Cell type conversion

Abstract

The present disclosure relates to a method of somatic cell nucleus reprogramming to alter cell types, comprising preparing a GV extract, permeating somatic cells, culturing the somatic cells with the GV extract to alter their cell types and resealing somatic cell membranes, where the GV extract does not comprise oocyte cytoplasm. The present disclosure also relates to a GV extract comprising a reprogramming factor.

Description

Technical Field

[0001] The present invention relates to changing the cell type of somatic cells. More specifically, the present invention relates to using germinal vesicle (GV) extracts to change the cell type of somatic cells.

Background Art

[0002] As cells and tissues age, the stability of cell differentiation is necessary to avoid malfunction. Here, we describe a procedure by which the differentiated state of cells can be reversed so that cells that have undergone differentiation will again exhibit pluripotent gene expression and new, unrelated cell types will form. To achieve this, cells of differentiated tissue are exposed to extracts of sufficient amphibian meiotic oocytes. Such reprogrammed cells can be induced to follow a new direction of differentiation and thus serve as replacements for malfunctioning mature cells in humans.

[0003] The discovery that four Yamanaka factors can generate ESC-like cells (induced pluripotent stem cells, iPSCs) when overexpressed in MEF 53 (mouse embryonic fibroblasts) has opened the way for deriving certain types of cell types from adult cells as sources of replacement cells in humans. Within a decade of their results, many laboratories have tried to determine the pathways by which this conversion can occur 3-5 . There are three reasons why there has been only limited success to date. They are (i) the procedure is slow, typically taking about 1 - 3 weeks for pluripotent cells to appear, (ii) the yield of pluripotent cells is small, i.e., only some of the treated cells are ancestors of the derived pluripotent cells, and (iii) DNA replication and cell division of the treated cells continue during the intervening period and may be necessary for reprogramming to occur.

[0004] Here, we describe a way to avoid these limitations and thus identify the earliest steps in this reprogramming process. This involves using oocytes (at the first meiotic prophase) rather than eggs (at the second meiotic metaphase) as recipients of transplanted mammalian somatic cell nuclei 2 . By comparison with previous attempts to figure out the earliest steps of iPSC reprogramming, oocyte nuclear transfer completely avoids the uncertain contributions of cell division and DNA synthesis 6、7 and long reprogramming times 8(For the earliest iPSC-derived markers, Thy1 down and SSEA1 up for 3 - 5 days) is greatly shortened to 24 hours at 18°C. Specifically, the method involves using GV extracts in the absence of oocyte cytoplasm. Thus, factors that may be undesirable for reprogramming and present in the oocyte cytoplasm are advantageously absent during downstream applications. In addition, the method achieves efficient reprogramming in the absence of other factors such as protease inhibitors. The GV extract can be modified as needed to express or overexpress factors that have the effect of further increasing the efficiency of reprogramming somatic cells. Additionally or alternatively, recombinant factors can be added during the culturing step, which also have the effect of further increasing the efficiency of reprogramming somatic cells.

[0005] For a long time, the need for cell division for nuclear reprogramming has been unclear. On the one hand, somatic cell nuclear transfer to metaphase eggs and interphase zygotes shows that the former is important for nuclear reprogramming to pluripotency due to its mitotic activity 10、11 . On the other hand, the fusion of somatic cells with embryonic stem cells shows that early reprogramming (indicated by the activation of Oct4) is independent of DNA replication and cell division, but the completion of subsequent reprogramming may depend on it 12、13 . Induction of pluripotency by Yamanaka factors is accompanied by several rounds of cell division over several weeks and only a small fraction of cells become induced pluripotent stem cells 1、5 . Induction of pluripotency by Yamanaka factors (OSK) (combined with the culture of MEF in metaphase and interphase egg extracts) shows that nuclear reprogramming requires mitosis to remodel the nucleus 14 . Here, we evaluated the completion of nuclear reprogramming by nuclear transfer to meiotically inactivated oocytes and developed a procedure for reprogramming human cells into a totipotent-like state for potential use in cell therapy.

Summary of the Invention

[0006] According to a first aspect of the present invention, there is provided a method for somatic cell nuclear reprogramming for changing cell type, which comprises preparing a GV extract, permeabilizing somatic cells, culturing the somatic cells with the GV extract to change their cell type, and resealing the somatic cell membrane, wherein the GV extract does not contain oocyte cytoplasm.

[0007] In a specific embodiment, changing the cell type may comprise reprogramming the somatic cells into progenitor cells or stem cells.

[0008] In a specific embodiment, the progenitor cells or the stem cells include unipotent stem cells, oligopotent stem cells, multipotent stem cells, pluripotent stem cells or totipotent stem cells.

[0009] In one specific embodiment, the method may further include differentiating the precursor cell or the stem cell to change its cell type.

[0010] In one specific embodiment, differentiating the precursor cell or the stem cell may produce somatic cells or their precursor cells, where the somatic cells or their precursor cells are of different lineages. In one specific embodiment, the differentiation may include culturing the precursor cell or stem cell in a differentiation medium. In one specific embodiment, the differentiation may include adding one or more differentiation factors.

[0011] In one specific embodiment, the GV extract may include one or more reprogramming factors. In one specific embodiment, the reprogramming factors may be factors that change chromatin accessibility. In one specific embodiment, the reprogramming factors may be DNA modifying enzymes, histone variants, histone modifying enzymes, chromatin remodelers, chromatin modifiers, and transcription factors. In one specific embodiment, the DNA modifying enzymes may be AID or Mbd3. In one specific embodiment, the histone modifying enzymes may be kdm4 or kdm6. In one specific embodiment, the chromatin remodelers may be Brg1. In one specific embodiment, the transcription factors are Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, mouse mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs.

[0012] In one specific embodiment, the isolated GV may be derived from a meiotic oocyte.

[0013] In one specific embodiment, the GV may be isolated from Xenopus, mammals, or ray-finned fish. In one specific embodiment, the mammal may be human, mouse, pig, or bovine. In one specific embodiment, the ray-finned fish may be salmon or sturgeon.

[0014] In one specific embodiment, the somatic cells may be mammalian cells. In one specific embodiment, the mammalian cells are human cells, mouse cells, bovine cells, pig cells, dog cells, cat cells, or horse cells.

[0015] In one specific embodiment, changing the cell type may further include adding one or more exogenous reprogramming factors.

[0016] In one specific embodiment, the somatic cells, the GV extract, and optionally one or more exogenous reprogramming factors can be cultured for any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours. In one specific embodiment, the somatic cells, the GV extract, and optionally one or more exogenous reprogramming factors can be cultured for any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0017] In one specific embodiment, the progenitor cells or stem cells can be cultured in a differentiation medium for any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 days or for 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months in the differentiation medium culture.

[0018] In one specific embodiment, the somatic cells can contain selectable or reportable markers, including totipotency genes, including one or more of Nelfa, Top2a, Gata2, Eif3h, Dppa2 / 4, and Atr, pluripotency genes, including one or more of Jun, Sox2, Myc, Klf4, Mycn, Klf2, Pou5f1, Utf1, and Sall4, trophoblast genes, including one or more of Tfap2c, Hand1, Msx2, Csf1r, and Gcm1, and gene markers for somatic cells, including Neun, Tubb3, Myod1, Myog, and Itga7.

[0019] In one specific embodiment, the somatic cells can be permeabilized using streptolysin O, digitonin, lysophosphatidylcholine, or a mixture thereof.

[0020] In one specific embodiment, the cell membranes can be resealed using CaCl2.

[0021] In one specific embodiment, the step of preparing the GV extract can include mechanical dissociation.

[0022] In a specific embodiment, the method may further comprise the step of modifying the GV extract to express genes encoding proteins selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, mouse mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs. In a specific embodiment, the GV extract overexpresses genes encoding proteins selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xklf2, xpou60, mouse mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs. In a specific embodiment, the step of modifying the GV extract comprises injecting mRNA into the cytoplasm or culturing the oocytes with rare-cut endonucleases such as TALEN, ZFN, or CRISPR / Cas9 and guide RNA, where the mRNA encodes proteins selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, mouse mSox2, mFoxa1, human hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs, or the guide RNA contains genes encoding: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xklf2, xpou60, mouse mSox2, mFoxa1, human hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs.

[0023] According to a second aspect of the present invention, there is provided a GV extract comprising reprogramming factors, where the reprogramming factors are factors that can change chromatin accessibility, if necessary.

[0024] In a specific embodiment, the reprogramming factors may be DNA modifying enzymes, histone variants, histone modifying enzymes, chromatin remodelers, chromatin modifiers, and transcription factors. In a specific embodiment, the DNA modifying enzymes may be AID or Mbd3. In a specific embodiment, the histone modifying enzymes may be kdm4 or kdm6. In a specific embodiment, the chromatin remodelers may be Brg1. In a specific embodiment, the transcription factors are Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, murine mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs.

[0025] In a specific embodiment, the GV may be isolated from Xenopus, mammals, or ray-finned fish. In a specific embodiment, the mammal is human, mouse, pig, or cow. In a specific embodiment, the ray-finned fish may be salmon or sturgeon.

[0026] In a specific embodiment, the GV extract is modified, where the GV is modified as needed to express a gene encoding a protein selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, murine mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, or their homologs.

[0027] In a specific embodiment, the GV is modified to overexpress a gene encoding a protein selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, murine mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, or their homologs.

[0028] According to a third aspect of the present invention, a cell is provided that is derived from the method according to the present invention.

[0029] According to a fourth aspect of the present invention, a cell is provided that has an altered cell type derived from the method according to the present invention.

[0030] According to a fifth aspect of the present invention, there is provided a kit comprising a permeabilizing agent, a GV extract and / or one or more reprogramming factors and, optionally, instructions for use. In a specific embodiment, the kit may further comprise a differentiation medium and / or one or more differentiation factors. In a specific embodiment, the GV extract is modified, optionally wherein the GV is modified to express or overexpress a gene encoding a protein selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, murine mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1 or homologs thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention is further described in the following non-limiting drawings.

[0032] Figure 1 Chromatin changes and nuclear envelope breakdown in transplanted MEFs injected into the GV of non-dividing oocytes. Figure 1 a Schematic diagram showing the procedure of nuclear transfer into oocytes and subsequent analysis. Figure 1 b Heat map showing similar oocyte-induced transcriptional changes of genes in donor cell types and nuclear grafts 48 hours after nuclear transfer. Pluripotency genes are indicated on the right side of the heat map. Figure 1 c Image showing chromatin (green) dispersion and loss of HP1α (magenta) in MEFs after nuclear transfer. Scale bar indicates 10 μm. Figure 1 d Bar graph showing a 2-fold chromatin dispersion in MEFs in the region 24 hours after nuclear transfer. Error bars represent ±SD. *p < 0.05, by Student's t-test, n = 3. Figure 1 e Bar graph showing loss of HP1α of chromatin at three density levels in MEFs starting from 4 hours. At all three time points, the denser the chromatin, the larger the area occupied by HP1α in the chromatin. Error bars represent ±SD. *p < 0.05, **p < 0.01, by Student's t-test, n = 3. (f) Image showing breakdown of the nuclear envelope (magenta) and bursting of chromatin (green) in the nucleus of the injected MEF after nuclear transfer. Chromatin bursting was observed at the breakdown of the nuclear envelope (white arrow) 4 hours after nuclear transfer. Scale bar indicates 10 μm. ​

[0033] Figure 2 :Completion of transcriptional reprogramming of oocytes to a totipotent-like state within 24 hours after nuclear transfer Figure 2 a, b Pluripotency genes (a) and trophectoderm genes (b) in the transplanted MEFs were activated by oocyte factors on the first day after nuclear transfer and maintained at the same expression level on the second day. Figure 2 c Sox2 transcripts (white dots) were activated by oocyte factors in all transplanted MEFs 24 hours after nuclear transfer. TO-PRO-1 staining of chromatin for MEFs is shown in red. The area outlined in blue shows the background area without hybridization probes for the MEF cell nuclei. Figure 2 d Venn diagram shows similar gene induction by oocyte factors in three cell types 48 hours after nuclear transfer. Figure 2 e, f Bar graphs show different responses between cell types for induction of pluripotency genes (e) and inhibition of myogenic genes (f) by oocyte factors 48 hours after nuclear transfer.

[0034] Figure 3 :Retention of gene resistance to oocyte reprogramming in differentiated MYO Figure 3 a Venn diagram shows that differentiated MYO nuclei are more resistant to oocyte reprogramming compared to embryonic ESCs and MEFs. Among the genes that can be induced by oocytes, differentially expressed genes (fold change ≥ 4) in certain cell types with lower gene expression after nuclear transfer indicate gene resistance. Considering that the transcriptional activity of donor cells may affect the results, genes that were downregulated by oocytes and shown in the oocyte resistance genes were excluded. Figure 3 b Heat map shows that three cell types respond similarly to oocyte reprogramming regarding the top 1500 highly expressed oocyte-inducible genes in MYO-NT. The top 1500 highly expressed genes in MYO-NT include pluripotency genes, Jun, Klf4, and Myc. Figure 3 c Box plot shows the transcriptional increase by number (TPM) of the top 1500 highly expressed oocyte-inducible genes in MYO-NT. Figure 3 ​​The heatmap shows the different responses to oocyte reprogramming among three cell types regarding the oocyte resistance genes in MYO-NT. The oocyte resistance genes in MYO-NT include six pluripotency genes, Klf2, Sox2, Pou5f1, Sall4, Utf1, and Mycn. Figure 3 The box plot shows the transcriptional changes of the resistance genes in MYO-NT by number (TPM).

[0035] Figure 4 : Potential applications for humans. Figure 4 a bar graph shows the induction of pluripotency genes in mouse cells (ESC, MEF, and MYO) and human cells (hNEU) at day 2 after nuclear transfer. ESC, n = 3; MEF, n = 4; MYO, n = 3; hNEU, n = 3. Figure 4 b bar graph shows the activation of pluripotency genes in human lung stem cells after nuclear transfer. Figure 4 c bar graph shows the increased gene expression of neural differentiation markers caused by GV extract after neural differentiation from reprogrammed hLB. GV, treatment with GV extract; ND, treatment with neural differentiation medium. Figure 4 d hDF forms and maintains in the form of embryoid bodies at day 15 after treatment with GV extract. The dark areas in the following figure are embryoid bodies. Figure 4 e hDF treated with GV extract shows the expression of neural differentiation markers at day 21 after neural differentiation. NEUN (green) and TUBB3 (red) are markers for neural differentiation. The nuclei are stained with DAPI (blue). Scale bar indicates 50 μm. Figure 4 f image shows an unfertilized egg 8 hours after injection of the nucleus of a mature human lung stem cell. Figure 4 g image shows an oocyte in prophase of meiosis 3 days after injection of the nucleus of a mature human lung stem cell. Figure 4 h bar graph shows the activation of pluripotency genes to similar levels in mouse cells (ESC and MEF) and human cells (hNB) at day 2 after nuclear transfer.

[0036] Figure 5 : Quantitative chromatin changes show chromatin dispersion / relaxation, also seen in ESC and MEF. Figure 5 a image shows the quantification of chromatin area per cell. The yellow circle encloses the area of chromatin (green) of each cell.​​ Figure 5 b Images show the quantification of the area of chromatin classified into three density levels and the quantification of the area of HP1α. Injection of one MEF into the GV of an enlarged oocyte is shown to detail the classification of chromatin into three density levels. The areas of dense, intermediate, and relaxed chromatin are circled by the yellow line, between the yellow and orange lines, and between the orange and purple lines, respectively. The area of HP1α covered by chromatin at the three density levels is circled by the magenta line. Figure 5 c Images show the dispersion of chromatin and the loss of HP1α in transplanted ESCs and MYOs after nuclear transfer. The same experimental procedures as in Figure 1 b are applied. Scale bar indicates 10 μm.

[0037] Figure 6 : Global gene expression shows that the induction of a totipotency-like state in MEFs by Xenopus oocytes is achieved within 24 hours after nuclear transfer. Figure 6 a Heat map shows the transcriptional pattern of donor MEFs reprogrammed by Xenopus oocytes within 24 hours after nuclear transfer (columns 1 - 3 vs. columns 4 - 7). Regarding the expression pattern of donor MEF cells, we show that the expression pattern of MEFs is almost identical with or without BrUTP capture (column 1 vs. column 2); the expression pattern of the MEF cell line is highly similar to the reference total transcript of primary MEFs in the Expression Atlas (column 2 vs. column 3). Figure 6 b Scatter plot shows that the expression patterns of 21,683 oocyte-induced genes in the MEF nucleus are almost identical at 24 and 48 hours after nuclear transfer (r = 0.78, Pearson correlation coefficient). Within 24 hours, pluripotency genes (black circles, o) and trophectoderm genes (black crosses, x) are induced by the oocyte and then maintained at the same expression level. From 24 to 48 hours after nuclear transfer, 18,706 genes (86% of 21,683 genes) maintain the same expression (4 > fold change > 0.25, orange dots); 1,390 genes are upregulated (fold change ≥ 4, red dots) and 1,587 genes are downregulated (fold change ≤ 0.25, green dots).

[0038] Figure 7 : Redirection of cell differentiation. Bar graph shows that functions related to differentiation are enriched in MYOs at 48 hours after nuclear transfer in the GV extract after overexpression of Xklf2 (Gene Ontology, adjusted p-value < 0.05, n = 3).

[0039] Figure 8 ​:Enhanced oocyte reprogramming caused by overexpression of xklf2. 9.2 μg of in vitro transcribed xklf2-HA mRNA (SEQ ID NO: 1) was injected into the cytoplasm of oocytes 24 hours before transferring mammalian cell nuclei into Xenopus oocytes to enhance oocyte reprogramming in the transplanted cells. In ESCs (a) and MEFs (c), overexpression of xklf2 regulates pluripotency gene expression. Klf2 and Myc are upregulated and Jun is downregulated in ESCs (b, p < 0.05, n = 3); Klf2, Sall4, and Mycn are upregulated and Pou5f1 is activated in MEFs (d, p < 0.05, n = 4). In MYOs (e), pluripotency genes resistant to oocyte reprogramming are activated by overexpression of xklf2, including Mycn, Sox2, Klf2, Pou5f1, Sall4, and Esrrb (f, p < 0.05, n = 3).

Embodiments

[0040] Specific embodiments of the present invention will now be further described. In the following paragraphs, different specific embodiments are described. Each aspect so defined can be combined with any other one or more aspects, unless clearly stated to the contrary.

[0041] Generally, the nomenclature and techniques associated with cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used by those of ordinary skill in the art to which the invention pertains. The methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art to which the invention pertains and described in various general and more specific documents cited and discussed throughout this specification, unless otherwise indicated. See, for example, Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, N.Y. (2012).

[0042] According to a first aspect of the present invention, there is provided a method for somatic cell nuclear reprogramming for changing cell type, which comprises preparing a GV extract, permeabilizing somatic cells, culturing the somatic cells with the GV extract to change their cell type, and resealing the somatic cell membrane, wherein the GV extract does not contain oocyte cytoplasm. As such, factors that may be undesirable for reprogramming and present in oocyte cytoplasm are advantageously absent during downstream applications. Since such factors do not contribute to reprogramming or are harmful to reprogramming, they are considered to be undesirable.

[0043] In addition, since there are no unwanted oocyte cytoplasmic contaminants, the desired factors contained in the GV extract are cultured with the somatic cells at a higher concentration.

[0044] In one specific embodiment, the changing of the cell type may include reprogramming the somatic cells into progenitor cells or stem cells.

[0045] In one specific embodiment, the method for reprogramming the somatic cell nucleus is an in vitro, in vivo or ex vivo method.

[0046] In one specific embodiment, the progenitor cells are unipotent stem cells, oligopotent stem cells, multipotent stem cells, pluripotent stem cells or totipotent stem cells.

[0047] As such, somatic cells cultured with the GV extract can be reverted to progenitor cells, unipotent stem cells, oligopotent stem cells, multipotent stem cells, pluripotent stem cells or totipotent stem cells.

[0048] In one specific embodiment, the method may further include differentiating the progenitor cells and stem cells to change their cell type.

[0049] In one specific embodiment, the differentiating of the progenitor cells and stem cells may produce somatic cells or their progenitor cells, wherein the somatic cells or their progenitor cells are of different lineages.

[0050] In one specific embodiment, the differentiation may include culturing the progenitor cells and stem cells in a differentiation medium. In one specific embodiment, the differentiation may include adding one or more differentiation factors. There are various methods for differentiating progenitor cells or stem cells to produce somatic cells and / or their progenitor cells and such are known to those of ordinary skill in the art to which the invention pertains. Such established methods are considered routine and in no way limit the present invention. For example, when the progenitor cells or stem cells are pluripotent or totipotent cells, ectoderm, mesoderm and endoderm can be produced 37 .

[0051] In the presence of differentiation factors, these progenitor cells or stem cells can be differentiated from the original cells into completely different cell types. Thus, when cultured with one or more differentiation factors in the presence of the GV extract and other reprogramming factors, somatic cell nuclear reprogramming can be achieved to change its cell type. Changing the cell type has many applications in both research and clinical settings.

[0052] In one specific embodiment, the present invention may thus include the steps of creating progenitor cells or stem cells after culturing the somatic cells with the GV extract and differentiating the said progenitor cells or stem cells to change their cell type.

[0053] The present invention for changing the cell type of cells can be carried out in a single step. Reverting such cells to a progenitor cell or stem cell state or transdifferentiating the cells into another differentiated state can be carried out simultaneously by culturing such cells with GV extract and other differentiation factors at the same time. The present invention can also be carried out in two stages: 1) reverting such cells to a progenitor cell or stem cell state and 2) after such cells have reached the progenitor cell or stem cell state, adding differentiation factors to cause such cells to differentiate into a new cell type.

[0054] Therefore, the present invention can be applied to personalized medicine and regenerative medicine. Using the present invention, it is possible to generate embryonic stem cells that can be used to create different cell types to treat many diseases and dysfunctions in humans and animals. For example, cells with an altered cell type generated using the present invention can be used to create healthy cells, tissues or organs to assist in restoring normal function to the human or animal body.

[0055] In one specific embodiment, the reprogramming factors are factors that alter chromatin accessibility. The reprogramming factors can be DNA modifying enzymes, histone variants, histone modifying enzymes, chromatin remodelers, chromatin modifiers and transcription factors.

[0056] In some specific embodiments, the DNA modifying enzymes are AID or Mbd3. In some specific embodiments, the histone modifying enzymes are kdm4 or kdm6. In some specific embodiments, the chromatin remodelers are Brg1. In some specific embodiments, the transcription factors are Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1 or xklf2.

[0057] In one specific embodiment, the isolated GV is derived from a meiotic oocyte. In one specific embodiment, the GV can be isolated from Xenopus, mammals or ray-finned fish. In one specific embodiment, the GV can be derived from mouse, pig, bovine, human, salmon, sturgeon or Xenopus oocytes. In one specific embodiment, the GV can be derived from human oocytes. In one specific embodiment, the GV can be derived from salmon or sturgeon oocytes. In one specific embodiment, the GV can be derived from Xenopus oocytes.

[0058] GV extract can be used to revert any cell type to a progenitor cell or stem cell state and the reprogramming factors can be used to change the identity of the cells into different cell types. This would allow (for example) taking skin cells from an individual and changing them into neurons, and they can be returned to the same individual without any rejection of the implanted cells.

[0059] In one specific embodiment, the changing of cell types may further include adding one or more exogenous reprogramming factors. These exogenous reprogramming factors can be recombinantly produced. In one specific embodiment, these reprogramming factors can be co-cultured with the somatic cells before, simultaneously with, or after culturing with the GV extract.

[0060] In some specific embodiments, these exogenous reprogramming factors can be the DNA modifying enzymes, histone variants, histone modifying enzymes, chromatin remodelers, chromatin modifiers, and transcription factors of the present invention or can be other factors. As such, the cultured somatic cells and GV extract can contain additional factors. In some specific embodiments, the cultured somatic cells and GV extract are in the absence of exogenous reprogramming factors.

[0061] Although it will be understood that the culturing of the somatic cells and the GV extract and, optionally, one or more exogenous reprogramming factors can be carried out for any period of time required to effect the change in cell type, in certain specific embodiments, a preferred period of time can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours, any one of them. Alternatively, in certain specific embodiments, a preferred period of time can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, any one of them.

[0062] In one specific embodiment, the progenitor cells or stem cells can be cultured in a differentiation medium for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 days, any one of them, or cultured in a differentiation medium for 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months. As mentioned above, there are various methods for differentiating progenitor cells or stem cells to produce somatic cells and / or their progenitor cells available and they are known to those of ordinary skill in the art to which the invention pertains. Such established methods are considered routine and in no way limit the present invention. As such, the present invention extends to variations of the above culturing durations.

[0063] In one specific embodiment, the somatic cells may contain selectable or reportable markers, which may include totipotency genes, which may include one or more of Nelfa, Top2a, Gata2, Eif3h, Dppa2 / 4, and Atr, pluripotency genes, which may include one or more of Jun, Sox2, Myc, Klf4, Mycn, Klf2, Pou5f1, Utf1, and Sall4, which may include trophectoderm genes, which may include one or more of Tfap2c, Hand1, Msx2, Csf1r, and Gcm1, and which may include gene markers for somatic cells, including Neun, Tubb3, Myod1, Myog, and Itga7. The markers advantageously allow the cells to be monitored and selected during the method.

[0064] The somatic cells may be permeabilized using streptolysin O, digitonin, lysophosphatidylcholine, or a mixture thereof. Permeabilization is an important step in the procedure to allow the factors from the GV extract to enter the cells and cause nuclear reprogramming. In addition to the agents listed above, any known permeabilizing agent and transfection reagent may be used in the context of the present invention.

[0065] The cell membranes are resealed using CaCl2. Calcium may be used to reseal the membranes after completion of the method. The use of calcium chloride is particularly advantageous for resealing the cells.

[0066] In one specific embodiment, the step of preparing the GV extract may include mechanical dissociation. In one specific embodiment, the step of mechanical dissociation may include centrifugation. The step of mechanical dissociation may occur in mineral oil.

[0067] In a specific embodiment, the method may further comprise the step of modifying the GV extract to express genes encoding proteins selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, mouse mSox2, mFoxa1, human hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs. In a specific embodiment, the GV extract may overexpress genes encoding proteins selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, mouse mSox2, mFoxa1, human hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs. As used herein, "x" represents "Xenopus", "h" represents "human", and "m" represents "mouse".

[0068] In a specific embodiment, the step of modifying the GV extract may comprise injecting the cytoplasm with mRNA or culturing the oocytes with rare-cut endonucleases such as TALEN, ZFN, or CRISPR / Cas9 and guide RNA. In a specific embodiment, the mRNA may encode proteins selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xklf2, xpou60, mouse mSox2, mFoxa1, human hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs. In a specific embodiment, the guide RNA may comprise genes encoding the following: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xklf2, xpou60, mouse mSox2, mFoxa1, human hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs.

[0069] Thus, the GV extract can be modified to express or over-express certain factors that may be useful in reprogramming somatic cells. Such factors can include the factors identified above. The methods by which such modification occurs are not limited to specific techniques; suitable techniques include (for example) injecting the GV with mRNA encoding such factors or genetic modification by using suitable techniques such as CRISPR Cas9 gene editing. This has the advantage of increasing the concentration of the desired reprogramming factors in the GV extract. Conversely, the modification of the GV can involve reducing or silencing the inhibitors of reprogramming. For example, siRNA can be injected into the GV extract to reduce or silence the inhibitors of reprogramming.

[0070] The step of modifying the GV extract can occur prior to culturing the GV extract with the somatic cells. For example, the step of modifying the GV extract can occur approximately 24 hours prior to culturing the GV extract with the somatic cells. Optionally, the GV extracts can be tested for the expression of such exogenous factors by routine methods (such as qPCR) prior to culturing with the somatic cells.

[0071] In a specific embodiment, reprogramming factors can be identified using any suitable method. Examples include (but are not limited to) experimental screening, computer methods, and artificial intelligence. For example, methods such as DeepAccess and diffTF identified in Hammelman et al. 38 can be used.

[0072] In a further aspect of the invention, a GV extract comprising reprogramming factors is provided. In a specific embodiment, the GV can be derived from mouse, pig, bovine, human, salmon, sturgeon, or Xenopus oocytes. In a specific embodiment, the GV can be derived from human, mouse, pig, or bovine oocytes. In a specific embodiment, the GV can be derived from salmon or sturgeon oocytes. In a specific embodiment, the GV can be derived from Xenopus oocytes. In a specific embodiment, the GV can be derived from Xenopus, mammals, or ray-finned fish.

[0073] In a specific embodiment, the reprogramming factors are factors that alter chromatin accessibility. The reprogramming factors can be DNA-modifying enzymes, histone variants, histone-modifying enzymes, chromatin remodelers, chromatin modifiers, and transcription factors.

[0074] In some specific embodiments, the DNA-modifying enzyme system is AID or Mbd3. In some specific embodiments, the histone-modifying enzyme system is kdm4 or kdm6. In some specific embodiments, the chromatin remodeler is Brg1. In some specific embodiments, the transcription factors are Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xklf2, xpou60, murine mSox2, mFoxa1, human hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs.

[0075] In another aspect of the present invention, a cell derived from the method of the first aspect of the present invention is provided.

[0076] In another aspect of the present invention, a cell having an altered cell type derived from the method of the first aspect of the present invention is provided.

[0077] In yet another aspect of the present invention, a kit is provided that includes a permeabilizing agent, a GV extract, and / or one or more reprogramming factors and instructions for use as needed. In one specific embodiment, the kit may further include a membrane resealing agent. In one specific embodiment, the membrane resealing agent may be CaCl2.

[0078] As used herein, the term "GV extract" refers to germinal vesicles (GVs) extracted from oocytes. The GV extract may include intact GVs or lysates of GVs. In one specific embodiment, the GV extract may include additional components, such as common factors found in growth media. In other specific embodiments, the GV extract does not include other components, such as common factors found in growth media or protease inhibitors. In additional specific embodiments, the media may be CA-free 2+ media. In one specific embodiment, the GV may be derived from Xenopus oocytes. The GV may be manually dissected in mineral oil. In one specific embodiment, the GV extract may be derived by rapid freezing and thawing of the dissected GVs or mechanical disruption of the GVs (e.g., pipetting the GVs to rupture the dissected GVs). In one specific embodiment, the GV extract may be filtered prior to use. As will be understood, intact GVs or lysates of GVs contain a variety of factors useful for reprogramming.

[0079] As used herein, the term "totipotent" refers to a cell that is capable of developing into any cell type or a complete organism.

[0080] As used herein, the term "pluripotent cell" or "pluripotent stem cell (PSC)" refers to a cell that has complete differentiation pluripotency, such as the ability to grow into any of the approximately 260 cell types of the mammalian body. Pluripotent cells can self-renew and can remain dormant or quiescent within tissues. Different from totipotent cells (such as fertilized, diploid oocytes), pluripotent cells (even pluripotent embryonic stem cells) generally cannot form new blastocysts.

[0081] As used herein, the term "multipotent cell" refers to a cell that can differentiate into a limited number of specialized cell types. As used herein, the term "oligopotent cell" refers to a cell that can differentiate into an even more limited number of specialized cell types. As used herein, the term "unipotent cell" refers to a cell that can differentiate into a single lineage.

[0082] As used herein, the term "progenitor cell" refers to a lineage-restricted cell that has limited proliferative capacity.

[0083] As used herein, the term "cell type" is a classification used to identify cells that share the same morphological or phenotypic identity.

[0084] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meaning generally understood by one of ordinary skill in the art to which this invention pertains. Although the foregoing disclosure provides a general description of the subject matter covered by this disclosure, including methods of making and using this disclosure and their best mode embodiments, the following examples are provided to further enable one of ordinary skill in the art to which this invention pertains to practice this disclosure. However, one of ordinary skill in the art will understand that the details of these examples should not be construed as limiting the present invention, and the scope of the present invention should be understood based on the appended claims of this disclosure and their equivalents. Based on this disclosure, various other aspects and embodiments of this disclosure will be apparent to one of ordinary skill in the art to which this invention pertains.

[0085] The entire contents of all documents mentioned in this specification are incorporated herein by reference, including references to gene accession numbers, scientific publications, and patent publications.

[0086] As used herein, "and / or" shall be understood to specifically disclose each of the two specifically recited features or components, with or without the other. For example, "A and / or B" shall be understood to specifically disclose each of the following: (i) A, (ii) B, and (iii) A and B, as if each were individually set forth herein. Unless the context otherwise requires, the foregoing descriptions and definitions of the features are not limited to any particular aspect or embodiment of the invention and are equally applicable to all the recited aspects and embodiments.

[0087] For the purposes of this text, the term "comprising" (or "comprises") means including one or more of the specifically recited components, but not excluding the presence of other components. The term "consisting essentially of" (or "consists essentially of") means including the specifically recited components, but excluding other components except for substances present as impurities, all inevitable substances resulting from the processes used to provide such components, and the like.

[0088] The term "consisting of" (or "consists of") means including the specifically recited components, but excluding other components.

[0089] Where appropriate, depending on the context, the use of the term "comprising" (or "comprises") may also be understood to include the meaning "consisting essentially of" and may also be understood to include the meaning "consisting of".

[0090] The optional features set forth herein may be used individually or, where appropriate, in combination with each other, and particularly in the combinations set forth in the appended claims. Where appropriate, the optional features of the aspects or exemplary embodiments of the invention set forth herein are also applicable to all other aspects or exemplary embodiments of the invention. In other words, those of ordinary skill in the art to which the invention pertains, upon reading this specification, should consider the optional features of the aspects or exemplary embodiments of the invention to be interchangeable and combinable among the different aspects and exemplary embodiments. Item

[0091] 1. A method for reprogramming the nuclei of somatic cells to change their cell types, comprising: Preparing a GV extract; Permeabilizing the somatic cells; Culturing the somatic cells with the GV extract to change their cell types; and Resealing the somatic cell membranes, wherein the GV extract does not contain oocyte cytoplasm.

[0092] 2. The method according to item 1, wherein changing the cell type comprises reprogramming the somatic cells into progenitor cells or stem cells.

[0093] 3. The method of item 2, wherein the progenitor cell or the stem cell line is a unipotent stem cell, an oligopotent stem cell, a pluripotent stem cell, a totipotent stem cell or a pluripotent stem cell.

[0094] 4. The method of any one of items 2 or 3, further comprising differentiating the progenitor cell or stem cell to change its cell type.

[0095] 5. The method of item 4, wherein differentiating the progenitor cell or stem cell produces a somatic cell or its progenitor cell, wherein the somatic cell or its progenitor cell is of a different lineage.

[0096] 6. The method of item 4 or 5, wherein the differentiation comprises culturing the progenitor cell or stem cell in a differentiation medium.

[0097] 7. The method of item 6, wherein the differentiation medium further comprises the addition of one or more differentiation factors.

[0098] 8. The method of any one of the foregoing items, wherein the GV extract comprises one or more reprogramming factors.

[0099] 9. The method of item 8, wherein the reprogramming factors are factors that alter chromatin accessibility.

[0100] 10. The method of item 9, wherein the reprogramming factors are DNA modifying enzymes, histone variants, histone modifying enzymes, chromatin remodelers, chromatin modifiers and transcription factors.

[0101] 11. The method of item 10, wherein the DNA modifying enzymes are AID or Mbd3.

[0102] 12. The method of item 10, wherein the histone modifying enzymes are kdm4 or kdm6.

[0103] 13. The method of item 10, wherein the chromatin remodeler is Brg1.

[0104] 14. The method of item 10, wherein the transcription factors are Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, murine mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1 and their homologs.

[0105] 15. The method of any one of the foregoing items, wherein the isolated GV is derived from a meiotic oocyte.

[0106] 16. The method according to any one of the preceding clauses, wherein the GV is isolated from Xenopus, mammals or ray-finned fish.

[0107] 17. The method according to clause 16, wherein the mammal is a human, mouse, pig or cow and the ray-finned fish is a salmon or sturgeon.

[0108] 18. The method according to any one of the preceding clauses, wherein the somatic cells are mammalian cells.

[0109] 19. The method according to clause 18, wherein the mammalian cell lines are human cells, mouse cells, pig cells, cow cells, dog cells, cat cells or horse cells.

[0110] 20. The method according to any one of the preceding clauses, wherein the altered cell type further comprises adding one or more exogenous reprogramming factors.

[0111] 21. The method according to any one of the preceding clauses, wherein the somatic cells, the GV extract and, optionally, one or more exogenous reprogramming factors are cultured for any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 hours.

[0112] 22. The method according to any one of clauses 1 to 20, wherein the somatic cells and the GV extract are cultured for any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0113] 23. The method according to any one of clauses 6 to 22, wherein the progenitor cells are cultured in a differentiation medium for any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 days or cultured in a differentiation medium for 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months.

[0114] 24. The method of any one of the preceding clauses, wherein the somatic cells comprise selectable or reportable markers, comprise totipotency genes, comprise one or more of Nelfa, Top2a, Gata2, Eif3h, Dppa2 / 4, and Atr, comprise pluripotency genes, comprise one or more of Jun, Sox2, Myc, Klf4, Mycn, Klf2, Pou5f1, Utf1, and Sall4, comprise trophectoderm genes, comprise one or more of Tfap2c, Hand1, Msx2, Csf1r, and Gcm1, and comprise gene markers for somatic cells, comprise Neun, Tubb3, Myod1, Myog, and Itga7.

[0115] 25. The method of any one of the preceding clauses, wherein the somatic cell lines are permeabilized using streptolysin O, digitonin, lysophosphatidylcholine, or a mixture thereof.

[0116] 26. The method of any one of the preceding clauses, wherein the cell membranes are resealed using CaCl2.

[0117] 27. The method of any one of the preceding clauses, wherein the step of preparing the GV extract comprises mechanical dissociation.

[0118] 28. The method of any one of the preceding clauses, wherein the method further comprises the step of modifying the GV extract to express a gene encoding a protein selected from the list comprising: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, murine mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and homologs thereof.

[0119] 29. The method of any one of the preceding clauses, wherein the GV extract overexpresses a gene encoding a protein selected from the list comprising: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xklf2, xpou60, murine mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and homologs thereof.

[0120] 30. The method of item 28 or item 29, wherein the step of modifying the GV extract comprises injecting mRNA into the cytoplasm or culturing oocytes with rare-cut endonucleases such as TALEN, ZFN or CRISPR / Cas9 and guide RNA. Optionally, the mRNA encodes a protein selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, mouse mSox2, mFoxa1, human hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1 and their homologs, or the guide RNA comprises a gene encoding: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xklf2, xpou60, mouse mSox2, mFoxa1, human hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1 and their homologs.

[0121] 31. A GV extract comprising reprogramming factors, optionally wherein the reprogramming factors are factors that alter chromatin accessibility.

[0122] 32. The GV extract of item 31, wherein the reprogramming factors are DNA modifying enzymes, histone variants, histone modifying enzymes, chromatin remodelers, chromatin modifiers and transcription factors.

[0123] 33. The GV extract of item 32, wherein the DNA modifying enzymes are AID or Mbd3.

[0124] 34. The GV extract of item 32, wherein the histone modifying enzymes are kdm4 or kdm6.

[0125] 35. The GV extract of item 32, wherein the chromatin remodeler is Brg1.

[0126] 36. The GV extract of item 32, wherein the transcription factors are Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, mouse mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1 and their homologs.

[0127] 37. A GV extract according to any one of items 31 to 36, wherein the GV is isolated from Xenopus, mammals or ray-finned fish.

[0128] 38. The GV extract according to item 37, wherein the mammal is human, mouse, pig or cow and the ray-finned fish is salmon or sturgeon.

[0129] 39. A GV extract according to any one of items 31 to 38, wherein the GV extract is modified, and optionally the GV is modified to express a gene encoding a protein selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, mouse mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1 or homologs thereof.

[0130] 40. The GV extract according to item 39, wherein the GV is modified to overexpress a gene encoding a protein selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, mouse mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1 or homologs thereof.

[0131] 41. A cell derived from the method according to items 1 to 30.

[0132] 42. A cell having an altered cell type derived from the method according to items 1 to 30.

[0133] 43. A kit comprising a permeabilizing agent, a GV extract and / or one or more reprogramming factors and, optionally, instructions for use.

[0134] 44. The kit according to item 43, further comprising a differentiation medium and / or one or more differentiation factors.

[0135] 45. A kit according to item 43 or 44, wherein the GV extract is modified, and optionally the GV is modified to express or overexpress a gene encoding a protein selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, murine mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, or homologs thereof. Example

[0136] The present invention is further described in the following non-limiting examples. Example 1 - Early chromatin changes and nuclear envelope breakdown in MEF cell nuclei transplanted into Xenopus oocytes.

[0137] Here, our procedure is as follows ( Figure 1 a). Inject 10 nanoliters of approximately 500 cell nuclei from ESC (mouse embryonic stem cells), MEF, or MYO (mouse myoblasts) into the GV of the oocytes and culture them for up to 48 hours for subsequent analysis. We have used several different females as donors of our oocytes. We have confirmed that the results we obtained are not affected by differences in the source of the oocytes from one female compared to another. Whenever we saw a difference in gene expression between one donor cell nucleus preparation and another, we saw the same difference in the oocytes of other females. For subsequent experiments on RNA-seq, six different females were used to provide recipient oocytes ( Figure 1 b). When comparing donor cell nucleus preparations from ESC, MEF, and MYO, there were substantial differences in gene expression, but the results for a given nucleus preparation were very similar for different oocyte sources ( Figure 1 b, compare columns 1 - 3 with columns 4 - 7 and 8 - 10). Therefore, our results are not affected by the source of the oocytes.

[0138] In previous studies of cell nuclei injected into oocytes, we saw significant nuclear enlargement and chromatin dispersion on the 3rd or 4th day after nuclear transfer 15 . We now explore on what time scale these effects occur initially; do they occur before or after changes in gene expression? To quantify chromatin changes, the chromatin of the transplanted MEF was labeled with the DNA dye TO-PRO-1 and heterochromatin protein 1α (HP1α) ( Figure 1 c). Changes in chromatin structure in the injected oocytes were as Figure 1as shown in c-e. At 24 hours after MEF nuclear transfer, the chromatin volume of the injected nuclei was dispersed approximately 3-fold, as judged by a 2-fold increase in the chromatin area of each cell ( Figure 1 c-d and Figure 5 a). The time-dependent decrease in HP1α in chromatin indicates relaxation of heterochromatin ( Figure 1 c, 1e, and Figure 5 b). This change shows that chromatin dispersion and heterochromatin relaxation occur within 24 hours of nuclear transfer ( Figure 1 c-e). These chromatin configuration changes were also seen in ESC and MYO ( Figure 5 c). We conclude that chromatin dispersion and relaxation begin with the early entry of GV factors into the oocyte and seem to reflect changes in gene expression induced by nuclear transfer into the oocyte.

[0139] The dramatic changes in chromatin further raise the question of how the nuclear envelope of the transplanted MEF responds to the three-fold dispersion of chromatin after nuclear transfer. To answer this, we used an anti-lamin-A / C antibody to label the nuclear envelope of the transplanted MEF and observed the changes in the nuclear envelope accompanying chromatin dispersion ( Figure 1 f). The nuclear envelope was intact at 0 hours ( Figure 1 f); at 4 hours, the nuclear envelope of the transplanted MEF began to decompose (lower middle, white arrow, Figure 1 f). From where it occurred, the chromatin contained in the donor nucleus burst open (upper middle, white arrow, Figure 1 f). At 24 hours after nuclear transfer, most of the nuclear envelope of the transplanted MEF had disappeared and the chromatin was dispersed ( Figure 1 f).

[0140] Here, we show that HP1α is removed during oocyte reprogramming. HP1α is known to be essential for the higher-order structure of constitutive heterochromatin and binds to H3K9me3 for gene repression and silencing 16、17 . Nuclear transfer of somatic cell nuclei into eggs and oocytes shows that gene resistance is marked by H3K9me3, which is alleviated by the ectopic H3K9me3 demethylase Kdm4d 18、19 . In addition, we show that nuclear envelope decomposition occurs during oocyte reprogramming, accompanied by the disappearance of lamin-A / C (which forms the nuclear lamina with other intermediate filaments). The nuclear lamina formed by lamin-A / C is tethered to the peripheral heterochromatin marked by HP1-H3K9me3 and is functionally linked to gene repression / silencing 20、21In summary, the removal of HP1α and the disappearance of lamin-A during oocyte reprogramming indicate the forced decomposition of H3K9me3-dependent heterochromatin and may lead to transcriptional reprogramming after nuclear transfer. Example 2 Completion of transcriptional reprogramming to a totipotent-like state in Xenopus oocytes within 24 hours.

[0141] Nuclear envelope breakdown and chromatin alterations are similar in nuclear transfer to eggs and oocytes. Are transcriptional alterations also similar in both types of nuclear transfer? We have measured transcriptional alterations of oocyte-induced genes in the donor cell nucleus by BrUTP incorporation after nuclear transfer. We determined newly synthesized BrUTP transcripts at 24 and 48 hours after nuclear transfer and compared these BrUTP transcripts with RNA-seq data of gene expression patterns of publicly available normally cultured cells from ESC, MEF, and MYO. For ESC and MEF, these publicly available results are from Expression Atlas, EMBL-EBI, E-GEOD-27843; for MYO, they are from ENCODE, ENCSR000AHY. Table 1 summarizes the comparison between donor cells and nuclear grafts. Table 1 Enhanced means an increase in transcripts with TPM ≥ 1 Activated means an increase in transcripts with TPM < 1 Reduced means a decrease in transcripts to TPM ≥ 1 Silenced means a decrease in transcripts to TPM < 1 % of inducible mouse genes created by Xenopus oocytes

[0142] From our experiments, there are 29,195 genes whose transcript sequences are different for Xenopus tropicalis compared to the mouse genome (see Materials and Methods). For approximately 30% of the analyzed MEF genes (6197 / 23525), there was an increase in transcripts during the 24-hour period after mouse nuclear transfer to Xenopus oocytes and a decrease in expression was seen in others (Table 1). Thus, approximately 40% of the MEF genes were induced to have transcriptional alterations (up or down) by the combined effect of these oocyte components and no differences were seen between samples cultured for 24 and 48 hours after nuclear transfer (Table 1). These similar alterations were seen in ESC and (almost as many) in MYO (Table 1). Overall gene expression also shows that oocyte components change the gene expression pattern in donor MEF to an oocyte-inducible gene expression state, which is similar at 24 and 48 hours after nuclear transfer (Figure 6 a and 6b). As seen in MEF cells, most of these gene expression changes occur within the first 24 hours after nuclear transfer, including pluripotency genes and trophectoderm genes ( Figure 2 a and 2b). Therefore, we conclude that the oocyte induces a totipotent-like state in the transplanted MEF within 24 hours after nuclear transfer, and to this extent, the gene expression changes induced in the egg and oocyte are similar.

[0143] The rate and extent of these gene expression changes are very significant compared to what is believed to occur in iPSC experiments 8、22 . If we accept the fact that oocyte nuclear transfer is cultured at 18 °C for amphibians rather than 37 °C for mammals, this difference is even more obvious. We have considered the possibility that only a very small number of the injected nuclei in the oocyte can progress to the stage where their pluripotency genes are expressed. In contrast, this is what occurs in iPSC experiments. In iPSC experiments, usually only a small percentage of the DNA-transfected mature cells switch to pluripotency and behave like embryos. The unequal cell division and selection during the growth time of the DNA-transfected cells mean that most of the DNA-transfected cells do not progress to the pluripotent ESC state. Does a low percentage response also occur in oocytes with injected nuclei?

[0144] We have tested this by performing in situ hybridization with a probe against Sox2 and we have explored which part of the injected nuclei contains the Sox2 transcript. Since there is no replication or division of the nuclei injected into the oocyte, we expect a high % of the injected nuclei to contain the pluripotency gene transcript, indicating that a high proportion of them would have been transformed to pluripotency. Figure 2 c shows the results, from which it can be seen that almost all or possibly all of the injected and analyzed nuclei contain some Sox2 transcript (marked in white, Figure 2 c). This means that after transfer to the oocyte, most of the nuclei are transformed to pluripotency.

[0145] Overall, we have seen strong similarities in how three donor nucleus types respond to their transplantation into the oocyte ( Figure 1 b, Table 1). We now explore whether this similarity extends to the selection of individual genes expressed in these cell types. There is considerable but not complete overlap between the gene expressions of the three donor cell types ( Figure 2 d, 14,552 out of a total of 24,936 genes that can be induced by the oocyte, see also Table 2). Table 2 ESC MEF MYO Number of inducible genes 19,921 21,886 17,101 Figure 2 d Number of resistance genes 885 693 2,339 Figure 3 a

[0146] Figure 2 Examples of individual gene expression values for pluripotency genes in e. We see that the further the donor cells are from the undifferentiated state, the higher the proportion of their genes that are activated, i.e., in the order of ESC, MEF, and MYO. Many of the most strongly activated genes are present in all three donor cell types, such as Jun, Klf4, and Myc, as the cells become more differentiated. If we restrict our attention to the individual myogenic gene transcript values, the results are similar for all three donor cell types and most myogenic genes show decreased expression after nuclear transfer ( Figure 2 f). Thus, Xenopus oocytes induce global changes and suppress cell type-specific genes representing the differentiated state in the transplanted nuclei within 24 hours after nuclear transfer, as occurs in nuclear transfer to enucleated eggs. 3. Retention of gene resistance to reprogramming in differentiated mouse myoblasts after nuclear transfer.

[0147] As cells approach the differentiated state, their nuclei become increasingly resistant to reprogramming. This has been known since the earliest nuclear transfer experiments in amphibian eggs 23 (King and Briggs, 1955). We now explore whether the same phenomenon can also be seen in somatic cell nuclear reprogramming by Xenopus oocytes. For the three donor cell types used here, this gene resistance was also seen in this nuclear transfer experiment to oocytes, which occurs in the absence of cell division (Table 2). In our present results ( Figure 3 a), compared to ESC (885 resistant genes) and MEF (693 resistant genes), the nuclei of the more differentiated MYO cells have a greater number of resistant genes (2339 genes).

[0148] The transcriptional changes of genes in MYO that can be induced by oocytes are shown as heat maps and box plots ( Figure 3 b-c). For all three cell types, the top 1500 most highly expressed genes in MYO nuclear grafts that can be induced by oocytes are strongly upregulated by oocyte factors ( Figure 3 b, columns 11-13 compared to columns 1-10, blue to red). In the donor cells, these genes have low to high expression levels ( Figure 3 c, median, 16 for MYO; 50 for ESC; 35 for MEF). After nuclear transfer, for the three cell types, these genes are induced by oocytes to very high expression levels with similar median values, and there are different fold changes because the initial expression levels are different among the donor cell types ( Figure 3 c). In contrast, the transcriptional changes of the oocyte-resistant genes in MYO ( Figure 3d, columns 8 - 11) with the same gene set of most of the ESCs and MEFs successfully reprogrammed by oocyte factors ( Figure 3 d, columns 1 - 7 and 12 - 13) is very different. Therefore, our results have established a strong resistance of differentiating MYO to the reprogramming factors of oocytes (the opposite of nuclear reprogramming), which has no such effect on ESCs or MEFs.

[0149] Among these three donor cell types, most of the genes that can be induced by oocytes respond similarly to oocyte factors ( Figure 3 c). However, there are differences in the oocyte - inducibility expression of genes resistant to oocyte reprogramming ( Figure 3 e). Therefore, the MYO nucleus behaves very differently from the ESC or MEF nucleus in terms of how it behaves after nuclear transfer. The MYO nucleus retains more resistance to oocyte reprogramming (2339 genes) after nuclear transfer; the ESC and MEF nuclei have less resistance to oocyte reprogramming because they have fewer resistant genes (885 genes and 693 genes) ( Figure 3 a). Overall, we draw the following conclusion: The process of cell differentiation is accompanied by increasing resistance to oocyte - induced reprogramming, as seen in both oocyte nuclear transfer and oocyte - nucleus transfer experiments. Example 4. Potential human applications.

[0150] We have been pursuing the idea that nuclear reprogramming to a totipotent - like state by the GV extract of Xenopus meiotic oocytes may be useful for human therapy. This would certainly be the case if different types of mature human cells become dysfunctional (as can occur with aging and disease). Following previous reports 24 two recent papers have described the derivation of human pluripotent cells, one by Yamanaka factors and the other by chemicals 25、26 . Human fibroblasts can be grown from most human mature tissues. Nucleated human blood cells (such as lymphocytes and human hematopoietic stem cells) can be easily obtained and converted into growth cultures, as has been done for many other cell types. These cultured cell types can be reprogrammed by our GV extract procedure and can then form new cell types.

[0151] To apply our GV extract procedure to human cells, we wanted to confirm that the activation systems of mouse and human pluripotency genes during transcriptional reprogramming are comparable. We have shown that pluripotency genes are activated to similar levels in both mouse cells (ESCs and MEFs) and human neuroblastoma cells (hNB) after nuclear transfer by Xenopus meiotic oocytes ( Figure 4a). Activation of pluripotency genes and completion of transcriptional reprogramming in both mouse and human cells allowed us to test human lung stem cells directly derived from human embryos. The pluripotency genes of human lung stem cells were successfully activated by nuclear transfer into oocytes ( Figure 4 b), and these may be related to alleviating human respiratory dysfunction.

[0152] We thus developed our procedure to apply our oocyte GV extract to cultured human cells and demonstrated activation of pluripotency genes in human hematolymphoblasts (hLB). We then tested cell type switching by exploring whether hLB adopted the gene expression of neural cells after being cultured in neural differentiation medium for 3, 10, or 17 days ( Figure 4 c). This was indeed the case, as Figure 4 shown in c, where hLB was seen to express the neuronal marker genes NES and SOX2 after treatment with GV extract and neuronal differentiation medium. This change in expression is exactly what we are looking for if the oocyte-reprogrammed cells are to have therapeutic value.

[0153] However, the change in gene expression did not enable the suspended hLB to transform into morphologically connected neuronal cells. We therefore used another cell type, human dermal fibroblasts (hDF), to develop our method. Following the same reprogramming procedure induced by GV extract, the reprogrammed hDF successfully formed embryoid bodies in culture after 4 days and maintained the function of embryonic stem cells for many days thereafter ( Figure 4 d). We then treated hDF with neural differentiation factors for three weeks. The GV-reprogrammed hDF differentiated morphologically into neuron-like cells and expressed two neuronal gene markers, NEUN and TUBB3 ( Figure 4 e).

[0154] The following is remarkable: Xenopus oocyte GV extract can activate gene expression in mammalian nuclei, even though the mammalian nuclei injected into the oocytes ( Figure 4 f, in metaphase of mitosis) undergo highly abnormal cell division and do not allow normal development. The best explanation for this is that the oocyte ( Figure 4 g, in prophase of meiosis) completely lacks the egg components that induce cell division but already contains the components that transform oocyte chromatin into a transcriptionally active state. This active chromatin state allows gene expression but does not cause chromosomal damage such as that induced by cell division. This is an astonishing switch in the activity of oocytes, as they change from meiotic activity to mitotic activity, which is normally caused by progesterone hormone activity over a time course of about 24 hours.

[0155] We draw the following conclusions: Culturing human cells in Xenopus oocyte GV extracts mimics the reprogramming process by nuclear transfer into GV oocytes. Transcriptionally and functionally, GV-reprogrammed human cells express pluripotency genes and acquire the functions of embryonic stem cells. Addition of differentiation factors allows GV-reprogrammed human cells to acquire new functions distinct from the original cell type.

[0156] In summary, these examples show that Xenopus meiotic oocytes and GV extracts can be used to reprogram mouse nuclei to totipotency and to study the benefits of using Xenopus meiotic oocytes to reprogram cells. Different from the iPSC method and nuclear transfer to mature eggs, nuclear transfer to meiotic oocytes does not require pioneer transcription factors or DNA synthesis / cell division but uses maternal factors to increase chromatin accessibility to promote transcriptional reprogramming of hundreds of transplanted nuclei within hours.

[0157] We first compared the results of nuclear transfer to oocytes as described herein with nuclear transfer to eggs. Are the types of genes activated by these procedures the same? There are certainly great differences in this regard. We found that almost 40% of the mouse genes tested here underwent changes in gene expression during nuclear transfer to oocytes, in most cases towards a more pluripotent level of gene expression. Some previous studies have been done on the differences between nuclear reprogramming caused by oocytes and eggs. Alberio et al. (2005) showed that the remodeling of nuclear lamins is similar in both oocyte and egg reprogramming; however, the transcriptional activities are different27. The main difference between nuclear transfer to oocytes and the iPSC method lies in the timing of the induced changes. Most of the changes seen in nuclear transfer to oocytes occur within 24 hours, and the changes at 48 hours are very small compared to those at 24 hours. The rate of change is similar for all donor cell types used here. Compared to the iPSC method 28 , oocytes show the ability to rapidly and efficiently induce a reprogrammed state of quasi-pluripotency.

[0158] We now explore whether the components of the oocyte required for reprogramming for oocyte nuclear transfer are the same as those for the iPSC method. We believe that most are not. Most of the molecules required for oocyte nuclear transfer have not been identified, but may be related to DNA synthesis and chromatin dispersion. There is no DNA synthesis in the oocyte, but the components required for chromatin structure alteration may be similar in the oocyte and the egg. These may include nucleoplasmin, DNA demethylase, and so on. Finally, we explore whether the mechanisms used for reprogramming are similar for oocytes and other pathways. Here, we have shown that DNA synthesis and cell selection are not required in the oocyte lineage but may be very important for the iPSC method and nuclear transfer into the egg. Other reprogramming components of the egg (including Gli129 and Brg124) are not known to be required for reprogramming by the oocyte. Previously, it has been shown that Xenopus Wave1 is necessary for nuclear reprogramming in the oocyte 30 . Our future research will aim to use the antibody procedure of Clift et al. (2017) 31 to identify other oocyte reprogramming components.

[0159] The main conclusions from the nuclear transfer experiments from somatic cells to oocytes and the GV extract treatment are that the most effective somatic cell reprogramming factors are present in the germinal vesicles of meiotic oocytes and do not require cell division or cell selection. Using maternal factors of the oocyte is different from using Yamanaka factors to reprogram cells. Possibly, oocyte reprogramming factors can be combined with other factors to enhance somatic cell nuclear reprogramming and effect cell type transformation. Example 5 GV Modification

[0160] 9.2 μg of xklf2-HA mRNA (SEQ ID NO: 1) was injected into the cytoplasm of the oocyte ( Figure 8 ) 24 hours prior to nuclear transfer of mammalian nuclei into Xenopus oocytes. Overexpression of xklf2-HA upregulates 1000 - 2000 genes in mouse cells, ESCs, MEFs, and MYOs. Among these genes, the reprogramming-resistant genes (Pou5f1 in MEFs and Sox2, Mycn, Pou5f1, Klf2, Sall4, and Esrrb in MYOs) are activated by overexpression of xklf2-HA. Materials and Methods

[0161] Isolation of oocytes from the oocyte material of female frogs can be obtained from the ovaries of any vertebrate. Xenopus laevis is particularly suitable because a normal female (weighing 170 g) has approximately 5,000 oocytes in its ovaries (weighing 30 g). The mildest and least painful procedure for obtaining oocytes from Xenopus laevis is to subcutaneously inject the female frog with 3 g of ethyl 3-aminobenzoate methanesulfonate (also known as MS222). This is preferably prepared in 0.5 ml of water. The injected frog is placed on its back on ice for 15 min, after which it should be completely anesthetized and unable to right itself properly. The ovarian material is removed from the frog and shredded to expose each oocyte to the culture medium. The required amount of ovarian material (usually approximately 3 ml of ovarian tissue without culture medium) is added to a substance called liberase (250 μl, 28 U / ml, in H2O, Roche, 5401020001) and this is prepared in 12.5 ml of MBS medium and placed on a slow shaker to expose the ovarian material completely to liberase. This is placed on the shaker at room temperature for 2 hours, after which almost all of the oocytes should be free of ovarian material, although each is covered by a monolayer of follicle cells (which helps to protect the oocyte for injection). Next, the liberase is washed off the oocytes with 1X MBS and stage V / VI oocytes with a diameter in the range of 1 to 1.2 mm are selected 32 for use in subsequent experiments. The selected oocytes are placed in a culture dish at 16 °C in 1X MBS and the follicle cell layer will separate during overnight culture. The resulting oocytes are then placed in MBS medium (containing 0.1% bovine serum albumin and antibiotics). The oocytes can be kept at 16 °C for several days. Cell culture

[0162] Mouse embryonic stem cells are cultured in gelatin-coated flasks in G-MEM BHK-21 (Gibco, 21710-025), 20% fetal bovine serum (Gibco, 10439-024), 1000 U / ml leukemia inhibitory factor (Chemicon, ESG1107), 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol, and 10 mM sodium pyruvate. A mouse embryonic fibroblast cell line 33 and a mouse myoblast cell line (C2C12) are cultured in DMEM (Sigma, D5671) with 10% fetal bovine serum (ThermoFisher Scientific, 26140079). The human lung stem cell line is derived from the fetal lung epithelial tip and grows in the form of long-term self-renewing organoids 34The human lymphoblastoid cell line K-562 (Merck, 89121407-1VL) was cultured in IMDM (ThermoFisher Scientific, 31980030) with 10% fetal bovine serum. The human adult dermal fibroblasts (ThermoFisher Scientific, C0135C) were cultured in Medium 106 (ThermoFisher Scientific, M106500) with a low serum growth supplement kit (ThermoFisher Scientific, S003K). Cell permeabilization

[0163] The cells were cultured to sub-confluence, washed twice with PBS and detached by trypsinization at 37 °C for 5 min. The trypsin was neutralized with medium containing BSA and the cells were centrifuged at 500 rpm for four minutes. The supernatant was discarded and the cells were resuspended in PBS. The cells were centrifuged at 2000 rpm for one minute and the PBS was replaced with SuNaSp solution (250 mM sucrose, 75 mM NaCl, 0.5 mM spermidine, 0.15 mM spermine). The cells were centrifuged at 2000 rpm for one minute and the supernatant was discarded. 20 μl of streptolysin O (SLO, 20,000 units / ml, in PBS, containing 0.01% BSA and 5 mM DTT, Sigma-Aldrich, S5265) and 100 μl of SuNaSp solution were added to 3 - 6 x 106 cells. The cells were then resuspended by pipetting and placed in a water bath at 37 °C for one minute to permeabilize them. The cells were incubated on ice and some cells were taken to check the permeabilization efficiency (95 - 99%) by trypan blue staining under a microscope. The SLO reaction was stopped by adding SuNaSp BSA. The cells were centrifuged at 2000 rpm for one minute, the supernatant was discarded and the cells were then resuspended in SuNaSp BSA solution to a cell concentration of ~500 nuclei / 10 nl. The cells in SuNaSp BSA solution were then aliquoted, snap-frozen on dry ice and stored in a -70 °C freezer. Cytoplasmic injection

[0164] For overexpression of xklf2, xklf2 mRNA (SEQ ID NO: 1) was synthesized in vitro from the linearized pCS2-xklf2-HA plasmid using MEGAscript SP6 (Invitrogen, AM1330). xklf2 mRNA (9.2 ng) was injected into the cytoplasm of oocytes 24 hours before nuclear transfer. BrUTP was injected into the cytoplasm 2 hours after nuclear transfer and 5 nl of BrUTP solution (100 mM, in H2O, Sigma, B7166) was injected into the cytoplasm of each oocyte. Nuclear transfer

[0165] Mix permeabilized cells with plasmid DNA encoding membrane GFP35. 9.2 nl of cell suspension (∼500 cells) and 5 pg of plasmid of membrane GFP were injected into the blastomeres of each oocyte. Permeabilized cells were injected into the GV of oocytes using a Drummond nanosyringe. For oocytes collected more than 24 hours after nuclear transfer, GFP-positive oocytes were selected; within 24 hours, no GFP selection was made. Samples were snap-frozen on dry ice and stored in a -70 °C freezer. Confocal microscopy and analysis

[0166] Nguyen T et al., 201936 mentioned the immunofluorescence of HP1α and lamin-A / C. After nuclear transfer, the oocytes were fixed in low-FG fixative and then post-fixed with MeOH / EGTA. The oocytes were rehydrated in the order of 25%, 50%, 75%, and 100% TBS / MeOH and the oocytes were bisected. The oocytes were bleached with a bleaching aqueous solution (1% H2O2, 5% formamide, 150 mM NaCl, 16 mM sodium citrate, adjusted to pH 7.0 with NaOH). The transplanted nuclei were stained with an anti-HP1α antibody (AlexaFluor 647, Abcam, ab198391) or an anti-lamin-A / C antibody (Alexa Fluor 680, Santa Cruz, sc-376248), and the chromatin was stained with the DNA dye TO-PRO-1 (Thermo Fisher, T3602). The oocytes were clarified with Murray's clearing solution for imaging by a Zeiss LSM880 confocal microscope. The chromatin density was quantified using a custom script for Fiji that applied a multi-level Otsu threshold, where the histogram used for calculation was restricted to values greater than or equal to the threshold from the previous iteration (https: / / github.com / gurdon-institute / DNA_Density / blob / main / Wen-Butler_Chromatin_Density.py). The area of the nucleus was measured by segmenting the Huang threshold mask of the TO-PRO-1 image using a recursive algorithm that used a decreasing distance map tolerance to distinguish sub-regions and extract objects of individual nuclei small enough to be considered to be in each stage (https: / / github.com / gurdon-institute / DNA_Density / blob / main / Wen-Butler_Recursive_Watershed.py). The region overlapping with the chromatin of the injected nucleus was marked, and each pixel in this region was scored for the signal from the TO-PRO-1-labeled chromatin. This DNA dye TO-PRO-1 was used to divide the chromatin into three density levels, which were relaxed, intermediate, or dense. Heterochromatin was indicated by an antibody against heterochromatin protein 1α, and the signal was scored for the heterochromatin area. For single-molecule RNA fluorescence in situ hybridization nuclear transfer, the oocytes were fixed in 1% MEMFA and then immersed in -20°C 100% MeOH for at least 48 hours. The oocytes were rehydrated in a series of TBST / MeOH (25%, 50%, 75%, and 100%), with at least 30 min for each solution twice. The oocytes were bisected and bleached with 1% H2O2, 5% formamide, and 1x SSC for 16 hours.Wash the oocytes twice with TBST for 30 min and replace with Stellaris Wash Buffer A (LGC Biosearch Technologies) for 30 min at RT. Remove Stellaris Buffer A and immerse the oocytes in Stellaris Hybridization Buffer (LGC Biosearch Technologies) and let the oocytes stand for 5 min. Remove the hybridization buffer and immerse the oocytes in a 125 nM working solution of Sox2 probe (probe for Stellaris RNA FISH Sox2, LGC Biosearch Technologies) at 37 °C in the dark for 16 h. Remove the probe working solution and wash with hybridization buffer. Stain chromatin with 5 μm TO-PRO-1 stained diluted with Wash Buffer A. Wash twice with TBST for 15 min. Post-fix with 1% formaldehyde / TBST at RT for 1 h. Wash twice with TBST for 30 min and dehydrate the oocytes with 100% MeOH for 16 h. Remove MeOH and add Murray's clearing solution. Let the oocytes settle at the bottom of the tube and image the oocytes using a Zeiss LSM880 confocal microscope. RNA Extraction

[0167] Collect oocyte-NT samples in groups of ten for RNA extraction. Use the Qiagen RNeasy Mini kit (QIAGEN, 74104) for RNA extraction and modify the procedure for our purposes. Briefly, lyse the oocyte-NT samples with 900 ul of RLT buffer and vortex for 4 min at 4 °C. Add 900 ul of 70% ethanol and transfer the mixture to an RNeasy spin column. Centrifuge at 10,000 rpm for 30 s and discard the flow-through. Add 350 ul of RW1 buffer and centrifuge at 10,000 rpm for 30 s. Discard the flow-through and add 80 ul of DNase I incubation mixture. Incubate at room temperature for 15 min, add 350 ul of RW1 buffer and centrifuge at 10,000 rpm for 30 s. Discard the flow-through and add 500 ul of RPE buffer and centrifuge at 10,000 rpm for 2 min. Place the RNeasy spin column in a new 2 ml collection tube and centrifuge at full speed for 1 min. Place the RNeasy spin column in a new 1.5 ml eppendorf and add 50 ul of nuclease-free H2O. Centrifuge at 10,000 rpm for 1 min and measure the RNA concentration with a Nanodrop. Quick-freeze the RNA extract on dry ice and store the RNA extract in a -70 °C freezer. Reverse Transcription and QPCR

[0168] The Superscript III reverse transcriptase kit (ThermoFisher, 18080) was used for reverse transcription. The SYBR Green JumpStart Taq premix (Sigma-Aldrich, S9939) was used for QPCR. Primer sets for pluripotency genes are listed in the paper by Halley-Stott35. 50 ng of cDNA was used per well of the QPCR plate and Gapdh was used for normalization. Immunoprecipitation of RNA incorporated with BrUTP

[0169] The experimental protocol was adapted from the published protocol19. The anti-BrUTP conjugated agarose beads (Santa Cruz Biotechnology, sc-32323AC) were washed twice with Buffer I (0.5X SSPE with 0.05% Tween 20 and 0.1% polyvinylpyrrolidone) and blocked with blocking buffer (Buffer I with 1 mg / ml RNase-free BSA) for 1.5 h at 4 °C. The bead solution was centrifuged at 3000 rpm for 3.5 min at 4 °C and the supernatant was removed. 25–50 μg of RNA extract was used per sample and 2.5 μl of SUPERase·In RNase inhibitor (ThermoFisher, AM2696) was added to each sample. The RNA extract was heated at 65 °C for 5 min, incubated on ice for at least 1 min and centrifuged. RNA was immunoprecipitated overnight at 4 °C with 200 μl of RIP buffer (anti-BrUTP beads in 0.5X SSPE with 0.05% Tween 20). The RNA–bead mixture was washed twice with low-salt buffer (0.2X SSPE with 0.05% Tween 20), twice with high-salt buffer (0.5X SSPE with 0.05% Tween 20 and 150 mM NaCl) and once with TET buffer (10 mM Tris, 1 mM EDTA, pH 8, with 0.05% Tween 20). The immunoprecipitated RNA was eluted with elution buffer (5 mM Tris, pH 7.5, with 300 mM NaCl, 1 mM EDTA, 0.1% SDS, 20 mM dithiothreitol) by incubating at room temperature for 1 min. It was centrifuged at 3000 rpm for 4 min and the supernatant was collected. The elution step was repeated 4 times. The eluted RNA was extracted by phenol / chloroform extraction and ethanol precipitation. The RNA extract was purified with the Qiagen RNeasy Plus Micro kit.

[0170] RNA-seq libraries were prepared and sequenced using the Single Cell RNA-seq System (NuGEN, part number 0342) to prepare RNA-seq libraries from newly synthesized RNA. For each sample preparation, 10 ng of newly synthesized RNA was used. The steps provided by the manufacturer were followed. Subsequently, cDNA reverse transcribed from the newly synthesized RNA was obtained and amplified in the form of an RNA-seq library. The RNA-seq library was verified by Agilent 2200 TapeStation and sequenced for SE50 on Illumina HiSeq 2000 and 4000. Filtering and mapping of sequencing data

[0171] Fasta files from mouse (mm10) and from Xenopus laevis (xla9.1) were concatenated one after another to create a mixed large mouse - Xenopus genome. To distinguish Xenopus laevis chromosomes in the fasta file, they were renamed to "xla_chr" instead of simply "chr". Similarly, gtf files containing annotations of all transcripts from mouse (mm10) and all primary transcripts from Xenopus laevis (xla9.1) were concatenated. The FastQ files were processed with cutadapt (version 1.9.1, option -q 10 -O3) for adapter trimming. Subsequently, the filtered reads were aligned to the mixed mouse - Xenopus genome using tophat (version 2.1.1). Transcripts were assigned to genes and counted using htseq-count (HTSeq - 0.5.4p3). In vitro reprogramming by GV extract

[0172] Oocytes were released from the ovaries of Xenopus laevis (see above, isolation of oocytes from female frogs). The GV was manually dissected in mineral oil and the GV was collected in a tube. The GV was quickly frozen on dry ice and stored in a -70 °C freezer. Calcium-free IMDM medium (United States Biological, I8750 - 08) was added and centrifuged at 16,100 g for 10 min at 4 °C. The supernatant was taken and the GV extract was placed on ice (or at -20 °C) for later use. Human lymphoblastoid cell line K-562 and human mature dermal fibroblasts were permeabilized with SLO in PBS (see above, cell permeabilization). Subsequently, the permeabilized cells were cultured in calcium-free IMDM medium with GV extract for 6 hours and medium with CaCl2 (final 2 mM) was added to reseal the membrane. Neural differentiation

[0173] The experimental protocol for neural differentiation was modified from a previously published paper 28(Giulitti et al., 2019). The cells reprogrammed with the GV extract were seeded on Matrigel-coated dishes at a density of 530 cells / mm2 and cultured in mTeSR1Plus medium (STEMCELL Technology, 100 - 0276) for six days. From day 0 to 2, the cells were cultured in neural medium (N2B27, 1% NEAA, 200 ng / ml L-ascorbic acid) supplemented with 20 ng / ml bFGF (STEMCELL Technology, 78003.1) and 0.1 μM LDN 193189 (STEMCELL Technology, 72147). Then on day 3, the cells were cultured in neural medium supplemented with 0.1 μM LDN 193189 and 10 μM SB431542 (STEMCELL Technology, 72234). For the next 6 days, the medium used on day 3 was supplemented with 1 μM all-trans retinoic acid (Merck, R2625) and 1 μM SAG (Merck, 566661). From day 10 to 15, the cells were cultured in neural medium supplemented with 5 μM DAPT (Merck, D5942), 4 μM SU-5402 (Merck, SML0443), 1 μM all-trans retinoic acid and 1 μM SAG. On day 16, the cells were seeded on glass-bottomed chamber slides and cultured in maturation medium (which contains neural medium supplemented with 20 ng / ml BDNF, 10 ng / ml GDNF, 10 ng / ml CNTF (PeproTech, 450 - 02, 450 - 10, 450 - 13) and 10 μM ROCK inhibitor (STEMCELL Technology, 72302)). The cells were cultured in the maturation medium up to day 22 and fixed for immunostaining. All media were changed daily. References

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Claims

1. A method for reprogramming the nucleus of a somatic cell to change its cell type, comprising: Preparing a GV extract; Permeabilizing the somatic cell; Culturing the somatic cell with the GV extract to change its cell type; and Re-sealing the somatic cell membrane, wherein the GV extract does not contain oocyte cytoplasm.

2. The method according to claim 1, wherein changing the cell type comprises reprogramming the somatic cell into a progenitor cell or a stem cell.

3. The method according to claim 2, wherein the progenitor cell or the stem cell is a unipotent stem cell, an oligopotent stem cell, a pluripotent stem cell, a totipotent stem cell or a pluripotent stem cell.

4. The method according to any one of claims 2 or 3, further comprising differentiating the progenitor cell or the stem cell to change its cell type.

5. The method according to claim 4, wherein differentiating the progenitor cell or the stem cell produces a somatic cell or its progenitor cell, wherein the somatic cell or its progenitor cell is of a different lineage.

6. The method according to claim 4 or 5, wherein the differentiation comprises culturing the progenitor cell or the stem cell in a differentiation medium.

7. The method according to claim 6, wherein the differentiation medium further comprises the addition of one or more differentiation factors.

8. The method according to any one of the preceding claims, wherein the GV extract comprises one or more reprogramming factors.

9. The method according to claim 8, wherein the reprogramming factor is a factor that changes chromatin accessibility.

10. The method according to claim 9, wherein the reprogramming factor is a DNA modifying enzyme, a histone variant, a histone modifying enzyme, a chromatin remodeler, a chromatin modifier and a transcription factor.

11. The method according to claim 10, wherein the DNA modifying enzyme is AID or Mbd3.

12. The method according to claim 10, wherein the histone modifying enzyme is kdm4 or kdm6.

13. The method according to claim 10, wherein the chromatin remodeler is Brg1.

14. The method according to claim 10, wherein the transcription factor is Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, mouse mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1 and their homologs.

15. The method according to any one of the preceding claims, wherein the isolated GV is derived from a meiotic oocyte.

16. The method according to any one of the preceding claims, wherein the GV is isolated from Xenopus, mammals or ray-finned fish.

17. The method according to claim 16, wherein the mammal is human, mouse, pig or cow and the ray-finned fish is salmon or sturgeon.

18. The method according to any one of the preceding claims, wherein the somatic cell is a mammalian cell.

19. The method according to claim 18, wherein the mammalian cell is a human cell, a mouse cell, a pig cell, a bovine cell, a dog cell, a cat cell, or a horse cell.

20. The method according to any one of the preceding claims, wherein the altering cell type further comprises adding one or more exogenous reprogramming factors.

21. The method according to any one of the preceding claims, wherein the somatic cell and the GV extract and, optionally, one or more exogenous reprogramming factors are cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

22. The method according to any one of claims 1 to 20, wherein the somatic cell and the GV extract are cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

23. The method according to any one of claims 6 to 22, wherein the progenitor cell is cultured in a differentiation medium for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 days or is cultured in a differentiation medium for 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months.

24. The method according to any one of the preceding claims, wherein the somatic cell comprises a selectable or reportable marker, comprises a pluripotency gene, comprises one or more of Nelfa, Top2a, Gata2, Eif3h, Dppa2 / 4, and Atr, comprises a multipotency gene, comprises one or more of Jun, Sox2, Myc, Klf4, Mycn, Klf2, Pou5f1, Utf1, and Sall4, comprises a trophectoderm gene, comprises one or more of Tfap2c, Hand1, Msx2, Csf1r, and Gcm1, and comprises a gene marker for somatic cells, comprises Neun, Tubb3, Myod1, Myog, and Itga7.

25. The method according to any one of the preceding claims, wherein the somatic cell is permeabilized using streptolysin O, digitonin, lysophosphatidylcholine, or a mixture thereof.

26. The method according to any one of the preceding claims, wherein the cell membrane is resealed using CaCl2.

27. The method according to any one of the preceding claims, wherein the step of preparing the GV extract comprises mechanical dissociation.

28. The method according to any one of the preceding claims, wherein the method further comprises the step of modifying the GV extract to express genes encoding proteins selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, murine mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs.

29. The method according to any one of the preceding claims, wherein the GV extract overexpresses genes encoding proteins selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xklf2, xpou60, murine mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs.

30. The method according to claim 28 or claim 29, wherein the step of modifying the GV extract comprises injecting mRNA into the cytoplasm or culturing oocytes with rare-cut endonucleases such as TALEN, ZFN, or CRISPR / Cas9 and guide RNA, wherein, as needed, the mRNA encodes a protein selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, murine mSox2, mFoxa1, human hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs, or the guide RNA comprises a gene encoding: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xklf2, xpou60, murine mSox2, mFoxa1, human hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs.

31. A GV extract, which comprises reprogramming factors, wherein, as needed, the reprogramming factors are factors that alter chromatin accessibility.

32. The GV extract according to claim 31, wherein the reprogramming factors are DNA modifying enzymes, histone variants, histone modifying enzymes, chromatin remodelers, chromatin modifiers, and transcription factors.

33. The GV extract according to claim 32, wherein the DNA modifying enzyme is AID or Mbd3.

34. The GV extract according to claim 32, wherein the histone modifying enzyme is kdm4 or kdm6.

35. The GV extract according to claim 32, wherein the chromatin remodeler is Brg1.

36. The GV extract according to claim 32, wherein the transcription factor is Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, mouse mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, and their homologs.

37. The GV extract according to any one of claims 31 to 36, wherein the GV is isolated from Xenopus, mammals, or ray-finned fish.

38. The GV extract according to claim 37, wherein the mammal is human, mouse, pig, or cow and the ray-finned fish is salmon or sturgeon.

39. The GV extract according to any one of claims 31 to 38, wherein the GV extract is modified, and optionally the GV is modified to express a gene encoding a protein selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, mouse mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, or their homologs.

40. The GV extract according to claim 39, wherein the GV is modified to overexpress a gene encoding a protein selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, mouse mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1, or their homologs.

41. A cell derived from the method according to claims 1 to 30.

42. A cell having an altered cell type derived from the method according to claims 1 to 30.

43. A kit comprising a permeabilizing agent, a GV extract, and / or one or more reprogramming factors, and optionally instructions for use.

44. The kit according to claim 43, further comprising a differentiation medium and / or one or more differentiation factors.

45. A kit according to claim 43 or 44, wherein the GV extract is modified, and optionally wherein the GV is modified to express or overexpress a gene encoding a protein selected from the list consisting of: Gli1, FoxA, Gata4, Ascl1, Brn2, Myt1l, PU.1, xklf2, xsox2, xpou60, murine mSox2, mFoxa1, hOCT4, hKLF4, Oct3 / 4, Sox2, Klf4, MyoD, Gata2 / 3, Foxa1, Hnf4a, Hnf1a, Pax4, Pdx1 or homologs thereof.

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