WO2017014513A1 - Procédé de conservation et système de banque de cellules nt - Google Patents

Procédé de conservation et système de banque de cellules nt Download PDF

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WO2017014513A1
WO2017014513A1 PCT/KR2016/007795 KR2016007795W WO2017014513A1 WO 2017014513 A1 WO2017014513 A1 WO 2017014513A1 KR 2016007795 W KR2016007795 W KR 2016007795W WO 2017014513 A1 WO2017014513 A1 WO 2017014513A1
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cells
cell
protein
oocytes
hla
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Korean (ko)
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차광렬
이동율
정영기
송지환
엄진희
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Cha Biotech Co Ltd
Industry Academic Cooperation Foundation of College of Medicine Pochon CHA University
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Cha Biotech Co Ltd
Industry Academic Cooperation Foundation of College of Medicine Pochon CHA University
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Priority to CN201680042142.1A priority Critical patent/CN108026544A/zh
Priority to JP2018521814A priority patent/JP6707636B2/ja
Priority to US15/745,301 priority patent/US12091679B2/en
Priority to EP16828017.0A priority patent/EP3327130A4/fr
Priority claimed from KR1020160090711A external-priority patent/KR20170009793A/ko
Publication of WO2017014513A1 publication Critical patent/WO2017014513A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the present invention is a cell prepared by using somatic cell nuclear transfer (NT) technology of somatic cells having a homozygous genotype in genes such as HLA (human leukocyte antigen) -A, HLA-B and HLA-DR It relates to the storage method and banking system.
  • NT somatic cell nuclear transfer
  • Cell therapy is a field that is emerging as a new paradigm of the pharmaceutical industry by enabling 'fundamental treatment' through cells, especially stem cells, which were considered to be limitedly treated through drugs or surgical procedures.
  • regenerative medicine is a new technology field that fights diseases by restoring or replacing tissues and organs damaged or degraded by aging, disease, accident, etc. It is a field that is emerging as a.
  • HLA-A HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR
  • MHC Major Histocompatibility Antigen Complex
  • HLA surface antigens The role of HLA surface antigens is to display fragments of the proteins present in the cell on the surface of the cell so that infections or mutations that may have occurred in vivo are detected by the immune cells, which is why antigen-providing proteins ( Also known as antigen presenting protein.
  • the Somatic Cell Nuclear Transfer (SCNT) technology refers to a technique of removing a nucleus of a somatic cell and transplanting the nucleus of an egg to replicate.
  • somatic cells By using somatic cells to separate the nucleus of the somatic cells and injecting the isolated nucleus into the nucleus from which the nucleus is removed to prepare a stem cell line that maintains the genetic characteristics of the somatic cells.
  • This method has the advantage of eliminating the immune rejection reaction in that it uses the patient's own somatic cells, and enables the patient-specific treatment.
  • NT's technology is advancing, the rate of formation from reconstructed oocytes to blastocysts is still very low. Therefore, various attempts have been made and demanded to increase this formation rate.
  • the largest disorder of NT embryos is zygotic gene activation (ZGA), which occurs in 4- to 8-cell phases in large mammals, including humans.
  • ZGA zygotic gene activation
  • the present inventors have led to the present invention by developing a cell therapeutic agent or a cell for transplantation that does not cause immunorejection from homozygous cells.
  • an object of the present invention is to provide a method for storing NT cell-derived stem cells applicable to various diseases of the same kind or different kinds.
  • the present invention comprises the steps of: a) screening for homozygous (Homozygous) from a plurality of donation tissue (donation);
  • d) provides a method for storing stem cells derived from an immunocompatible NT cell comprising cryopreserving a plurality of stem cells.
  • the present invention comprises the steps of a) screening homozygous (Homozygous) from a plurality of donation tissue (donation) tissue;
  • the present invention comprises the steps of a) screening homozygous (Homozygous) from a plurality of donation tissue (donation) tissue;
  • It provides a method for producing a cell differentiated from the stem cells, which are derived from an immunocompatible NT comprising: d) preparing the differentiated cells for cell transplantation from the prepared stem cells.
  • the present invention also includes a) means for collecting a plurality of donor tissues, b) means for screening collected tissues, c) means for preparing stem cells from tissues, and d) means for cryopreserving stem cells, It provides a banking system of immunocompatible NT-derived stem cells.
  • Implantable cell and tissue materials can be provided for the treatment of various diseases such as diabetes, osteoarthritis and Parkinson's disease, and in particular offer the possibility of fundamental treatment of cell type specific defects.
  • the homozygous cells can provide a therapeutic approach that can reduce the risk of immune rejection and immune tolerance.
  • 1 is a diagram showing that when the number of cells less than 700 million according to the law on cord blood management and research is classified for disposal.
  • Figure 2 shows the chromosome test results of the donor cells of Example 1.
  • Figure 3 shows the chromosome test results of NT cells prepared in Example 3.
  • Figure 4 shows (a) genomic DNA test results and (b) mitochondrial DNA test results of NT cells prepared in Example 3 compared with donor somatic cells and donor oocytes.
  • Figure 5 shows the results of immunochemistry analysis of stem cell markers of NT cells prepared in Example 3.
  • Figure 6 shows the results of RT-PCR analysis of stem cell markers of NT cells prepared in Example 3.
  • Figure 8 shows that there is no difference as a result of comparing the markers of (a) morphology of embryonic stem cell-derived RPE cells and NT-derived RPE cells prepared in Example 3 and (b) RPE cells.
  • immunocompatibility homozygous means that each of the HLA-A, HLA-B, and HLA-DR genes inherited from the donor's paternal and maternal genomes is completely identical and has three HLA genotypes instead of six. Means that.
  • tissue cell refers to any tissue cell in the body except sex cells or precursors thereof.
  • stem cells are capable of self-renewal (having the ability to pass multiple cell division cycles while maintaining an undifferentiated state) and exhibit at least one multidifferentiation capacity (the ability to differentiate into one or more specialized cells). It means a cell that can.
  • long term culture means proliferation of cells under controlled conditions for at least two months or longer than at least 10 passages.
  • the long term culture is cultured for at least 4 months, at least 6 months or at least 1 year.
  • the long term culture is passaged for at least 15 passages, at least 18 passages or at least 20 passages.
  • the duration of long term culture depends mainly on individual cells and can vary from cell line to cell line.
  • mature refers to a process consisting of coordinated biochemical steps leading towards finally differentiated cell types.
  • differentiation refers to the adaptation of the cell to a particular form or function.
  • differentiated cell includes any somatic cell that is not pluripotent in its original form, as the term is defined herein.
  • differentiated cells also refers to partially differentiated cells, such as multipotent cells, or cells that are stable, non-potentially partially reprogrammed or partially differentiated cells produced using any of the compositions and methods described herein. It includes.
  • the differentiated cells are cells that are stable intermediate cells, such as non-pluripotent, partially reprogrammed cells. It should be noted that placing a large number of primary cells in the culture may result in some loss of fully differentiated properties.
  • telomeres are also extended passaged without loss of growth potential as compared to parent cells with lower likelihood of having generally only a limited number of divisions in culture. Has the ability to suffer.
  • the term “differentiated cells” also refers to cells of less specialized cell types (ie, increased likelihood) (eg, from undifferentiated cells or reprogrammed cells), where the cells are an intracellular differentiation process. Refers to cells of more specialized cell types (ie, reduced likelihood of occurrence) derived from).
  • hematopoietic stem cells muscle cells, cardiomyocytes, liver cells, chondrocytes, epithelial cells, urinary organ cells, adipocytes, kidney cells, vascular cells, retinal cells, mesenchymal stem cells (MSC) and neuronal cells It is selected from the group consisting of but not limited to.
  • the "immunocompetent cell” refers to a cell in which the HLA-A, HLA-B and HLA-DR genes are homozygous-like, without being particularly limited thereto, and all HLA genotypes in which only three of the six pairs are identical. It may have the advantage of being transplantable to the beneficiaries of the combination.
  • bank means a storage location of stem cells, and may be used as it is or may be differentiated from the stored cells to the individual or another individual for therapeutic, clinical or research purposes as needed.
  • administration means introducing a certain substance into a patient in any suitable way and the route of administration of the substance can be administered via any general route as long as it can reach the target tissue.
  • administration can be performed by any device capable of moving to a target cell.
  • the present invention comprises the steps of: a) screening for homozygous (Homozygous) from a plurality of donation tissue (donation); b) isolating nuclei from Homozygous cells to produce NT cells; c) generating stem cells from the prepared NT cells; And d) cryopreserving a plurality of stem cells; provides a method for storage of stem cells derived from immunocompatible NT cells.
  • Cells made via NT may carry the genetic material of the patient's nucleus and are in this respect individual patient specific. Thus, cell transplantation into patients with a significantly reduced risk for autologous transplantation, i.e. allogeneic rejection, is possible.
  • the homozygous cells of the present invention are cells that match the HLA antigen type, and can be transplanted to a heterogeneous person without anti-HLA antibodies. That is, the NT-derived stem cells have a homozygous type and can be transplanted into immunocompatible cells.
  • the screening in step a) is preferably selected that the genes of human leukocyte antigen (HLA) -A, HLA-B and HLA-DR is homozygous (homozygous).
  • HLA genotyping screens find 140 donors with different immunocompatible homozygotes, it has been announced that it is possible to secure immune-compatible cell lines that can be transplanted to over 90% of Japan's population. (A more efficient method to generate integration-free human iPS cells, Nature Methods 8, 409-412 (2011))
  • homozygous (homozygous) cells were screened based on data from a primary hospital donor umbilical cord blood bank. According to the current umbilical cord blood management and research law, if the number of cells is less than 700 million cells, the Ministry of Health and Welfare Umbilical Cord Blood Committee is authorized to discard them so that they can be used for research (see FIG. 1).
  • the cells to be used in this study can be used primarily for cryopreservation blood (with less than 700 million cells), and in addition to all donor blood for research, registered with the Center for Organ Transplantation Management (KONOS). It may include. In addition, samples obtained by the hematopoietic stem cell donor network and medical institutions affiliated with the Cha Hospital can be used.
  • KONOS Center for Organ Transplantation Management
  • the NT cell production method in step b) is the step of denuclearizing oocytes; Fusing the nucleus of the somatic cell to denucleated oocytes; And culturing the fused oocytes in post activation medium.
  • the method for producing stem cells derived from NT comprises the steps of removing the nuclei of oocytes, adding one or more nuclei of one or more donor cells to generate nuclear transplanted (NT) oocytes, and incubating the NT eggs by incubation in an activation medium. Activating blast cells, and generating blastocysts from the activated NT oocytes.
  • removing the nucleus of the oocyte includes removing the mid-phase II (MII) stage egg spindle.
  • the first pole body 1PBE is removed.
  • the method includes the step of stripping the cumulus cells before completion of maturation.
  • oocytes are observed by non-UV light based observation on 1PBE in real time.
  • the observation takes place in the absence of a staining or labeling agent such as Hoechst staining. In one embodiment, this involves the use of a polscope, eg, Research Instruments (CRi) Oosight TM image system.
  • a polscope eg, Research Instruments (CRi) Oosight TM image system.
  • removing the nuclei of oocytes comprises the use of contoured micropipettes that allow the removal of 1 PBE from the oocytes and the pores of the oocyte membranes.
  • removing the nuclei of oocytes comprises the use of a piezoelectric drill.
  • the step of removing nuclei of oocytes is carried out in a denucleation medium containing cytocalin B and optionally, a protein phosphatase inhibitor such as caffeine.
  • Caffeine is a protein phosphatase inhibitor that inhibits premature activation and improves the growth of cloned embryos, thereby increasing the rate of blastocyst formation. Therefore, preferably, the denucleation of the oocytes is made in a medium containing a protein phosphatase inhibitor, and the protein phosphatase inhibitor may be caffeine.
  • generating the nuclearly transplanted (NT) oocytes by adding one or more nuclei of one or more donor cells may comprise implanting the donor nuclei.
  • Implanting the donor nucleus involves the use of agents that modify the oocyte membrane structure.
  • removing the nucleus of the oocyte through the use of an agent to modify the oocyte membrane structure comprises fusion with somatic cells.
  • implanting a donor nucleus may comprise providing 3 to 4 donor cells with an injection pipette (eg, 12 ⁇ m diameter), paramyxovirus or paramyxovirus proteins such as Sendai virus, Releasing the donor cells in a predetermined amount of a solution containing the envelope protein or extract thereof.
  • the recovery of the pipette does not interfere with the contact between the yolk sac and the donor cell.
  • the cells are fused after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more minutes of donor cell insertion. In various embodiments, the cells are fused 10 minutes after donor cell insertion. Optionally, the process is repeated for cells that have not been successfully fused.
  • polyscopes such as the Oosight TM image system, are used throughout the process.
  • the method of implanting a donor nucleus may be performed by a direct injection method.
  • the step of implanting a donor nucleus may comprise electrical cell manipulation, eg, electrofusion.
  • the method may comprise separating the nucleus of the somatic cell nuclear donor, the stem cell nuclear donor, and the sperm cell nuclear donor.
  • the method comprises inserting one or more donor nuclei via a pipette or piezoelectric injection after the step of separating the somatic nuclei for NT.
  • the donor nucleus is derived from a cell, such as dermal fibroblasts, white blood cells, hair follicles, or other somatic cell nuclear donors.
  • the present invention discloses a method comprising isolation and preparation of a nucleus from sperm cell donation.
  • the separation of the nucleus may be tissue biopsy, transfusion, or other method for obtaining a tissue sample, mechanical separation, collagenase digestion, washing, centrifugation based density gradient separation, and / or standard culture medium. Processing of tissues through culture.
  • the step of fusing the nucleus of the somatic cells is made in a medium containing Sendai virus or Sendai virus extract.
  • the fused oocytes After fusing the nucleus of the somatic cell to the denucleated oocytes, the fused oocytes are transferred to a post activation medium and undergo an activation process.
  • NT-hPSC cell lines C) generating stem cells from the prepared NT cells, specifically, activating NT oocytes by incubating nuclear transplanted (NT) oocytes in an activation medium, and generating blastocysts from the activated NT oocytes. And isolating endothelial cell (ICM) cells from the blastocyst, wherein the ICM cells can be further cultured as NT-hPSC cell lines.
  • ICM endothelial cell
  • MFP Metaphase Promoting Factor
  • MII I block of cells is hampered by a cell change in calcium ions (Ca 2 +) level due to the entry of sperm. Thereafter, targeted degradation of cyclin B (MPF regulatory subunit) occurs, which releases oocytes from cell cycle blocking, pronuclear formation, and meiosis and mitosis processes.
  • Oocyte activation relies on an artificial calcium-change strategy to release cultured oocytes from cell cycle blockade. Examples include the addition of calcium ion carriers, fat soluble molecules that transfer ions through the lipid bilayer, such as ionomycin and A23817. Alternative strategies rely on electrical activity or direct implantation of ions.
  • oocyte activation using calcium alteration techniques also occurs.
  • kinase inhibitors such as 6-dimethylaminopurine (6-DMAP)
  • protein synthesis inhibitors such as ethanol and cycloheximide (CHX)
  • TSA Histone deacetylase inhibitors
  • Treatment of TSA can promote the formation of blastocysts.
  • an electrical pulse may be applied during fusion and activation of somatic cells. Electrical activation includes electrical pulses in electrical cell fusion media.
  • the electrical cell fusion the medium is from 0.1 to 0.5 M mannitol, 0.01 to 1 mM MgSO 4 .7H 2 O, 0.01 to 1 mg / ml of polyvinyl alcohol, 1 to 10 mg / ml human serum albumin, 0.005? 0.5 mM CaCl 2 .2H 2 O.
  • nuclear transplanted oocytes are treated in a post-activation medium for complete activation.
  • the activated reconstructed nuclear transplanted oocytes are then incubated in post-activation medium.
  • the post-activation medium is HEPES-removing medium, protein-removing medium, G1 or G2 medium, eggplant medium, eggplant aid medium, IVF medium, blastocyst forming medium, or global human embryo culture medium.
  • the post-activation medium is 6-DMAP, puromycin, ethanol, cycloheximide (CHX), trichostatin A (TSA), and cytochalasin B It may include any one or more selected from (CB).
  • the activated oocytes are 30 to 45, 45 to 60, 60 to 90, 90 to 120, 120 to 150, 150 to 180, 180 to 210, 210 to 240, 240 to 240 in post-activation medium. Incubate for less than 270, 300 to 330, 330 to 360, 360 to 390 minutes, or more than 390 minutes. In certain embodiments, the activated oocytes are incubated for 240, 300, or 360 minutes. In various embodiments, the activation and post-activation steps are performed under hypoxic conditions. In certain embodiments, hypoxic conditions are about 80-85%, 85-90%.
  • low oxygen conditions include about 90% N 2 , about 5% O 2 , and about 5% CO 2 .
  • the post-activation medium is 1, 2, 3, 4, 5, 5, or in a gas mixture, such as about 90% N 2 , about 5% O 2 , and about 5% CO 2 . 1, 2, 3, 4, 5, 5, or more mM 6-DMAP in eggplant media, incubated at 37 ° C. for longer time.
  • post-activated oocytes are incubated in wash medium.
  • the wash medium is HEPES-removing medium, protein-removing medium, G1 or G2 medium, eggplant medium, eggplant aid medium, IVF medium, blastocyst forming medium.
  • the culture medium does not require continuous medium exchange as global human embryo culture medium.
  • the wash medium comprises TSA.
  • post-activated oocytes are incubated for 240, 300, or 360 minutes in wash media comprising TSA.
  • the post-activated reconstructed nuclear transplanted oocytes are washed and further cultured.
  • the post-activated reconstituted nuclear transplanted oocytes are washed in 6-DMAP removal medium.
  • various disclosed media such as HEPES-removing media, protein-removing media, G1 or G2 media, eggplant media, eggplant aid media, IVF media, blastocyst forming media, or global human embryo culture media,
  • growth factors such as GM-CSF or IGF1.
  • the growth factor may be added at 1, 2, 3, 4, 5, 6, 7, or later days after nuclear transfer.
  • the activating and / or post-activating step comprises adding a factor, derivatives and extracts isolated from sperm.
  • the human sperm factor is injected into the reconstructed ovum activated using any of the described infusion methods.
  • the human sperm factor is injected into the post-activated reconstructed egg using any of the described infusion methods.
  • post-activated reconstituted nuclear transplanted oocytes are transferred to eggplant medium.
  • the post-activated reconstructed nuclear transplanted oocytes are transferred to eggplant medium.
  • the sperm factor includes a factor that is isolated from, for example, a cellular protein present inside or outside the sperm cell.
  • the total sperm extract is obtained using mechanical mixing of the surfactant and ejaculated sperm.
  • the whole cell extract is treated with DNAase I and RNAase.
  • the crude extract is washed with buffer and centrifuged (20,000 g for 2 hours).
  • fresh ejaculated human sperm is imported and centrifuged at 900 g for 10 minutes to remove semen plasma, followed by Sperm-TALP containing 5 mg / mL bovine serum albumin.
  • the post-activated reconstructed nuclear transplanted oocytes are further cultured into blastocysts.
  • the post-activated reconstituted nuclear transplanted oocytes are further cultured in SAGE eggplant medium, eg, Quinn's medium.
  • medium for example 3i medium (Neuro basal medium 50%, DMEM / F-12 50%, N2 additive 1/200 v / v, B27 additive 1/100 v / v, 100 mM L-glutamine 1/100 v / v, 0.1M ⁇ -ME 1/1000 v / v, SU5402 (FGFR inhibitor) 2 ⁇ M, PD184352 (ERK cascade inhibitor) 0.8 ⁇ M, CHIR99021 (GSK3 inhibitor) 3 ⁇ M) or modified 3i medium (including 0.4 ⁇ M PD0325901 (MAPK inhibitor)) promotes pluripotency.
  • the additional culture is for at least 1, 2, 3, 4, 5, or 5 days.
  • additional cultures are provided in a culture medium having a reprogramming factor and / or methylation-modifying agent.
  • the additional culture is for 3 days in G2 medium to which any of CARM1 CARM1, Esrrb, Kdm4a, Kdm4b and Kdm4d Esrrb is added.
  • CARM1 and / or Esrrb may be provided at concentrations in medium of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 or more ⁇ g / ml, respectively.
  • CARM1 and / or Esrrb are each provided at a concentration in medium of 2 ⁇ g / ml.
  • further incubation into blastocysts and induction of pluripotent stem cells (pSCs) from blastocysts comprises treating the cultured blastocysts with acidic Tyrode's solution to remove the zona pellucida (ZP). Include. In various embodiments, the treatment is for a few seconds (eg, 1 to 5). In various embodiments the removal of ZP is followed by a wash in HEPES-HTF medium. In various embodiments, isolation of the internal cell mass (ICM) comprises discarding the trophoblast of the blastocyst. In various embodiments, ICM cells are plated in a mouse embryonic feeder (MEF) prepared one day prior to plating.
  • MEF mouse embryonic feeder
  • the whole blastocyst is plated on MEFs.
  • the method includes stripping the zona pellucida of the blastocyst.
  • the method removes the zona pellucida of the blastocyst with 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1% pronase in Hepes-HTF medium. Steps.
  • the method comprises removing the zona pellucida of the blastocyst with 0.5% pronase in Hepes-HTF medium.
  • the method comprises pronase in TH3 medium (SAGE blastocyst medium) for 1-10, 10-20, 20-30, 30-60, 60-120, 120-180, or> 180 seconds. It includes the step of applying. In yet another embodiment, applying 0.5% pronase in HTF medium for 30-60 seconds.
  • the blastocyst is derived from reconstructed nuclear transplanted oocytes obtained from somatic cell nuclear transfer (NT) of donor cell nuclei into recipient oocytes.
  • the stem cell line is a somatic cell nuclear transfer human pluripotent stem cell (NT-hPSC) cell line.
  • the invention includes a method for immunosurgery comprising mechanical dispersion of an internal cell mass (ICM) from trophectodermal cells.
  • ICM internal cell mass
  • the exfoliated blastocysts are treated with rabbit anti-human spleen serum at 37 ° C. for about 10, 20, 25, 30, 35, 40, 45, or 60 minutes.
  • the method comprises washing the exfoliated blastocyst with TH3 (SAGE blastocyst medium) and incubating in guinea pig complement reconstituted with HECM-9 (SAGE blastocyst medium) at 37 ° C. for 30 minutes. do.
  • the zona pellucida of the expanded blastocyst is removed with slight exposure (45-60 seconds) to 0.5% pronase or acidic tirod solution in TH3 (hepes-HTF) medium.
  • the method is laser assisted incubation using small bore pipetting, Ehsms zilos-tk unit (Hamilton Thorne) to selectively separate endocytosis from trophectoderm cells. mechanically spreading the cells using a hatching method.
  • the post activation medium is made from a medium containing TSA, and the post activation medium is 6-DMAP. More preferably, the cells are cultured in a medium containing 6-DMAP at the time of post activation, and then further cultured in a medium containing TSA.
  • the nuclei of at least one donor cell can be altered by focusing epigenetic modifying agents to increase successful NT production.
  • Epigenetic regulators specifically alter the state of methylation or acetylation of specific proteins or DNA to increase transcriptional efficiency and consequently to increase NT efficiency.
  • Targets of these epigenetic regulators include histone acetyl transferase (HAT) proteins, histone deacetylase (HDAC) proteins, lysine dimethylase (KDM) domain proteins, and protein methyl transfer.
  • RNA small interfering RNA
  • NT oocytes are cultured in the presence of agents that alter epigenetic status. Specific examples of these formulations are described in Tables 2-5 below.
  • Histone acetyl transferase (HAT) protein, histone deacetylase (HDAC) protein, lysine dimethylase (KDM) domain protein, protein methyl transferase (PMT) protein It is not limited to the examples below.
  • Histone acetyl transferase (HAT) protein Target_ID (
  • Histone deacetylase protein Target_ID (
  • Lysine Dimethylase (KDM) Domain Protein Target_ID (
  • methyl transferases can be inhibited by co-substrate analogues.
  • co-substrate analogues Three kinds of co-substrate analogues that inhibit various kinds of methyltransferases are known.
  • Sinefugin a structurally similar antibiotic compound to S-adenosylmethionone (SAM), dimethylated co-substrate SAH and methylthioadenosine as feedback inhibitors.
  • Lysine methyl group inhibitors include the first identified chaetocin and G9a (KMT1C) inhibitor Bix-01294. These are optional for SUV39H1 and PRM1.
  • Bix-01338 is a rather nonselective inhibitor with no selectivity between lysine and arginine methyltransferase, showing an IC50 of 5 mM for G9a and an IC50 of 6 mM for PRMT1.
  • UNC0224 has been proposed as a new inhibitor with IC50 15 mM against lysine methyltransferase G9a.
  • Inhibitors of histone methyltransferases such as EPZ5676, EPZ005687 and GSK126 also show anticancer activity in various cancer animal models.
  • Protein arginine methylation is accomplished by PRMTs and is divided into two groups. Type I methyl transferases form asymmetrically substituted arginine residues, and type II methyl transferases form symmetrically substituted arginine residues.
  • CARM1 shows affinity with proline-glycine-methionine-arginine (the so-called PGM motif).
  • PRMT5 is also known to methylate the PGM motif.
  • Cosubstrate analogs such as sinefungin can also be used as inhibitors of arginine methyltransferases (also known as AMIs as arginine methyltransferase inhibitors).
  • AMI-1 is the most active inhibitor of PRMT1 with IC50 9 mM. Inhibitors of allatodapsone and stilbamidine induce hypomethylation in H4R3.
  • DNA methyltransferases preferentially methylate the CpGnucleotide sequence of DNA.
  • methylation of these promoter sites inhibits the expression of genes by preventing transcription factors from binding to DNA.
  • methylated DNA is bound by a methyl-CpG binding domain protein, which attracts histone modeling enzymes, consequently condensing chromatin structure to express genes. May induce a mechanism to suppress DNMT inhibitors can increase the efficiency of NT by preventing gene expression from being inhibited.
  • Some examples include chlorogenic acid, mithramycin, azacytide, bisdemethoxycurcumim, decitabine, lomegutatrib, benzylguanine, sorafenib and sorafenib tosylate.
  • HDAC histone deacetylase
  • Type IV HDACs (11 subtypes) are found in both the nucleus and cytoplasm and are primarily located in the brain, heart, and muscle cells. HDACs inhibitors have anticancer activity when administered in combination with other chemotherapy drugs. HDAC inhibitors can promote DN transcription, resulting in increased NT efficiency.
  • Epigenetic modifying agents such as epigenetic chromatin and ⁇ histone modification agents, and / or DNA modifiers may be included in the post activation medium.
  • it is a protein arginine methyl-transferase (PRMT1) and a coactivator-associated arginine methyltransferase 1 (CARM1 / PRMT4), a nuclear orphan receptor estrogen.
  • receptor estrogen may be selected from related receptor ⁇ (Esrrb) proteins and may also be selected from Lysine (K) -Specific Demethylase 4A (Kdm4a), Lysine-specific dimethylase 4B (Lysine (K)).
  • CARM1 and Esrrb may be a 7X arginine (7R) -cell-penetrating peptides (CPPs), or one of skill in the art, to enhance the penetration of proteins and peptides through cell and nuclear membranes, and to bind and / or transactivate DNA. May be modified with other proteins known to increase.
  • the method uses transcript talent-based reprogramming to allow octamer binding transcription factor-4 (Oct-4), sex determination site Y-box-2 (sex determining). region Y-box-2) (Sox-2), nanog, Kruppel-like factor-4 (Klk-4), MyoD, c-Myc, zinc finder protein-42 protein-42) (Rex-1 / Zfp-42), lefty A, teratocarcinoma-derived growth factor (Tdgf), and / or telomeric repeating binding epigenetic reprogramming of nuclear donor cells with factor) (Terf-1).
  • the method comprises direct piezoelectric injection, viral injection, liposome injection, or other intracellular injection.
  • the transcription factor can be delivered in the form of mRNA, protein, and / or cell extracts that can be applied prior to nuclear transfer into nucleated oocytes.
  • the method may comprise the use of HDAC inhibitors (Class I, II, and III), or DNMT3a and DNMT3b inhibitors.
  • the present invention preferably post activation medium comprises epigenetic modifying agents. More preferably, epigenetic modifying agents are histone acetyl transferase (HAT) proteins, histone deacetylase (HDAC) proteins, lysine dimethylase (KDM) domain proteins, Protein methyl transferase (PMT) domain proteins and DNA methyl transferases (DNA) are involved in any one or more selected from the group comprising DNA transferases (DNMTs).
  • HAT histone acetyl transferase
  • HDAC histone deacetylase
  • KDM lysine dimethylase
  • PMT Protein methyl transferase domain proteins
  • DNA DNA methyl transferases
  • the method for producing NT cell-derived stem cells is the step of activating NT cells to generate blastocysts; Isolating endocytosis (ICM) cells from the resulting blastocysts; And further culturing the isolated inner cell population cells with stem cells.
  • ICM Isolating endocytosis
  • Stem cells of the present invention can be cryopreserved for future use.
  • the cryopreservative comprises one or more cryoprotectants including but not limited to dimethyl sulfoxide (DMSO), ethylene glycol, glycerol and propanediol;
  • DMSO dimethyl sulfoxide
  • One or more culture media including but not limited to DMEM, MEM and the patented media disclosed above;
  • the stem cells are cryopreserved in a solution comprising one or more additional substances, including but not limited to sucrose, dextran, serum substitutes and HEPES buffer.
  • the solution comprises CryoStor TM CS-10 media (BioLife Solutions Inc., Botel, Washington).
  • the serum replacement is Knockout serum replacement (Invitrogen 10828-028).
  • Cryopreservation of the prepared stem cells freezes the cells at a controlled rate or in a "manual" process.
  • the controlled rate freezing procedure begins by turning on a controlled rate freezer and setting up a freezing program for tissue or cell freezing.
  • the controlled rate freezer will use liquid nitrogen to reduce the temperature in the inner chamber (which will reduce the temperature of any contents of the chamber).
  • the freezing program for cells begins by cooling the inner chamber to 4 ° C. and maintaining the temperature until it is stimulated to continue the procedure. While the controlled rate freezer cools, the cells are suspended in cryopreservation medium cooled to 4 ° C.
  • the cell suspension is aliquoted into frozen vials in an amount of 1 ml per cryovial.
  • the frozen vials are then labeled and placed in a controlled rate freezer chamber to stimulate the program to continue.
  • the temperature of the chamber is maintained at 4 ° C. for an additional 10 minutes.
  • the chamber is cooled at a rate of -1 ° C / min until the temperature reaches -80 ° C.
  • the chamber is then cooled at a rate of -50 ° C / min until the chamber reaches a temperature of -120 ° C.
  • the temperature of the frozen cells will equilibrate to -120 ° C.
  • the frozen vials of the frozen cells are then transferred to liquid nitrogen Dewar for long term storage.
  • the present invention comprises the steps of a) screening homozygous (Homozygous) from a plurality of donation tissue (donation) tissue;
  • the screening in step a) is that the genes of human leukocyte antigen (HLA) -A, HLA-B, and HLA-DR are homozygous, and in step b), NT production may include denuclearizing oocytes; Fusing the nucleus of the somatic cell to denucleated oocytes; Comprising a step of culturing the fused oocytes in a post activation medium, more preferably, the denucleation of the oocytes is made in a medium containing a protein phosphatase inhibitor (protein phosphatase inhibitor).
  • HLA human leukocyte antigen
  • the step of fusing the nucleus of the somatic cells is made in a medium containing Sendai virus or Sendai virus extract
  • the post-activation medium preferably comprises a histone deacetylase inhibitor (distone deacetylase inhibitor), It is more desirable to include TSA.
  • the post activation medium preferably contains epigenetic modifying agents, more preferably the epigenetic modifying agents include histone acetyl transferase (HAT, histone acetyl). at least one selected from the group consisting of transferase (HDC) protein, histone deacetylase (HDAC) protein, lysine dimethylase (KDM) domain protein, and protein methyl transferase (PMT) domain protein.
  • HDC transferase
  • HDAC histone deacetylase
  • KDM protein methyl transferase
  • the present invention comprises the steps of a) screening homozygous (Homozygous) from a plurality of donation tissue (donation) tissue;
  • It provides a method for producing cells differentiated from stem cells derived from immunocompatible NT cells, comprising the steps of: preparing the differentiated cells for cell transplantation from the prepared stem cells.
  • the stem cells may be optionally added to cryopreservation, in which case, the step of thawing the cryopreserved cells prior to step d) may be selectively added.
  • Differentiated cells are derived from stem cells, hematopoietic stem cells, muscle cells, cardiomyocytes, liver cells, chondrocytes, epithelial cells, urinary organ cells, adipocytes, kidney cells, vascular cells, retinal cells, mesenchymal stem cells (MSCs) and neurons.
  • stem cells hematopoietic stem cells
  • muscle cells cardiomyocytes
  • liver cells chondrocytes
  • epithelial cells urinary organ cells
  • adipocytes adipocytes
  • kidney cells vascular cells
  • retinal cells mesenchymal stem cells (MSCs) and neurons.
  • MSCs mesenchymal stem cells
  • Such a banking system can reduce the continuous supply of new oocytes by replacing NT production with autologous cells and enable a wide range of autologous or heterologous cell transplants.
  • the screening step for xenotransplantation from these banking systems and the preparation of specific differentiated cells from the selected stem cells expand the range of use as more and more various cell therapies. It is possible to apply variously to the field of cell transplantation treatment that is used in the past.
  • the differentiation into vascular endothelial cells can lead to vascular related diseases, retinal pigment epithelial cells, and retinal related diseases through differentiation into neurons.
  • the field of treatment such as neurodegenerative diseases through differentiation will not be limited.
  • the present invention is to provide a cell population comprising an immune-compatible stem cells prepared by the method for producing an immunocompatible NT cell-derived stem cells.
  • the present invention also provides a composition comprising a cell population of NT cell-derived stem cells for the treatment of various diseases.
  • Cell composition of the present invention with respect to the total weight of the composition of the present invention contains 0.1 to 99.9% by weight as an active ingredient, may comprise a pharmaceutically acceptable carrier, excipient or diluent.
  • compositions of the present invention may be in various oral or parenteral formulations.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.
  • Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin.
  • lubricants such as magnesium stearate, talc and the like are also used.
  • Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.
  • As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • the pharmaceutically effective amount is 0.0001 to 100 mg / kg, 0.001 to 10 mg / kg, but is not limited thereto.
  • the dosage may vary depending on the weight, age, sex, health condition, diet, duration of administration, method of administration, elimination rate, severity of disease, and the like of the particular patient.
  • composition can be administered orally or parenterally during clinical administration and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or intrathoracic injection during parenteral administration. And can be used in the form of general pharmaceutical formulations.
  • 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.
  • the present invention provides a means for collecting a plurality of donor tissue
  • Means for cryopreserving stem cells provides an immunocompatible NT cell-derived stem cell banking system comprising a.
  • the present invention provides a stem cell bank for storing NT cell derived stem cells obtained from multiple donors of an individual.
  • Stored stem cells can be used as a source of cells for restoring a particular cell population of an individual's body for health reasons or for the treatment or clinical use of another individual.
  • Stored stem cells can also be used for research applications.
  • Stored stem cells may be thawed and used as stem cells after long-term preservation, or may be used or administered to a patient after differentiation into specific cells.
  • Genomic DNA was extracted using Gentra Puregene TM Blood Kits (QIAGEN, Hilden, Germany) for HLA-A, B and DRB1 genotyping, and then SeCore A, B and DRB1 Locus Sequencing Kit (Invitrogen, Brown Deer, WI, USA) Sequence-based typing was used.
  • exon 2-4 for HLA-A and B and exon 2 for HLA-DRB1 were amplified using a locus specific primer included in the kit, and ABI3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA) , USA) was used to sequence the PCR product formed, and the data using HLA SBT u-type software v3.0 (Invitrogen) and Sequencher (Gene Codes Corp., Ann Arbor, MI, USA) The analysis was carried out. In the end, HLA homozygous donor cells (A * 33: 03-B * 44: 03-DRB1 * 13: 02) (haplotype frequency: 4.6%) were most frequently used in donor blood If found, NT-cells could cover about 9% of the total population.
  • hematopoietic cells of HLA AB-DRB1 haplotype were selected as donor cells and cultured in a cell culture flask under 5% CO 2 at 37 ° C.
  • DMEM culture medium containing 10% DMSO and 30% FBS is used as a freezing solution, frozen in cryo vials and stored in a liquid nitrogen tank until use.
  • FBS chromosome test
  • Ovarian stimulation was performed according to established clinical IVF guidelines (Tachibana et al., 2013). The women were sedated with Midazolam 5-7.5 mg (Versed, Roche, and Nutley. NJ, USA) and Fentanyl 50-75 ug (Abbott Pharmaceutical, Abbott Park, Ill. USA) 36 hours after Lupron or hCG injection. And oocytes were then recovered using the previously described ultrasound map.
  • COCs cumulus-oocyte cell complexes
  • IVF medium Quinn's IVF medium, SAGE Biopharma, Bedminster, NJ
  • SSS serum substitutes
  • HTF-Hepes medium Global Medium
  • COCs were treated with hyaluroniase (100 IU / ml, Sigma, St. Louis, Mo. USA), and then the oocytes were sorted according to maturity, and oocytes in the middle II (MII) phase were used for NT.
  • MII middle II
  • the denuclearization of oocytes can be carried out by a previously known method (Tachibana et al., 2013), the denuclearization of oocytes and nuclear replacement of somatic cells can be performed by stage warmers, narishige micromanipulators. ), An Oosight TM imaging system (poloscopic microscopy), and an inverted microscope equipped with a laser. An inverted microscope with piezo may optionally be used instead of an inverted microscope with a laser.
  • HTF-Hepes medium Global Medium
  • cytocarcin B 5 ⁇ g / ml
  • caffeine 1.25 mM
  • Caffeine is a protein phosphatase inhibitor that inhibits premature activation and improves the growth of cloned embryos, thereby increasing the rate of blastocyst formation.
  • the zona pellucida next to the spindle was drilled with a laser pulse, and the injection pipette was inserted through the drilled portion.
  • the injection pipette was aspirated with spindles in contact with a small amount of cytoplasm surrounded by a plasma membrane.
  • the transparent band may optionally use piezo pulses instead of laser pulses.
  • the donor cells were then aspirated into micropipettes and transferred to small drops containing Sendai virus envelope protein (HJV-E extract, Isihara Sangyo Kaisha). Then, the nuclear donor cells of Example 1 were inserted into a perivitelline space located opposite the first polar body.
  • Sendai virus envelope protein Sendai virus envelope protein
  • the prepared eggs were further incubated for 30 minutes or 2 hours in Global 10% SPS medium.
  • Activation was performed under 0.25 mM d-sorbitol buffer containing 0.1 mM potassium acetate, 0.5 mM magnesium acetate, 0.5 mM HEPES, 1 mg / ml fatty acid-free BSA (fatty-acid-free BSA). Electrical pulses (2 ⁇ 50 ⁇ s DC pulses, 2.7 kV / cm) were added to the test. Activated cells were incubated for 4 hours in Global Medium (excluding serum) containing 2 mM DMAP under 5% CO 2 , 37 ° C., and under 5% CO 2 , 5% O 2 , 90% N 2 , 37 ° C.
  • Example 2 The blastocyst cultured in Example 2 was treated with acidic Tyrode solution (pH 2.0) for several seconds to remove the zona pellucida (ZP). After removal of ZP, embryos were vigorously washed in Hepes-HTF medium to remove even trace amounts of Tyrode solution. Intracellular cell mass (ICM) was isolated using a laser-assisted blastocyst ablation system (Hamilton-Thorne Inc.) and the remaining portion of the blastocyst (nutrient membrane) was discarded to confirm that the blastocyst was no longer intact.
  • ICM Intracellular cell mass
  • ICMs were plated on MEFs prepared one day prior to plating, but whole embryos were plated if replicated blastocysts had indistinguishable ICMs.
  • hPSC induction medium was serum replacement (5% SR, Invitrogen), FBS (10%, Hyclone), plasmamate (5%), bFGF (32 ng / ml), and human LIF (2000 units / mI, Sigma) Knockout-DMEM supplemented with -Aldrich) was included. After incubation of ICM for 3 days in the same medium without change, on day 4 about 1/3 of the medium was replaced. 1 ⁇ 2 of the medium was changed every other day from day 6. Initial outgrowth was observed within 7 days after plating.
  • EBs embryonic bodies
  • FIG. 7 (a) and (b) In order to confirm the pluripotency in the body, stem cells were injected into the testis or subcutaneously of the immunodeficient mice to induce teratoma formation and histologically, the differentiation was confirmed through H-E and special staining. The results are shown in Figure 7 (c).
  • the cells prepared in Example 3 are sorted according to homozygous cells and stored and recorded in a document or program.
  • the information of the cell donor is stored together. It can be used directly for future autologous or allogenic recipients (patients) or stored for use as differentiated cells.
  • NT-derived stem cells were mechanically clump-form of NT-ES cells using a sterilized tip under an anatomical microscope (a fragment of approximately 300-600 undifferentiated embryos). Stem cell line). Clump-type NT-derived stem cells were supplemented with 15% (v / v) knockout TM DMEM (Thermo Scientific, CA, USA) supplemented in low attachment 6-well plates (Corning, CA, USA).
  • TM serum replacement (Thermo), 1% (v / v) glutamax (Thermo), 1% (v / v) NEAA (Thermo), 1% (v / v) penicillin-streptomycin (Thermo) and 0.1 mM ⁇ -mercaptoethanol (Thermo)) incubated for 4 days in a suspended state.
  • the cultured embryoid body is transferred to the culture dish and induces RPE differentiation while attached.
  • Embryos were transferred to a 6-well culture dish coated with 0.1% gelatin coating and allowed to stand in the incubator for 3 days for attachment. Then, EBDM culture medium was replaced every 2-3 days, and retinal pigment epithelial cells were grown. Incubated for about 50-55 days until it appears. To separate the color-coded RPE cells by pigmentation, the cells were washed twice with physiological saline (DPBS containing Ca 2 + Mg 2 + (Thermo)) and physiological saline containing collagenase type 4 (Type IV collagenase). (Thermo) in DPBS with Ca 2+ Mg 2+ (Thermo)) and incubated in an incubator maintained at 37 ° C., 5% CO 2 for 2 hours.
  • physiological saline DPBS containing Ca 2 + Mg 2 + (Thermo)
  • Type IV collagenase Type IV collagenase
  • detached cell clusters separated from the culture dish were collected in a 50 ml tube and washed twice with DMEM-FBS culture using a centrifuge (1500 rpm, 5 minutes). The cell masses were transferred to a 60 mm Petri dish, and the pigmented cell clusters were collected from other non-pigmented cell masses using a glass pipette drawn under a dissecting microscope.
  • Pigmented cell clusters are washed twice with physiological saline (DPBS without Ca 2 + Mg 2 + (Thermo)) without calcium and magnesium to isolate and culture single cells. : 1 mixture of 0.25% Trypsin-EDTA (Thermo) and Cell Dissociation Buffer (Thermo)) were isolated. Retinal pigment epithelial cells separated into single cells were washed with DMEM-FBS medium using a centrifuge (1500 rpm, 5 min), and then 200,000 cells / 4 well plate was prepared by releasing the cells with EGM2 medium (Lonza, PA, USA). In a well of 0.1% gelatin-coated 4well culture plate at a concentration of one well was incubated in EGM-2 culture until it is full.
  • the cells are filled with a culture plate with RPE differentiation medium (RGMM: 1: 1 mixture of EBDM and DMEM-FBS media) to show the shape and characteristics of retinal pigment epithelial cells.
  • RPE differentiation medium RGMM: 1: 1 mixture of EBDM and DMEM-FBS media
  • Retinal pigment epithelial cells differentiated from NT-derived stem cells were subcultured using the same separation enzyme solution as above, and functional retinal pigment epithelial cells were obtained through the proliferation process using EGM2 culture and the aging process using RGMM culture.
  • Some retinal pigment epithelial cells from passages 2 and 3 were obtained using 2 million cells / mL / cryo using freezing solution (90% (v / v) FBS (Thermo) and 10% (v / v) DMSO (Sigma)). Freezing at the concentration of vial was stored until use, and some of the retinal pigment epithelial cells were characterized.
  • Figure 8 shows that there is no difference in the morphology and differentiation markers of RPE cells of embryonic stem cell-derived RPE cells and NT-derived stem cells prepared in Example 3 (Fig. 8 (a), (b)).

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Abstract

La présente invention porte sur un procédé de conservation et un système de banque de cellules préparées au moyen de la technologie de transfert nucléaire (NT) de cellules somatiques de génotypes homozygotes dans des gènes d'antigène leucocytaire humain (HLA)-A, HLA-B, HLA-DR, etc. La mise en banque de cellules souches dérivées de cellules NT selon la présente invention peut être appliquée à des patients allogéniques ou hétérogènes et peut produire des cellules et des matières tissulaires transplantables pour le traitement de diverses maladies comme le diabète, l'arthrose, la maladie de Parkinson, etc.
PCT/KR2016/007795 2015-07-17 2016-07-18 Procédé de conservation et système de banque de cellules nt Ceased WO2017014513A1 (fr)

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CN201680042142.1A CN108026544A (zh) 2015-07-17 2016-07-18 Nt细胞的贮存方法和存储系统
JP2018521814A JP6707636B2 (ja) 2015-07-17 2016-07-18 Ｎｔ細胞の保管方法、及びそのバンキングシステム
US15/745,301 US12091679B2 (en) 2015-07-17 2016-07-18 Storage method and banking system of NT cell
EP16828017.0A EP3327130A4 (fr) 2015-07-17 2016-07-18 Procédé de conservation et système de banque de cellules nt

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CN113854280A (zh) * 2021-09-06 2021-12-31 创芯国际生物科技(广州)有限公司 一种新型的低温保存液及其制备方法和应用

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WO2008124142A1 (fr) * 2007-04-06 2008-10-16 International Stem Cell Corporation Lignes de cellules souches spécifiques au patient dérivées de blastocytes humains parthénogénétiques
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11116206B2 (en) 2018-10-01 2021-09-14 Cook Medical Technologies Llc Cryocontainer
CN113854280A (zh) * 2021-09-06 2021-12-31 创芯国际生物科技(广州)有限公司 一种新型的低温保存液及其制备方法和应用

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