pluripotency-associated gene 2 (Dppa-2). In other embodiments, the pn-hPSC does not comprise a recessive mortality rate. In other embodiments, the pn-hPSC possesses high alkaline phosphatase (AP) and / or telomerase activity. In other embodiments, the pn-hPSC can form a teratoma in an embryoid body and / or an immunodeficient animal in a suspension in an immunodeficient animal.

The step b) may be a suspension culture of hPSC. The suspension culture period may be one day, two days, three days, four days, or five days or more, and 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days Day or 6 days or less.

In one embodiment, hPSCs can be cultured in DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) medium supplemented with KSR (knockout-serum replacement) .

The hPSCs can be suitably separated before incubation and cultured, for example, by separating into 2 to 5 clumps. At this time, the hPSC can be separated using appropriate physical or chemical methods.

The step c) may be a step of culturing the EB formed in step b) in a medium supplemented with a TGF-beta inhibitor and / or a medium in a low glucose condition.

The low glucose condition may be that the medium contains glucose of 15 mM or less, preferably 10 mM or less, more preferably 7 mM or less, and most preferably 3 to 6 mM. For example, 5.5 mM D-glucose (1 g / L) may be included in the medium. MSC can be efficiently prepared by culturing under commonly used high glucose conditions (for example, containing about 25 mM glucose) under low glucose conditions.

In one embodiment, EBs can be cultured under low glucose conditions supplemented with a TGF-beta inhibitor, cultured for a period of time in a medium supplemented with a TGF-beta inhibitor, and cultured for a predetermined period in a medium with low glucose , It may be cultured in a low glucose condition for a certain period of time and then cultured in a medium supplemented with TGF-beta inhibitor for a certain period of time. Preferably, the EB may be cultured in a medium supplemented with a TGF-beta inhibitor and then cultured in a medium with low glucose conditions. For example, EBs can be cultured in DMEM / F12 medium supplemented with TGF-beta inhibitor and KSR, and then cultured in DMEM / F12 medium supplemented with serum and antibiotics under low glucose conditions. Fetal Bovine Serum (FBS) may be used as the serum, but the present invention is not limited thereto. The antibiotic may be at least one selected from the group consisting of penicillin and streptomycin, but is not limited thereto. EB may be cultured for 10, 11, 12 or 13 days or more in a medium supplemented with TGF-beta inhibitor and cultured for 18 days, 17 days, 16 days, or 15 days or less have. In addition, the EB may be cultured for 12, 13, 14 or 15 days or more in a medium with low glucose conditions, and cultured for 20 days, 19 days, 18 days or 17 days or less have. In other embodiments, it may be by adherent culture of EBs in medium with low glucose conditions. Gelatin may be used for attachment culture, but is not particularly limited thereto.

In another embodiment, the EB formed in step b) may be cultured in a medium in which basic fibroblast growth factor (bFGF) is excluded. MSC (mesenchymal stem cells) can be efficiently produced by excluding bFGF as an undifferentiated state as a medium component. For example, the EB formed is cultured in KSR and DMEM / F12 medium without bFGF for a short period of time, for example, 1 to 3 days, followed by addition of TGF-beta inhibitor for 10 to 18 days and culturing for 12 to 20 days under low glucose conditions can do.

In another embodiment, the EB may be cultured in an MSC growth medium in which the EB is cultured in a medium supplemented with a TGF-beta inhibitor and / or a medium in a low glucose condition. For example, the EB may be cultured in a medium supplemented with a TGF-beta inhibitor and then cultured in an MSC proliferation medium to produce an MSC. The MSC growth medium may be DMEM medium supplemented with at least one member selected from the group consisting of serum, antibiotics, non-essential amino acids (NEAA) and? -Mercaptoethanol. Fetal Bovine Serum (FBS) may be used as the serum, but the present invention is not limited thereto. The antibiotic may be at least one selected from the group consisting of penicillin and streptomycin, but is not limited thereto.

In other embodiments, the EB product may be isolated and cultured in single cells prior to culturing in MSC growth medium. For example, the result of EB may be separated into single cells by physical or chemical methods, followed by adherent culture for a predetermined period, and then cultured in an MSC proliferation medium.

In another embodiment, the method comprises: d) separating a single cell from the prepared mesenchymal stem cell (MSC); And e) culturing the isolated single cells to produce single cell-derived MSCs.

In another embodiment, step d) may be to separate single cells from the mesenchymal stem cell population using a pipette (e.g., a glass pipette). The single cell separation may be performed by diluting mesenchymal stem cells into a culture medium and then isolating single cells with the aid of a microscope. Further, in isolating the single cells, a mesenchymal stem cell population of the early passage, for example, p1 to p9, p2 to p8, p3 to p8, p4 to p7, or p5 to p7, It may be to use groups.

In another embodiment, the step e) comprises: dividing a single cell per well into a well plate; And culturing the dispensed cells until a colony is formed. Also, in step e), it may be determined whether single cells per well have been dispensed and then the culture is started. The cultivation in step e) may be carried out under the same or similar culture conditions as in step c), or in subculture water. For example, the single cell may be cultured in a medium containing bFGF (for example, MSC proliferation medium supplemented with bFGF). The culturing may be performed for at least two days, five days, ten days, eleven days, twelve days, or thirteen days or more, but not limited thereto, at least until the period when the colonies are formed. The culture may also include subculture. The subculture may be performed at least 2 times, 5 times, 10 times or more.

In another embodiment, the prepared mesenchymal stem cells may be a substantially homogeneous population of cells. The substantially homogeneous cell population is derived from a single cell such that a plurality of cells (e.g., at least 70%, at least 80%, at least 90%, or 100%) in the cell population Or may have the same mentioned activity. Since the mesenchymal stem cells derived from the single cells have advantages in terms of safety to differentiate into different types of cells or induce teratomas and the like, they can be usefully used in clinical practice.

The mesenchymal stem cell (MSC) produced by the method of the present invention may be positive for at least one selected from the group consisting of CD29, CD44, CD90 and CD105.

For example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or 99% or more may be positive for CD29.

At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 96%, 97%, 98% or 99% or more of which is positive for CD44.

At least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 96%, 97%, 98% or 99% or more of which is positive for CD90.

MSC at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83% , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% It can be positive.

The mesenchymal stem cell (MSC) may be negative for at least one selected from the group consisting of TRA60, SSEA4, CD34 and CD45.

For example, at least 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7% 5%, 4%, 3%, 2%, 1%, 0.5% or 0.05% or less may be negative for TRA60.

MSC has at least 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7% , 4%, 3%, 2%, 1%, 0.5%, or 0.05% or less may be negative for SSEA4.

MSC has at least 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7% , 4%, 3%, 2%, 1%, 0.5% or 0.05% of the CD34 can be negative for CD34.

MSC has at least 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7% , 4%, 3%, 2%, 1%, 0.5%, or 0.05% of the CD45 is negative for CD45.

According to the production method of the present invention, the MSC may have a replication ability capable of progressing passage at least 5 times, 6 times, 10 times, 11 times, 12 times, 13 times, 14 times, or 15 times in the cell culture have.

The doubling time of the MSC prepared by the process of the present invention is 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours or 41 hours or more and 48 hours, 47 hours, 46 hours or 45 hours Or less.

MSCs produced by the method of the present invention have the ability to differentiate into differentiated cells. For example, it may have at least one differentiation ability selected from the group consisting of hematopoietic stem cells, muscle cells, cardiac muscle cells, liver cells, cartilage cells, epithelial cells, urinary tract cells, kidney cells, blood vessel cells, retinal cells and neuron cells.

The MSC of the present invention can be cryopreserved for future use. In one embodiment, the cryopreservative is one or more cryoprotectants including but not limited to dimethylsulfoxide (DMSO), ethylene glycol, glycerol, and propanediol; One or more culture media including, but not limited to, DMEM, MEM, and the patented media described above; The stem cells are cryopreserved in a solution containing 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 medium (BioLife Solutions Inc., Joobetel, Washington, USA). In another embodiment, the serum replacement is a knockout serum replacement (Invitrogen 10828-028).

Cryopreservation of MSC freezes cells at a controlled rate or freezes them to a "manual" process. The controlled rate freezing procedure is initiated by turning on the controlled rate freezer and setting the freezing program for tissue or cell freezing. The controlled rate freezer will use liquid nitrogen to reduce the temperature in the inner chamber (thereby reducing the temperature of any of the contents of the chamber). The freezing program for the cells is initiated by keeping the temperature until the inner chamber is cooled to < RTI ID = 0.0 > 4 C < / RTI > While the controlled rate freezer is cooling, the cells are suspended in cryopreserved media cooled to 4 占 폚. The cell suspension is aliquoted into a freezing vial in an amount of 1 ml per cryovial. The frozen vial is then labeled and placed in a controlled speed freezer chamber and stimulated to continue the program. First, the temperature of the chamber is maintained at 4 DEG C for an additional 10 minutes. Next, the chamber is cooled at a rate of -1 [deg.] C / min until the temperature reaches -80 [deg.] 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 be equilibrated to -120 ° C. The frozen vials of the frozen cells are then transferred to liquid nitrogen (Dewar) for long term storage.

The present invention also provides cells differentiated from the MSC.

The differentiated cells are not limited from stem cells such as hematopoietic stem cells, muscle cells, myocardial cells, liver cells, chondrocytes, epithelial cells, urinary tract cells, adipocytes, kidney cells, vascular cells, retinal cells and neuronal cells. Refers to any cell that is differentiated into any one or more cells from the group consisting of, and can be used as a therapeutic agent through cell transplantation without limitation. A step of screening for immunocompromised cells through HLA screening of heterologous patients from cryopreserved cell banking may be added. Immunoadjunction is to enable banking by enhancing the optimal supply of regenerative therapy as an implantable material.

The present invention also provides a population of cells comprising the MSCs prepared by the method of making the MSCs. The present invention also provides a composition comprising a population of cells of MSC for the treatment of various types of diseases.

The cell composition of the present invention contains from 0.1 to 99.9% by weight of the active ingredient, based on the total weight of the composition of the present invention, and may include a pharmaceutically acceptable carrier, excipient or diluent.

The compositions of the present invention may be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

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

The composition can be administered orally or parenterally at the time of clinical administration and can be administered orally or parenterally in the case of parenteral administration by intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine injection, intracerebral injection, And may be used in the form of a general pharmaceutical preparation.

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.

Best Mode for Carrying Out the Invention Hereinafter, the functions and effects of the present invention will be described in more detail through specific embodiments of the present invention. It is to be understood, however, that these embodiments are merely illustrative of the invention and are not intended to limit the scope of the invention.

Example 1. Preparation of nuclear transfer pluripotent stem cells (NT-hPSC)

Screening of homozygous cells from donor cells and selection of donor cells

Homozygous cells were screened for umbilical cord blood of less than 700 million donated cord blood from Cha Hospital. Genomic DNA was extracted using Gentra Puregene ^™ Blood Kits (QIAGEN, Hilden, Germany) for HLA-A, B and DRB1 genotyping and sequenced using SeCore A, B and DRB1 Locus Sequencing Kit (Invitrogen, Brown Deer, Sequence-based typing. In particular, exon 2-4 for HLA-A and B and exon 2 for HLA-DRB1 were amplified using the locus specific primers included in the kit. The ABI3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA (Invitrogen) and Sequencher (Gene Codes Corp., Ann Arbor, MI, USA) were used to perform sequencing of the PCR products generated using the HLA SBT u-type software v3.0 Analysis. HLA homozygous donor cells (A * 33: 03-B * 44: 03-DRB1 * 13: 02) (haplotype frequency: 4.6%) were the most frequent donors from Koreans And if they make NT-cells, they can cover about 9% of the total population.

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

1.2 Recovery and preparation of oocyte

The SCA (Stem cell Research Oversight) Committee of the CHA Regenerative Medicine Institute (CHARMI) and the Essex Institutional Review Board (EIRB).

They recruited 20-32 year-old women through web-based advertising and tested their reproductive, medical and psychological health status according to the American Society for Reproductive Medicine (ASRM) guidelines. The study was conducted on women with BMI <28 Kg / m ² who underwent both medical and psychological tests.

Ovarian stimulation was performed according to established clinical IVF guidelines (Tachibana et al., 2013). The women were sedated with Lupron or hCG for 36 hours with Midazolam 5-7.5 mg (Versed, Roche, and Nutley. NJ, USA) and Fentanyl 50-75 ug (Abbott Pharmaceutical, Abbott Park, And the oocytes were then recovered using the previously described ultrasonography map. Freshly-isolated cumulus-oocyte cell complexes (COCs) from IVF medium (Quinn's IVF medium, SAGE Biopharma, Bedminster, NJ) were pooled and mixed with 10% serum substitute (SSS; Quinns Advantage Serum, Cooper Surgical ) Were placed in supplemented HTF-Hepes medium (Global Medium) at 37 ° C. COCs were treated with hyaluronic acid (100 IU / ml, Sigma, St. Louis, Mo. USA), and oocytes were classified according to the degree of maturation, and oocytes in the middle stage II (MII) .

1.3 Enucleation of oocytes, nuclear replacement and activation of somatic cells

Enucleation of oocytes can be performed by a previously known method (Tachibana et al., 2013), enucleation of oocytes and nuclear replacement of somatic cells is accomplished using a stage warmer, a narishige micromanipulator ), An Oosight ^TM imaging system (poloscopic microscopy), and an inverted microscope equipped with a laser. An inverted microscope with a piezo instead of an inverted microscope with a laser may optionally be used.

The oocytes were placed in a droplet of HTF-Hepes Medium (Global Medium) containing cytochalasin B (5 ㎍ / ml) and caffeine (1.25 mM) And the temperature was kept at 37 占 폚 for 10 to 15 minutes. Caffeine is a protein phosphatase inhibitor that inhibits premature activation to improve the growth of cloned embryos and consequently increases the rate of blastocyst formation. Then the oocytes were fixed with a holding pipette so that the spindle was located close to the 2 to 4 o'clock position. Then, the zona pellucida was punctured with a laser pulse and the injection pipette was inserted through the open part. The infusion pipette was inhaled into the spindle in contact with a small amount of cytoplasm surrounded by a plasma membrane. Instead of a laser pulse, a piezo pulse may be selectively used for the transparent bar.

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

When the fusion was confirmed, the prepared oocytes were further cultured in Global 10% SPS medium for 30 minutes or 2 hours.

Activation was carried out in 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 (BSA) Followed by the addition of an electrical pulse (2 x 50 μs DC pulses, 2.7 kV / cm). Activated cells were cultured for 4 hours in Global Medium (except serum) containing 2 mM DMAP at 5% CO ₂ at 37 ° C, and cultured in 5% CO ₂ , 5% O ₂ , 90% N ₂ , Were cultured in Global Medium supplemented with 10% FBS, 12 μM BME (β-mercaptoethanol), CARM (2 μg / ml) and 10 nM TSA (Trichostatin A) for 12 hours. After pronuclear formation was confirmed, the cells were incubated for up to 7 days in Global Medium supplemented with 10% FBS and 12 μM BME without 5% CO ₂ , 5% O ₂ , 90% N ₂ , Lt; / RTI &gt; Then, CARM mRNA is injected into the kidney using microinjection system at the time of giving up the 4th year. HDAC1, SIRT2 or KDM4D can optionally be added instead of CARM.

1.4 Preparation and characterization of stem cell line

The blastocysts cultured in 2 above were treated with an acidic Tyrode solution (pH 2.0) for several seconds to remove ZP. After removal of ZP, the embryo was vigorously washed in Hepes-HTF medium to remove even the small amount of Tyrode solution. ICM was isolated using a laser-assisted bladder ablation system (Hamilton-Thorne Inc.) and the remaining part of the blastocyst (the trophoblast) was discarded to confirm that the blastocyst was no longer intact. One day before plating, the ICM was plated onto the prepared MEF, but when the cloned blastocyst had an indistinguishable ICM, the entire embryo was plated. The hPSC induction medium contained serum replacement (5% SR, Invitrogen), FBS (10%, Hyclone), plasmamate (5%), bFGF (32 ng / ml), and human LIF (2000 units / -Aldrich) supplemented with &lt; / RTI &gt; ICM was cultured in the same medium for 3 days without change, and about 1/3 of the medium was replaced on the fourth day. Half of the medium was replaced every day from day 6. Outgrowth was confirmed within 7 days after plating.

Colonies with ESC shape were selected, characterized and cytogenetic analysis for further propagation. Colonies were expanded and frozen before day 12. The characteristics of the cells were analyzed by karyotype (G-banding), DNA fingerprinting and mitochondrial DNA genotyping. The results are shown in Figures 3 and 4, respectively. In order to confirm the expression of stem cell markers of the cells, alkaline phosphatase activity was confirmed by AP staining and Oct4, SSEA-4, TRA 1-60 and TRA 1-81 were analyzed by immunocytochemistry. RT-PCR The expression of Oct4, Nanog and Sox-2 markers was analyzed. The results are shown in Figures 5 and 6. It was confirmed that the cells derived from these results were stem cells. In addition, in order to confirm the ability of differentiation into 3 germ layers derived cells, embryoid bodies (EBs) were formed in vitro and cultured. Immunochemistry and RT-PCR were performed to express the expression patterns of the germplasm-derived differentiation markers (Fig. 7 (a) and (b)). In order to confirm the differentiation ability in the body, stem cells were injected subcutaneously into the testis of immunodeficient mice to induce teratoma formation, and histolysis was confirmed by H-E and special staining. The results are shown in Fig. 7 (c).

1.5 Cryopreservation and banking of NT-hPSC

The cells prepared in the above 3 are classified and stored in accordance with homozygous cells and recorded in a document or a program. In particular, information on the cell donor is stored together. Can be used directly in an autologous or allogenic recipient (patient) or stored for use as differentiated cells.

1.6 Differentiation from NT-hPSC to functional retinal pigment epithelial cells (RPE)

In order to induce differentiation into RPE, NT-hPSC in undifferentiated state was mechanically divided into several clumps-form of NT-ES cells using a sterilized tip under a dissecting microscope (about 300-600 undifferentiated embryonic stem Cell line). The clump-shaped NT-hPSC was added to low attachment 6-well plates (Corning, CA, USA) and supplemented with 15% (v / v) knockout ^TM medium (EBDM; knockout ^TM DMEM (Thermo Scientific, (Thermo), 1% (v / v) glutamax (Thermo), 1% (v / v) NEAA (Thermo), 1% (v / v) penicillin-streptomycin (Thermo) and 0.1 mM β-mercaptoethanol Thermo)) for 4 days in a floating state. The cultured embryo bodies are transferred to a culture dish and induced to induce RPE differentiation.

1.7 Isolation of differentiated retinal pigment epithelial cells from NT-hPSC

The embryoid body was placed in a 6-well culture dish with 0.1% gelatin coating, allowed to stand in the incubator for 3 days, and then replaced with the EBDM culture medium every 2 to 3 days. Of the cells growing out of the embryoid body, Were incubated for about 50 to 55 days. In order to separate the RPE cell is color-separated pigmentation, cells are normal saline ^{(DPBS containing Ca 2 + Mg 2} + (Thermo)) with twice the normal saline solution containing a collagenase type 4 After washing (Type IV collagenase (Thermo) in DPBS with Ca ²⁺ Mg ²⁺ (Thermo)) and incubated for 2 hours at 37 ° C in a 5% CO ₂ incubator. To remove the enzyme, detached cell clusters were collected in a 50 ml tube and washed twice with DMEM-FBS culture medium using a centrifuge (1500 rpm, 5 min). The cell masses were transferred to a 60 mm Petri dish, and then pigmented cell clusters were separated from other cell masses that were not pigmented using a glass pipette that had been minutely pulled out under a dissecting microscope.

1.8 Maturation and proliferation of retinal pigment epithelial cells differentiated from NT-hPSC

The RPE cell mass (pigmented cell clusters) are then washed twice with normal saline with calcium and magnesium were removed (DPBS without Ca ^{2 +} Mg ^{2 +} (Thermo)) to the culture to separate a single cell sorting enzyme liquid (1 : 1 mixture of 0.25% Trypsin-EDTA (Thermo) and Cell Dissociation Buffer (Thermo)). The retinal pigment epithelial cells isolated by single cells were washed with a DMEM-FBS culture medium using a centrifuge (1500 rpm, 5 minutes), and then the cells were released with EGM2 medium (Lonza, PA, USA) The cells were cultured in EGM-2 medium until the cells were completely filled with 0.1% gelatin-coated 4-well culture dishes at a concentration of 1 well. After 3 to 4 days, the cells were incubated in RPE differentiation medium (RGMM; 1: 1 mixture of EBDM and DMEM-FBS media) to show cobble stone morphology and pigmentation Were replaced and cultured for 7 days.

The retinal pigment epithelial cells differentiated from NT-hPSC were subcultured using the same isolation enzymes as above, and functional retinal pigment epithelial cells were obtained through proliferation using the EGM2 medium and aging using the RGMM culture medium. The retinal pigment epithelial cells obtained from the 2nd and 3rd passages were stained with 2 million cells / mL / cryo (Sigma) using a frozen solution (90% (v / v) FBS (Thermo) and 10% (v / v) DMSO vials were kept frozen at the time of use and some of them were analyzed for retinal pigment epithelial cell characteristics. Fig. 8 shows that there is no difference in the morphology and differentiation markers of RPE cells derived from embryonic stem cells and the RPE cells derived from NT-hPSC prepared in Example 3 (Fig. 8 (a), (b)).

Example 2. Preparation of semi-aqueous body-derived pluripotent stem cells (pn-hPSC)

2.1 Unit Reproduction Ⅰ: Generation of heterozygous pn-hPSC

A common method for oocyte activation is to mimic the normally-induced calcium-altering effects of sperm entry. In one example, a heterozygous HLA pn-hPSC cell line can be generated by the application of ionomycin for oocyte activation followed by the addition of the 6-aimedylamino-purine (6-DMAP) By inhibiting the activation of H1 kinase, it prevents spontaneous ovarian collapse.

Sequential addition of ionomycin and 6-DMAP initiates calcium flow first, leading to inhibition of protein phosphorylation, which leads to progenitor formation without the completion of meiosis. Other approaches rely on electrical activation of oocytes and subsequent addition of ionomycin, and 6-DMAP. However, the addition of 6-DMAP for precursor formation common to these techniques prevents the release of the second polar body and induces the production of diploid cells. This normally does not allow for the production of homozygous hPSC cell lines due to the formation of keyamatas (paternity and maternal genomic recombination) during early embryogenesis. Thus, after the first meiosis, the remaining oocyte nuclei are heterozygous with both the maternal and paternal genomes.

More specifically, unit reproduction of oocytes for production of heterozygous pn-hPSC cell lines can be obtained through the following process:

A) Retrieval of oocytes and preparations

1. The cumulus-oocyte complex (COC) is treated with hyaluronidase (100 IU / ml, Sigma, St. Louis, Mo. USA) for 2 minutes and stripped with a small caliber micropipette (diameter 150 μm). Stripped oocytes with germinal vesicles (GV) or no first polar body are not used. A stripped oocyte with a single first polar body is classified as medium II (MII) oocytes.

Alternatively, COC can be treated with SynVitro Hyadase to remove cumulus cells, and then incubated for 30 minutes in IVF medium with a Paraffin overlay

B) Activation of oocytes

1. In the activated medium, the MII oocytes are treated for 5 min at 37 ° C (5 uM ionomycin) and washed thoroughly in the follicular medium.

2. Re-treat the oocytes in the medium (1-2 mM 6-DMAP in 5 nM tricostatin A (TSA) -containing medium) after activation for 4 hours.

3. Wash the activated oocytes in 6-DMAP-free medium and incubate for 6 hours in a confluent medium containing 5 nM TSA before transfer to normal culture medium.

4. After incubation for the first 2 days in SAGE medium, the cells are incubated in SAGE blastocyst medium for 3 days.

Alternatively, after continuous exposure to 5 uM ionomycin for 5 minutes and continuous exposure to 1-2 mM 6-DMAP for 4 hours, cultivation in fresh IVF medium with careful washing and paraffin overlay, in IVF medium with a paraffin overlay Activation can be performed, followed by incubation in a confluent medium the following day.

2.2 Unit Reproduction Ⅱ: Generation of homozygous pn-hPSC

In contrast, a direct approach for the generation of HLA homozygous hPSC cell lines is based on one progenitor oocyte oocyte) from an HLA homozygous oocyte donor. However, a major disadvantage of this approach is that relatively homogeneous homozygous donors are present in the population. Alternatively, an HLA homozygous hPSC cell line can be obtained from heterozygous donors by the method except for the 6-DMAP addition for the second polar release (2PBE).

Activator, for example, Ca ^{2 +} ionophore, A23817 or Io initial addition of Norma who is and, further, this approach that relies on the addition of Saito collar new B is furo rapamycin or no cyclohexanone polyimide can be the release of the second polar body To provide a haploid parthenote with only half of the mid-II chromosome set. This hemispheric subject can be further cultured with diploid blastocysts for the formation of homozygous genotypes.

More specifically, unit reproduction of the oocyte for the production of a homozygous pn-hPSC cell line can be obtained through the following process:

A) Recovery and preparation of oocyte, same as above.

B) Activation of oocytes

1. In activated medium, MII oocytes are treated for 5 min at 37 ° C (5 uM A23187) and washed thoroughly in streak media.

2. Re-treat the oocytes in post-activated medium (cyclohexamide without 10 ug / mL furomycin nail medium or cytochalasin B) for 4 hours.

3. Wash the activated oocytes and incubate for 24 h in medium to allow for development into embryos at 2 cell stage. The two halves are then electrofused by applying 160 mV of electricity for 20 microseconds using an electric fusion device (BTX 2000, Harvard Electric) in mannitol or sorbitol-based fusion media. Alternatively, a single pulse can be applied, which appears to be sufficient to fuse the halves. Fused oocytes are cultured for one additional day in SAGE medium and cultured in SAGE blastocyst for 3 days.

2.3. Production of pn-hPSC

Generation of semi-adulterated (through unit breeding) Next, subsequent cultures lead to blastocyst formation from which hPSCs can be obtained from endothelial cells through separation of the surrounding trophectodermal tissue.

1. Remove the zona pellucida (ZP) by treating the cultured blastocyst with the protease (0.5%, Sigma-Aldrich) or acidic tie rod solution (pH 2.0) for several seconds. After removal of ZP, the embryo is washed vigorously in Hepes-HTF medium to remove traces of the proteinase or tie-rod solution.

2. Separate intracellular debris (ICM) using a laser-assisted blastocyst removal system (Hamilton-Thorne Inc.). Discard the remaining part of the blastocyst (the nutrient barrier) to ensure that the blastocyst is no longer intact.

3. Plating ICM on top of MEF fabricated the day before plating. The hPSC induction medium contained serum replacement (10% SR.Invitrogen), FBS (5% Hyclone), plasmamate (5%), bFGF (32 ng / ml), and human LIF (2000 units / Aldrich) is composed of a supplemented knockout-DMEM.

4. Cultivate ICM for 3 days in the same medium without any changes.

5. Replace about 1/3 of the medium on the 4th day.

6. Replace 1/2 of the badge every 6 days from the next day.

7. The initial result is visible within 7 days of plating.

8. Expand colonies 12 days before and cryopreserve.

Example  3. Human Versatility  Stem Cells( hPSC ) from Intermediate lobe  Stem Cells( MSC ) Of  Produce

3.1 EB Formation

H9 and CHA-hES 15 cells as human embryonic stem cell (hESC) strains and CHA-hES NT4, CHA-hES NT5 and CHA-hES NT8 cells as NT-hPSC strains were prepared. Specifically, cells were co-cultured in a colony form on MEF culture supernatants prepared at a concentration of 7.5 × 10 ⁴ cells / well (0.1% gelatin coated dish, ⁴ well).

For EB formation, the hESC and NT-hPSC colonies were mechanically divided into several groups (2 to 4 clump forms) using a sterile tip under a dissecting microscope, and 20% KSR (knockout-serum replacement, Invitrogen) And cultivated on a 60 mm Petri dish of DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) for 5 days.

3.2 MSC Manufacturing

After the EB was formed, the cells were cultured for one day in 20% KSR + DMEM / F12 medium (EB medium: hESC medium except bFGF medium), and 1 μM TGF-Beta inhibitor (SB431542) was added to the EB medium for 2 weeks Day), and the medium was changed once every two days.

The cells were then transferred to a 6-well plate coated with 0.1% gelatin (30 min, air dry) to a density of 5 to 7 per well per well and cultured in 10% FBS and 1% P / S (penicillin- streptomycin, Invitrogen ) In DMEM (low glucose; 5.5 mM D-glucose (1 g / L)) for 16 days. Forty-eight hours after incubation, cells attaching to the EB and extending out of the EB were identified, and the medium was changed twice a week. After 16 days, the cells growing out of the EBs were separated by a TrypLE solution (Invitrogen; 500 μl TrypLE per well, 2 min incubation) and transferred to a 75% flask coated with 0.1% gelatin and added with 10% FBS and 1% P / S And cultured in DMEM (low glucose; 5.5 mM D-glucose (1 g / L)).

On the next day, MSC proliferation media 10% FBS, 1% P / S, 1% NEAA (1 g / L) in DMEM supplemented with 10% FBS and 1% P / nonessential amino acids, Invitrogen) and DMEM (high glucose) supplemented with 0.1% β-mercaptoethanol (Invitrogen). As shown in FIG. 10, the MSC showed a spindled-shaped form of elongated spindle.

MSCs were incubated with 0.05% Trypsin-EDTA (1.5 ml per 75 T flask, 2 min incubation) at 80% confluence. % FBS (Thermo) + 10% DMSO (Sigma)).

3.3 Manufacture of single cell-derived MSC

To prepare single cell-derived MSCs, CHA-hES 15 cells of Example 3.1 were differentiated into MSCs by the method of Example 3.2 above, and MSCs of p5 to p7 corresponding to the early passage were used. First, 10% FBS, 1% P / S, 1% NEAA (nonessential amino acids, Invitrogen) and 0.1% β-mercaptoethanol (Invitrogen) were added to 96-well plates coated with 0.1% The cells were pre-warmed by adding 4 ng / ml hrbFGF to DMEM (high glucose). Cells were harvested using 0.05% Trysin-EDTA (1.5 ml per 75 T, 2 min incubation) as in the case of subculturing the cells cultured at 75 T as described in Example 3.2, and then diluted in the culture medium and placed in a 60 mm dish. Thereafter, one cell per well was dispensed into the above-prepared 96-well plate using a glass pipette which was finely mined under a dissecting microscope. After confirming that the cells had entered one cell per well under an inverted phase contrast microscope, the culture was started and the cells were cultured until a colony was formed. Table 6 below shows the number of wells (fractionation efficiency) accurately divided into single cells when CHA-hES 15-derived MSCs (CHA-hES15-MPC) were divided into 96 wells as single cells as described above, And the number of wells grown (multiplication efficiency).

Cell line (passage) Dividing Efficiency Efficiency CHA-hES15-MPC (p5) 68/96 (71%) 23/68 (34%)

Subsequently, the cells were grown to fill the wells, then subcultured using 0.05% Tryinsin-EDTA (20 ul per well, 2 min incubation) and cultured in 4, 6, or 12 well culture dishes And then cultured at 75 T to prepare single cell-derived MSCs.

Example 4. Characterization of MSC

4.1 Evaluation of proliferative ability

MSCs derived from hESCs, MSCs derived from NT-hPSCs, and hBM-MSCs were monitored over time for their growth potential. The results are shown in FIG.

11A shows the doubling time of CHA-hES 15 derived MSC (CHA-hES 15 MPC) and CHA-NT4 derived MSC (CHA-NT4-MPC) (CHA-hES 15 MPC), CHA-NT4-derived MSC (CHA-NT4-MPC) and hBM-MSC, MSC (CHA-NT4-MPC) and hBM-MSC (Bar: mean ± SE, n = 3, triplicate).

As shown in Fig. 11, MSCs derived from hESCs and MSCs derived from NT-hPSCs were found to exhibit superior proliferative activity as compared to hBM-MSCs.

4.2 Surface marker analysis

MSC (passage 6) derived from hESC and passage 6 from NT-hPSC were analyzed by flow cytometry. As an example, CHA-hES 15-derived MSC (CHA-hES 15 MPC) and CHA-NT4 derived MSC (CHA-NT4-MPC) are shown in FIG.

MSC (CHA-hES 15 MPC) and CHA-NT4 MSC (CHA-NT4-MPC) derived from CHA-hES 15 were positive for MSC-related markers CD29, CD44, CD90 and CD105, And negative for ESC markers TRA1-60 and SSEA4 and hematopoietic cell markers CD34 and CD45.

4.3 Differential analysis

Lipogenesis (adipogenesis), bone formation (osteogenesis) and cartilage generation (chondrogenesis) the MSC and hBM-MSC derived from the MSC, NT-hPSC derived from, hESC to analyze the multipotential Each lipogenesis culture media (StemPro of ^® in Adipogenesis Differentiation Kit (GIB-A10070-01) ), bone formation, culture media ^{(StemPro ® Chondrogenesis Differentiation Kit (GIB} -A10071-01)) and cartilage generation culture media ^{(StemPro ® osteogenesis Differentiation Kit (GIB} -A10072-01)) The cells were cultured for 3 to 4 weeks and observed under a microscope. The results are shown in FIG.

MSC (CHA-hES 15 MPC), CHA-NT4 MSC (CHA-NT4-MPC) and hBM-MSC from CHA-hES 15 derived from adipogenesis, osteogenesis and It was confirmed that it has the ability to differentiate from chondrogenesis.

Expression of osteogenic markers (COL1, Runx2), lipogenic markers (PPARγ, C / EBPα) and cartilage development markers (COMP, Sox9) were analyzed by RT-PCR. And the results are shown in Fig.

Example 5. Characterization of single cell-derived MSCs

5.1 Evaluation of proliferative ability

The growth rate of the single cell-derived MSC (CHA-hES15 (single-derived) -MPC) derived from the hESC prepared in Example 3.3 was monitored over time and the results are shown in FIG.

Figure 15 (a) shows the cumulative cell counts of CHA-hES 15 single-derived MSCs (CHA-hES15 (single-derived) -MPC), CHA-hES 15 derived MSCs (CHA-hES 15 MPC), and hBM- cumulative cell numbers, and (b) cumulative doubling numbers of the cells.

As shown in Fig. 15, the single cell-derived MSCs derived from hESCs showed a similar ability to proliferate as MSCs derived from hESCs, and showed superior proliferative activity compared to hBM-MSCs.

5.2 Surface marker analysis

The surface markers were analyzed by flow cytometry on single cell derived MSC (CHA-hES15 (single-derived) -MPC) derived from the hESC prepared in Example 3.3, Respectively.

As shown in FIG. 16, the single cell derived MSC (CHA-hES15 (single-derived) -MPC) derived from hESC is positive for MSC-related markers CD29, CD44, CD90 and CD105, and the ESC marker TRA1 -60 and SSEA4 and hematopoietic cell markers CD34 and CD45, respectively.

Single cell-derived MSCs derived from hESCs are homogeneous populations, and there are few embryonic stem cells with some ability to differentiate and proliferate in the cell population, causing them to differentiate into different types of cells or induce teratomas And has an advantage in terms of safety.

As a result, the single cell-derived MSCs derived from hESCs are homogeneous cell groups having substantially the same advantages in terms of cell proliferating ability and surface markers, and MSC derived from hESCs, It can be seen that this is a potentially large cell.

Claims (63)

a) producing human pluripotent stem cells (hPSC);
b) forming said human pluripotent stem cells into embryoid body (EB); And
c) culturing the embryoid body to prepare mesenchymal stem cells (MSCs). 2. The stem cell according to claim 1, wherein the human pluripotent stem cell is selected from the group consisting of nuclear transfer pluripotent stem cells (NT-hPSC), hematopoietic-derived pluripotent stem cells (pn-hPSC), reprogrammed pluripotent stem cells A stem cell (ESC). The method according to claim 1, wherein the step (a) comprises separating nuclei from homozygous cells to generate nuclear transfer human pluripotent stem cells (NT-hPSC)
further comprising the step of screening for homozygous from a plurality of donation tissues prior to step a). [4] The method according to claim 3, wherein the screening is homozygous for the genes of human leukocyte antigen (HLA) -A, HLA-B, and HLA-DR. The method according to claim 1, wherein the step a) is a step of generating nuclear transfer human pluripotent stem cells (NT-hPSC)
The step a) includes the steps of removing nuclei of oocytes;
Adding one or more nuclei of one or more donor cells to produce nuclear-engrafted (NT) oocytes;
Incubating in a post-activated medium to activate said NT oocyte;
Generating a blastocyst from the activated NT oocyte; And
Separating the inner cell mass (ICM) cells from the blastocyst, wherein the ICM cells are further capable of culturing into NT-hPSC cells. 6. The method according to claim 5, wherein the step of removing the nuclei of the oocyte is performed in a medium containing a protein phosphatase inhibitor. 7. The method according to claim 6, wherein the protein phosphatase inhibitor is caffeine. 6. The method of claim 5, wherein adding one or more nuclei of the one or more donor cells comprises direct injection. 6. The method of claim 5, wherein the step of adding one or more nuclei of the one or more donor cells comprises somatic cell fusion. 6. The method of claim 5, wherein at least one nucleus of the at least one donor cell is contacted with epigenetic modifying agents. 12. The method of claim 11, wherein the epigenetic regulator is selected from the group consisting of a histone acetyl transferase (HAT) protein, a histone deacetylase (HDAC) protein, a lysine dimethylazase (KDM) A protein methyltransferase (PMT) domain protein, and the like. 12. The method of claim 11, wherein the epigenetic regulator is siRNA (small interfering RNA), a low molecular weight protein, a peptide, or an antibody. 12. The method according to claim 11, wherein the eugenic regulator is at least one selected from the group consisting of Oct3 / 4, Sox2, Klf4, c-Myc and Lin2. 6. The method according to claim 5, wherein the step of adding nuclear of at least one donor cell to produce nuclear-engrafted (NT) oocytes is in a medium comprising Sendai virus, a protein thereof or an extract thereof . 6. The method of claim 5, wherein the at least one donor cell comprises somatic cells or germ cells. 6. The method according to claim 5, wherein the post-activation medium in the step of activating the NT oocytes is selected from the group consisting of 6-DMAP, puromycin, ethanol, CHX (cycloheximide), TSA (trichostatin A) cytochalasin B). &lt; / RTI &gt; 6. The method according to claim 5, wherein the NT oocyte is cultured in the presence of an erogenous regulator. 18. The method of claim 17, wherein the epigenetic regulator is selected from the group consisting of a histone acetyl transferase (HAT) protein, a histone deacetylase (HDAC) protein, a lysine dimethylazase (KDM) A protein methyltransferase (PMT) domain protein, and the like. 18. The method of claim 17, wherein the epigenetic regulator is siRNA (small interfering RNA), low molecular weight, protein, peptide or antibody. 6. The method according to claim 5, wherein step (a) is performed in the absence of ultraviolet light. [7] The method of claim 5, wherein the step a) includes injecting sperm factors into an oocyte. The method according to claim 1, wherein the step a) is a step of generating hemihydrate-derived human pluripotent stem cells (pn-hPSC)
The step a)
Activating oocytes by applying electrical and / or chemical stimulation;
Incubating the activated oocytes in a post-activated medium to produce a hemihydrate;
Incubating the semi-adulterate in a culture medium to form a blastocyst; And
Separating the inner cell mass (ICM) cells from the blastocyst, wherein the inner cell mass is further cultivated with pn-hPSC. 23. The method according to claim 22, wherein the chemical stimulation in the step of activating the oocyte is incubation in an activated medium, and the activated medium comprises a calcium ion carrier. 24. The method of claim 23, wherein the calcium ion carrier comprises at least one member selected from the group consisting of ionomycin, A23187, beauvericin, X-537A, and avenaciolide . 23. The method of claim 22, wherein the post-activation medium in the step of generating the semi-adulterated body is selected from the group consisting of 6-DMAP, puromycin, ethanol, CHX cycloheximide), TSA (trichostatin A), and CB (cytochalasin B). 23. The method of claim 22, wherein the post-activation medium in the step of generating the semi-aqueous body comprises a compound that does not block the second polar body emission. 23. The method of claim 22, wherein the oocyte comprises middle stage II (MII) stage oocytes. 23. The method of claim 22, wherein the oocyte comprises middle stage I (MI) stage oocytes. 23. The method of claim 22, wherein the oocyte comprises a single pronuclear oocyte. 23. The method of claim 22, wherein the pn-hPSC is heterozygous. 23. The method of claim 22, wherein the pn-hPSC is homozygous. 23. The method of claim 22, wherein the pn-hPSC comprises pluripotent cells having at least 1% of the population and an immunocompetent human leukocyte antigen (HLA) haplotype. 33. The method of claim 32, wherein said HLA haplotype comprises HLA-A, HLA-B, and HLA-DR. 23. The method according to claim 22, wherein the step a) comprises injecting a sperm factor into an oocyte. 23. The method according to claim 22, wherein the oocyte is incubated in the presence of CARM1 and / or Esrrb. 2. The method according to claim 1, wherein the step b) comprises suspension culturing the hPSC. 37. The method of claim 36, wherein the incubation period is from 1 day to 14 days. 36. The method of claim 36, wherein step b) comprises culturing hPSC in DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) supplemented with knockout-serum replacement (KSR) . 37. The method according to claim 36, wherein the step b) comprises separating the hPSC into two or more clumps and culturing the hPSC. The method according to claim 1, wherein the step c) comprises culturing the EB in a medium supplemented with a TGF-beta inhibitor and / or low glucose. 2. The method of claim 1, wherein step c)
Culturing the EB in a medium supplemented with a TGF-beta inhibitor; And
Culturing said EB in a medium with low glucose conditions. 42. The method of claim 41, wherein step c)
Culturing the EB for 10 to 18 days in a medium supplemented with a TGF-beta inhibitor; And
And culturing said EB for 12-20 days in a medium with low glucose conditions. 40. The production method according to claim 40 or 41, wherein the EB is adhered and cultured in the medium with the low glucose condition. 42. The method of claim 41, wherein step c)
Culturing the EB in DMEM / F12 medium supplemented with a TGF-beta inhibitor and KSR; And
And culturing said EB in DMEM / F12 medium with low glucose conditions supplemented with serum and antibiotics. 41. The method of claim 40, wherein step c)
Culturing said EB in a medium supplemented with a TGF-? Inhibitor and / or in a medium with low glucose; And
Culturing the outgrowth of EB cultured in said medium in MSC proliferation medium. 42. The method of claim 41, wherein step c)
Culturing the EB in a medium supplemented with a TGF-beta inhibitor;
Culturing said EB in a medium with low glucose conditions; And
Culturing the resultant of EB cultured in said medium in MSC proliferation medium. 46. The MSC proliferation medium according to claim 45 or 46, wherein the MSC proliferation medium is DMEM medium to which at least one member selected from the group consisting of serum, antibiotic, non-essential amino acid (NEAA) and? -Mercaptoethanol is added . 48. The method according to claim 45 or 46, wherein the resultant EB is separated into single cells, followed by adherent culture, followed by culturing in an MSC proliferation medium. The method according to claim 1, wherein more than 80% of the MSCs are positive for the markers CD29, CD44 and CD90, and more than 20% are positive for the marker CD105. The method according to claim 1, wherein more than 80% of the MSCs are positive for the markers CD29, CD44, CD90 and CD105. The method according to claim 1, wherein less than 5% of the MSCs are negative for the markers TRA1-60, SSEA4, CD34 and CD45. The method according to claim 1, wherein less than 3% of the MSCs are negative for the markers TRA1-60, SSEA4, CD34 and CD45. The method according to claim 1, wherein the MSC has a replication ability capable of progressing passage at least 5 times in cell culture. 2. The method according to claim 1, wherein the MSC has a replication ability capable of progressing at least 10 passages in cell culture. 2. The method of claim 1, wherein the MSC has a replication ability capable of progressing at least 15 passages in cell culture. 2. The method of claim 1, wherein the doubling time of the MSC is from 35 to 48 hours. The method of claim 1, wherein the doubling time of the MSC is from 41 to 45 hours. The MSC of claim 1, wherein the MSC is selected from the group consisting of hematopoietic stem cells, muscle cells, cardiomyocytes, liver cells, chondrocytes, epithelial cells, urinary tract cells, kidney cells, blood vessel cells, retinal cells, Or more. A mesenchymal stem cell prepared by the method of claim 1. The method according to claim 1, wherein the step c) is performed under bFGF free conditions. 61. The method of claim 60, wherein step c)
culturing EB in a medium in which bFGF is excluded, followed by culturing with addition of a TFG-beta inhibitor;
And culturing said EB in low glucose conditions. The method of claim 1, further comprising: d) separating the single cell from the prepared mesenchymal stem cell (MSC); And e) culturing the isolated single cells to produce single cell-derived MSCs. 63. The method according to claim 62, wherein the mesenchymal stem cells of step d) are cells of P (Passage) 1 to P9.

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