Method for producing high-proportion chimeric living monkeys by using embryonic stem cells

Abstract

The invention provides a method for producing high-proportion chimeric living monkeys by using embryonic stem cells for the first time, in particular, the invention provides an in-vitro preparation method of chimeric embryos of non-human primates, and the chimeric embryos or chimeric non-human primates obtained by the method have the characteristic of high chimeric proportion. Moreover, the method provided by the invention can significantly improve the contribution rate of the immature stem cells, for example, the contribution rate of the immature stem cells is up to 21-92%.

Description

Technical Field

[0001] The present invention relates to the field of mammalian reproduction technology, and in particular to a method for producing high-ratio chimeric living monkeys using embryonic stem cells. Background Art

[0002] The transient pluripotent state of the inner cell mass (ICM) of the mammalian blastocyst can be captured by establishing embryonic stem cell lines (ESCs) or by induced reprogramming of somatic cells into pluripotent stem cells (iPSCs) in vitro. Scalable pluripotent stem cell (PSC) lines can be generated under different culture conditions to mimic different stages of embryonic development. Preimplantation ICM-like PSCs are often referred to as state, while the epiblast-like PSCs after implantation are called the “primed” state.

[0003] Complex genetic modification of NHPs is generally performed by injecting CRISPR / Cas9 reagents into early embryos, or by combining somatic cell nuclear transfer technology with in vitro editing of cultured fibroblasts, but both are limited by their efficiency. Another method is to use injection of homologous PSCs to produce high-contribution chimeras. However, the chimera technology of NHPs and other mammals lags behind that of rodents. Previous chimera studies of homologous PSCs in non-human primates, cynomolgus macaques, have produced aborted fetuses and offspring of chimeras, but the contribution of donor cells in chimeric tissues is very low (0.1-4.5%). It is generally believed that the reason for the inability to achieve high chimerism is that the development of donor cells is not synchronized with that of the host embryo, resulting in the gradual elimination of donor cells in the blastocyst or tissue in competition. Recent studies have shown that increasing cell survival by overexpressing exogenous factors (such as BCL2 or BMI1) can partially overcome PSC apoptosis and increase the proportion of interspecies chimeras, but overexpression has the risk of inducing genomic abnormalities. Therefore, improving culture methods to achieve more stable The pluripotent state is a suitable experimental method. PSCs culture medium, but its There are differences in the expression levels and genomic stability of pluripotency genes. Methods with high expression of pluripotency genes showed lower DNA methylation levels, which led to genomic instability and karyotypic abnormalities, which may induce differentiation of injected PSCs.

[0004] Therefore, there is an urgent need in the art to develop a method that can Early embryonic complementation of PSCs can generate chimeras with high ESC contribution in NHPs, paving the way for a new approach to the future generation of transgenic NHPs using gene-edited PSCs. Summary of the invention

[0005] The present invention aims to provide a method for Early embryonic complementation of PSCs can generate chimeras with high ESC contribution in NHPs, paving the way for a new approach to the future generation of transgenic NHPs using gene-edited PSCs.

[0006] In a first aspect, the present invention provides a method for preparing a chimeric embryo of a non-human primate in vitro, comprising the steps of:

[0007] (i) culturing primed pluripotent stem cells (PSCs) under a first culture condition to obtain primed stem cells or naive stem cells, wherein the first culture condition comprises a first culture medium selected from the group consisting of 4CL, 5iLAF, PXGL, RseT, LCDM, primed, or a combination thereof;

[0008] (ii) contacting the flat stem cells or naive stem cells obtained in step (i) with a non-human primate embryo to obtain a non-human primate chimeric embryo containing the flat stem cells or naive stem cells, and culturing the non-human primate chimeric embryo containing the flat stem cells or naive stem cells under a second culture condition to obtain a blastocyst stage chimeric embryo, wherein the second culture condition comprises a mixed culture medium in which a second culture medium and a third culture medium are mixed in a volume ratio of 1:0.5-1.5, wherein the second culture medium is selected from the group consisting of Primed, 4CL, 5iLAF, or a combination thereof, and the third culture medium comprises HECM9.

[0009] In another preferred embodiment, the first culture condition is a culture condition suitable for differentiation of flat stem cells or immature stem cells.

[0010] In another preferred example, the first culture medium is selected from the following group: Primed, 4CL, 5iLAF, or a combination thereof.

[0011] In another preferred embodiment, the pluripotent stem cells are derived from embryonic stem cells of non-human primates.

[0012] In another preferred embodiment, the immature stem cells include immature embryonic stem cells.

[0013] In another preferred example, the second culture condition includes a mixed culture medium in which the second culture medium and the third culture medium are mixed in a volume ratio of 1:1.

[0014] In another preferred embodiment, before step (i), the method further comprises the step of pre-culturing primed pluripotent stem cells (PSC):

[0015] Primed pluripotent stem cells (PSCs) are cultured in a culture system, wherein the culture system is a culture solution containing a fourth culture medium and an additive, wherein the additive includes Y27632, and the fourth culture medium includes an E8+K culture medium.

[0016] In another preferred embodiment, the embryo or chimeric embryo comprises a morula stage embryo.

[0017] In another preferred embodiment, the contact treatment includes:

[0018] The naive stem cells are injected into the embryos of the non-human primates.

[0019] In another preferred embodiment, the immature stem cells are stem cells within 5-10 generations.

[0020] In another preferred embodiment, 10-30 immature stem cells are injected into the embryo.

[0021] In another preferred embodiment, in step (i), primed pluripotent stem cells (PSCs) are cultured under the first culture condition for 48-96 h, preferably 60-96 h (eg 72 h).

[0022] In another preferred embodiment, in step (ii), the non-human primate embryo containing immature stem cells is cultured under the second culture condition for 48-96 hours, preferably, 60-96 hours (such as 72 hours).

[0023] In another preferred example, the second culture medium includes 4CL and 5iLAF.

[0024] In another preferred embodiment, the method further comprises step (iii): regenerating the chimeric embryo obtained in step (ii) to obtain a chimeric non-human primate.

[0025] In another preferred embodiment, the chimeric embryo has a high chimerism ratio.

[0026] In another preferred embodiment, the chimeric non-human primate is characterized by a high chimerism ratio.

[0027] In another preferred embodiment, the chimeric embryo or chimeric non-human primate has a characteristic selected from the following group:

[0028] The contribution rate of immature stem cells is high. Through paternity testing, genome sequencing, immunofluorescence, and single-cell sequencing analysis, the chimerism ratio was identified to be 21%-92%.

[0029] In another preferred embodiment, the regeneration is performed in the uterus of a non-human primate surrogate animal.

[0030] In another preferred embodiment, the species of the surrogate animal is the same as the species of the chimeric embryo.

[0031] In another preferred embodiment, the regeneration is carried out in an artificial uterus.

[0032] In another preferred embodiment, the step (iii) comprises:

[0033] The chimeric embryo is transplanted into the fallopian tube of a non-human primate, thereby obtaining the chimeric non-human primate.

[0034] In another preferred embodiment, the transplantation is non-invasive transplantation (ie, does not cause any trauma).

[0035] In another preferred embodiment, the non-human primates include monkeys and chimpanzees.

[0036] In another preferred embodiment, the non-human primate includes cynomolgus monkey.

[0037] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0038] The second aspect of the present invention provides a chimeric non-human primate prepared by the method described in the first aspect of the present invention.

[0039] The third aspect of the present invention provides a combination of a culture medium for producing chimeric embryos, comprising:

[0040] (a) a first culture medium for culturing and obtaining primed stem cells or naive stem cells, selected from the group consisting of 4CL, 5iLAF, PXGL, RseT, LCDM, primed, or a combination thereof;

[0041] (b) A mixed culture medium for obtaining chimeric embryos, the mixed culture medium comprising a second culture medium and a third culture medium, the volume ratio of the second culture medium to the third culture medium in the mixed culture medium is 1:0.5-1.5, the second culture medium is selected from the following group: Primed, 4CL, 5iLA, or a combination thereof; the third culture medium comprises HECM9.

[0042] In another preferred example, the first culture medium is selected from the following group: primed, 4CL, 5iLAF, or a combination thereof.

[0043] In another preferred example, the first culture medium includes 4CL and 5iLAF.

[0044] In another preferred example, the second culture medium is selected from the following group: 4CL, 5iLAF, or a combination thereof.

[0045] In another preferred example, the volume ratio of the second culture medium to the third culture medium in the mixed culture medium is 1:1.

[0046] The fourth aspect of the present invention provides a use of the combination of culture media described in the third aspect of the present invention for preparing chimeric non-human primates.

[0047] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 .Characteristics of monkey ESCs cultured under different conditions.

[0049] (A) Representative images of monkey ESCs cultured under the indicated culture conditions. Scale bar, 100 μm.

[0050] (B) Monkey ESCs under specified culture conditions Representative immunofluorescence images of (KLF17) and shared (SOX2 / NANOG) pluripotency markers. Scale bar, 25 μm.

[0051] (C) Heat map showing the expression of cytokines in monkey ESCs under the indicated culture conditions. Expression of shared and primed pluripotency genes. N = 2 technical replicates.

[0052] (D) Pseudo-global correlation analysis of scRNA-seq data of monkey ESCs cultured in E8+K, 4CL, 5iLAF, and PXGL with in vivo embryo data.

[0053] (E) UMAP expression comparing monkey embryos to ESCs cultured in E8+K, 4CL, 5iLAF, and PXGL.

[0054] (F) Violin plots show the comparison of DNA methylation levels of monkey ESCs cultured under the indicated conditions with those of ICMs.

[0055] (G) Violin plots showing the expression of primed and 4CL under different passage conditions. DNA methylation levels of monkey ESCs cultured under the same conditions. N = 2 technical replicates.

[0056] (H) Images of G-banded karyotypes of monkey ESCs cultured under the indicated conditions. Twenty metaphases were counted for each condition.

[0057] Figure 2. Optimization of the culture protocol for generating chimeric blastocysts using monkey ESCs.

[0058] (A) Schematic diagram of chimeric blastocyst generation.

[0059] (B) Schematic diagram of the injection of monkey ESCs into morulae using different protocols.

[0060] (C) Representative images showing GFP-labeled monkey ESCs integrated into host embryos at the blastocyst stage. Hrs, hours; Pr, plan. Scale bars, 100 μm (left), 50 μm (right).

[0061] (D) Summary of blastocyst development and GFP signal under different culture schemes.

[0062] (E) Representative images of GFP signal and OCT4 immunostaining (red) at the blastocyst stage in 4CL ESC-injected chimeric embryos. Scale bar, 50 μm.

[0063] (F) OCT4 in ICM of the three injection groups + GFP + / OCT4 + Quantification of cell percentage. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

[0064] Figure 3 .Contribution of monkey ESCs to embryos during delayed in vitro culture.

[0065] (A) Representative images and GFP signals of extended in vitro cultures of chimeric monkey embryos at the indicated time points during development. White lines indicate the ectoderm. dpf, day one postfertilization. Scale bar, 100 μm.

[0066] (B) Representative images and GFP signal of extended in vitro culture of uninjected control embryos. White line indicates ectoderm. Scale bar, 100 μm.

[0067] (C) Dynamic changes in the developmental rate of chimeric embryos generated from 4CL, 5iLAF and primed ESCs and control monkey embryos (control n=33; 4CL n=27; 5iLAF n=18, prime n=18).

[0068] (D) Histogram showing the percentage of GFP signals in the three groups of chimeric monkey embryos at D17.

[0069] (E) UMAP comparison of the development of 4CL ESC-injected IVC chimeric embryos in this study with in vivo and in vitro embryos.

[0070] (F) Based on Figure 3UMAP visualization of E shows GFP + Expression of E-PGC (TFAP2C) and Gast1 (SLC7A3) genes in cells.

[0071] (G) Based on Figure 3 UMAP visualization of E showing GFP from IVC mosaic embryos + (green) and GFP - (black) Distribution in integrated objects.

[0072] (H) Bar graph showing the expression of GFP in each identified cell type in IVC mosaic embryos. + The percentage of cells.

[0073] (I) Heat map of DEGs among key cell types in IVC mosaic embryos. Representative genes are shown for each cell type.

[0074] (J) Genes enriched in key cell types in 4CL ESC-injected IVC chimeric embryos. P values ​​were calculated using the hypergeometric test and adjusted for multiple testing using the Benjamini-Hochberg correction.

[0075] Figure 4 .Generate chimeric monkeys with high homologous ESC contribution.

[0076] (A) Summary of the results of in vivo chimera experiments.

[0077] (B) Genomic DNA PCR was used to detect GFP, SRY, and ACTIN sequences in ear tips of live-born offspring and other tissues of aborted fetuses. PC, positive control; NC, negative control.

[0078] (C) Blastocysts and GFP images of two chimeric embryos before transplantation (#9 and #10). Scale bar, 50 μm

[0079] (D) Green fluorescence signal images of different body parts of the chimeric monkey (#10) 3 days after birth.

[0080] (EF) STR analysis of two chimeric monkeys (#9 and #10) at the D6S2741 and D9S921 loci.

[0081] (G) Summary of chimeric embryo surrogate mothers and their delivery information.

[0082] Figure 5 .Comprehensive analysis of monkeys Contribution of ESCs to live births in chimeric monkeys.

[0083] (A) Genomic DNA PCR detection of GFP, SRY and ACTIN sequences in different tissue samples of chimeric monkey #10. (B) Identification of mitochondrial DNA SNPs and genomic DNA SNPs between oocyte donors and ESC donors of chimeric monkey #10.

[0084] (CD) Quantification of the contribution of ESCs in chimeric monkey #10 by deep sequencing of mitochondrial DNA SNPs and genomic DNA SNPs. Data are presented as mean ± SEM. n = 3 biological replicates.

[0085] (E) GFP fluorescence shows GFP-tagged 4CL ESCs differentiated into PBMCs and BMCs of chimeric monkey #10. Scale bar, 20 μm.

[0086] (F) FACS analysis showed GFP expression in PBMC and BMC of chimeric monkey #10 + The percentage of cells.

[0087] (G) GFP fluorescence shows GFP-tagged 4CL ESCs differentiated into different tissues in chimeric monkey #10. Scale bar, 100 μm.

[0088] (H) GFP immunofluorescence shows GFP-tagged 4CL ESCs differentiated into the indicated tissues of chimeric monkey #10. GFP was detected using GFP antibody + Cells. Scale bar, 25 μm.

[0089] (I) Whole-brain image of chimeric monkey No. 10. Scale bar, 1 cm.

[0090] (J) GFP-tagged 4CL Chimerism of ESCs in the brain of chimeric monkey #10. Anti-NeuN antibody was used to detect mature neurons in the brain. Scale bar, 25 μm.

[0091] (K) GFP expression in different tissues of chimeric monkey #10 + Quantification of cell percentage. Data are presented as mean ± SEM.

[0092] (L) GFP in NeuN-positive cells in the brain of chimeric monkey #10 + Quantification of cell percentage. Data are presented as mean ± SEM.

[0093] Figure 6 .Characterization of cell lineages in live-born chimeric monkeys.

[0094] (A) UMAP visualization showing GFP expression in different cell types in the bone marrow Smart-seq2 data of chimeric monkey #10+ (Green) Distribution of cells.

[0095] (B) UMAP visualization shows GFP in the bone marrow Smart-seq2 data of chimeric monkey #10 -+ Different cell types identified in GFP (left) and GFP (right) cells. GMPs, granulocyte-monocyte progenitors.

[0096] (C) Bubble plot showing the expression frequency and average expression of cell type-specific marker genes in the bone marrow Smart-seq2 data of chimeric monkey #10.

[0097] (D) GFP of each identified cell type in the bone marrow Smart-seq2 data of chimeric monkey #10 + Cell percentage.

[0098] (E) UMAP visualization showing GFP expression in different cell types identified in chimeric monkey #10 bone marrow DNBelab C4 data (droplet-based scRNA-seq) + Distribution of cells.

[0099] (F) UMAP visualization showing different cell types identified in the DNBelab C4 data of chimeric monkey #10 bone marrow. (G) Bar graph showing GFP expression in different cell types identified in the DNBelab C4 data of chimeric monkey #10 bone marrow. + and GFP - The proportion of cells.

[0100] (H) UMAP visualization showing GFP expression in different cell types in the Smart-seq2 data of the chimeric monkey #10 brain + (Green) Distribution of cells.

[0101] (I) UMAP visualization showing GFP in the Smart-seq2 data of the brain of chimeric monkey #10 -+ (left) and GFP (right) cell types.

[0102] (J) Bubble plots showing the expression frequency and average expression of cell type-specific marker genes in the Smart-seq2 data of chimeric monkey #10 brain.

[0103] (K) Bar graph showing GFP expression in different cell types identified in Smart-seq2 data from the brain of chimeric monkey #10 + percentage.

[0104] (L) UMAP visualization showing GFP expression in different cell types identified in the brain droplet-based snRNA-seq data of chimeric monkey #10 + (Green) Distribution of cells.

[0105] (M) UMAP visualization showing different cell types identified in the DNBelab C4 data of the brain of chimeric monkey #10. (N) Bar graph showing GFP expression in different cell types identified in the snRNA-seq data of the brain of chimeric monkey #10. - and the proportion of GFP cells + .

[0106] Figure 7 .monkey Mosaicism of ESCs in testis and placenta and genomic integrity and epigenetic status in offspring tissues.

[0107] (A) Representative immunofluorescence images showing GFP-tagged 4CL Contribution of ESCs in the testis of chimeric monkey #10. Combination of GFP and VASA antibodies. Scale bar, 25 μm.

[0108] (B) GFP + Cells as a percentage of total cells and VASA + The data are expressed as mean ± SEM.

[0109] (C) UMAP comparing chimeric monkey #10 testis (Smart-seq2) to age-matched wild-type monkey testis (droplet-based scRNA-seq) and highlighting GFP from chimeric monkey #10 testis and wild-type monkey testis cells (grey) - (green) and GFP + Distribution of cells (black).

[0110] (D) UMAP visualization showing GFP expression in the testes of #10 chimeric monkeys and age-matched wild-type monkeys + and GFP - The cell type of the cell.

[0111] (E) Bubble plots showing the frequency and average expression of specific marker genes in testes from #10 chimeric and wild-type monkeys.

[0112] (F) Bar graph showing GFP expression in different cell types in the Smart-seq2 data of the testis of chimeric monkey #10 + The percentage of cells.

[0113] (G) Bubble diagram showing the expression of the Sertoli cell-specific marker gene GFP in the testis of #10 chimeric monkey + and GFP + Cell expression.

[0114] (H) Representative immunofluorescence images showing GFP-tagged 4CL in chimeric monkey #10 Contribution of ESCs to the placenta and wild-type placenta. GFP antibody was used in combination with KRT7 antibody. Scale bar, 25 μm.

[0115] (I) Violin plot showing GFP - BMCs and GFP + Global DNA methylation levels of BMCs. N = 2 technical replicates.

[0116] (J)GFP - BMCs and GFP + Comparison of TSS and TES DNA methylation levels in BMCs. N = 2 technical replicates.

[0117] (K)GFP - BMCs and GFP + Heat map of promoter region DNA methylation level and gene expression level of the top 100 BMC highly expressed genes in BMCs [log 10 (Average TPM+1)].

[0118] (L) Heat map showing GFP - BMCs and GFP + DNA methylation levels in the promoter regions of imprinted genes and gene expression levels in BMCs [log2(mean TPM+1)].

[0119] (M) Detection of GFP using whole genome sequencing - / GFP + BMCs and GFP - / GFP + Copy number in ear apex fibroblasts.

[0120] Figure 8 .Characterization of monkey ESCs cultured under six different conditions, related Figure 1 .

[0121] (A) Representative images showing the isolation of primed ESCs from monkey blastocysts. Scale bars, 25 μm (left image), 100 μm (other images).

[0122] (B) Summary of the efficiency of establishing monkey primed ESC lines. Data are presented as mean ± SEM. N = 6 biological replicates. (C) Summary of the conversion of five cell lines from primed state under five different culture conditions. Status of the situation.

[0123] (D) Representative images of ESC clone morphology under the indicated conditions. Scale bar, 100 μm.

[0124] (E) AP after single cell isolation under the indicated culture conditions + Representative images of ESC clones. The cell seeding density (2x10 4 cells / well). Scale bar, 200 μm.

[0125] (F) AP after single cell isolation under the indicated ESC culture conditions + Quantitative analysis of clones. Data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. N=3 technical replicates.

[0126] (G) H&E staining of teratomas generated from ESCs under the indicated culture conditions. Representative images of the three germ layers are shown. Scale bar, 100 μm.

[0127] (H) Summary of the teratoma formation ratios of ESCs cultured under the indicated conditions.

[0128] (I) Representative images showing mitochondrial activity of live cells in each of the indicated ESC culture conditions by co-staining with a mitochondrial marker (MitoTracker) and TMRE. MitoTracker is a universal stain; TMRE staining is dependent on mitochondrial membrane activity. Scale bar, 20 μm.

[0129] (J) Immunofluorescence images of TFE3 (green) and OCT4 (red) in ESCs cultured under the indicated conditions. Nuclei were stained with DAPI. Scale bar, 25 μm.

[0130] (K) RT-qPCR detection of classical marker genes (OCT4 and NANOG) and Expression of pluripotency genes (DPPA3, KLF17, and TFCP2L1). Data are presented as mean ± SEM of fold change compared with primed ESCs. N = 3 technical replicates.

[0131] Fig. 9 .Transcriptome and epigenomic characteristics of monkey ESCs under different conditions, related Figure 1 .

[0132] (A) GO analysis of upregulated genes in monkey ESCs cultured in 4CL (left), 5iLAF (middle), and PXGL (right) compared with primed ESCs. P values ​​were calculated using the hypergeometric test and adjusted for multiple testing using the Benjamini-Hochberg correction.

[0133] (B) Comparison of DNA methylation levels at TSS (transcription start site) and TES (transcription end site) in monkey ESCs under the indicated culture conditions.

[0134] (C) Heat map showing DNA methylation levels in promoter regions of imprinted genes in monkey ESCs compared with monkey ICMs under the indicated culture conditions.

[0135] (D) Comparison between primed and 4CL DNA methylation levels in TSS and TES regions of monkey ESCs at passages 1, 3, 7, and 10 in culture. P channel.

[0136] (E) Comparison between primed and 4CL DNA methylation levels at different genomic regions in monkey ESCs at passages 1, 3, 7, and 10 in culture.

[0137] (F) Heat map showing the difference between primed and 4CL Primed genes and DNA methylation levels of pluripotency genes.

[0138] (G) Genomic loci show that in primed and 4CL DNA methylation levels at indicated pluripotency gene loci at 1, 3, 7, and 10 generations under culture conditions.

[0139] (H) Heat map showing primed and 4CL DNA methylation levels in the promoter regions of imprinted genes in ESCs at passages 1, 3, 7, and 10 in culture conditions.

[0140] (I) Transformation of 4CL by primed ESCs The two cells were cultured for 10 generations and scWGS sequencing was performed using the same primed ESCs cultured for 10 generations. Primed-P10, n=28; 4CL-P10, n=20.

[0141] (J) QQ plot of genome-wide copy number variation analysis. The Y axis is -log10 (observed P value) and the X axis is -log10 (expected P value). The plot is aligned with the diagonal line representing consistency with the global null hypothesis, which means that there is a positive correlation between the primed and Significant differences in CNVs detected between cells.

[0142] Fig.10 .Optimized 4CL Methods for generating chimeric blastocysts from monkey ESCs, related Figure 2 .

[0143] (A) GFP-labeled monkey ESCs (left) transformed into 4CL Image of monkey ESCs (center). Monkey ESCs were digested into single cells (right panel) for embryonic injection, and GFP fluorescence was detected at the single-cell level. Scale bar, 100 μm.

[0144] (B) Monkey ESCs injected into morula-stage embryos (left) and GFP + Image of a chimeric blastocyst (right). Scale bar, 100 μm.

[0145] (CE) Images of developing chimeric embryos using morula injection of GFP-labeled 4CL-ESCs (C), primed ESCs (D), and 5iLAF ESCs (E). Scale bar, 100 μm. Figure 2 One panel of Figure S3E, which shows the developmental time course of chimeric embryos under 5iLAF conditions, is also used in C as a representative image. Figure 2 C shows the endpoint of blastocyst development of chimeric embryos under the same conditions.

[0146] (F) Immunofluorescence images of GFP and OCT4 in primed ESC-injected and 5iLAF ESC-injected chimeric embryos at the blastocyst stage (red). Nuclei were stained with DAPI (blue). Scale bar, 50 μm.

[0147] (G) Localization of injected GFP ESCs in ICM and trophectoderm cultured under primed, 4CL, and 5iLAF conditions. White arrows indicate GFP + ESCs are located in the trophectoderm. Scale bar, 50 μm.

[0148] Fig.11 .Analysis of the developmental potential of delayed cultured chimeric embryos in vitro, related Figure 3 .

[0149] (AB) Images of embryonic development of primed ESC-injected (A) and 5iLAF ESC-injected (B) embryos at the indicated time points during delayed in vitro culture. White lines indicate the ectoderm. Scale bar, 100 μm.

[0150] (C) Images and GFP signals of three groups of injected embryos at D17 in vitro extended culture. Scale bar, 100 μm. IVC, in vitro extended culture.

[0151] (D) Images of single-cell dissociation of 5iLAF ESC-injected and 4CL ESC-injected embryos at D17. Scale bar, 50 μm.

[0152] (E) GFP in D17 embryos injected with 5iLAF ESCs and 4CL ESCs + Quantitative analysis of cells. Data are presented as mean ± SEM. N = 3 biological replicates.

[0153] (F) Based on Figure 3 E UMAP visualization shows the expression of lineage-specific marker genes for EPI, E-PGC, VE / YE, TE (trophectoderm), and EXMC. The color gradient from gray to blue indicates a change in expression value from low to high.

[0154] (G) t-SNE visualization shows GFP in Smart-seq2 data of 4CL ESC-injected IVC chimeric embryos + (Left) and GFP - (Right) Cell types. Cells are identified as EPI, Gast, E-PGC, VE / YE, EXMC, and TE and their derivatives. Cells are colored and labeled by cell type.

[0155] (H) Correlation analysis of Smart-seq2 scRNA-seq data of our IVC mosaic embryos with reported in vivo embryos (E16 and E17) and IVC embryos (dpf16 and dpf17) based on the top 3000 variable genes. Average linkage hierarchical clustering with Spearman correlation is shown. E-PGC, early primordial germ cell; Gast, gastrula; EPI, epiblast; VE / YE (visceral / yolk sac endoderm), EXMC (extraembryonic mesenchyme), TE.

[0156] Fig.12 .monkey Detailed analysis of ESC contribution to chimeric monkey #9, related Figure 5 .

[0157] (A) Detection of GFP, SRY and ACTIN sequences in different tissue samples of chimeric monkey #9 by sgRNA DNA PCR.

[0158] (B) Identification of mitochondrial DNA SNPs and genomic DNA SNPs between oocyte donors and ESC donors in chimeric monkey #9. (CD) Quantification of the contribution of ESCs in chimeric monkey #9 by deep sequencing of mitochondrial DNA SNPs and genomic DNA SNPs. The names of the tissues or organs detected are abbreviated with the first three letters. Data are expressed as mean ± SEM. N = 3 biological replicates.

[0159] (E) GFP fluorescence shows GFP-tagged 4CL ESCs differentiated into BMCs of chimeric monkey #9. Scale bar, 20 μm.

[0160] (F) GFP in BMC of aborted chimeric monkey #9 + Quantification of cells. Data are presented as mean ± SEM.

[0161] (G) GFP fluorescence shows GFP-tagged 4CL ESCs differentiated into different tissues in chimeric monkey #9. Scale bar, 100 μm.

[0162] (H) GFP immunofluorescence shows GFP-tagged 4CL ESCs differentiated into the indicated tissues of chimeric monkey #9. GFP was detected using GFP antibody + Cells. Scale bar, 25 μm.

[0163] (I) Representative images showing GFP-tagged 4CL Chimerism of ESCs in the brain of chimeric monkey #9. Anti-NeuN antibody was used to detect mature neurons in the brain. Scale bar, 25 μm.

[0164] (J) GFP expression in different tissues of chimeric monkey #9 + Quantitative analysis of cell percentage. Data are presented as mean ± SEM.

[0165] (K) GFP in NeuN-positive cells in the brain of chimeric monkey #9 + Quantitative analysis of cell percentage. Data are presented as mean ± SEM.

[0166] Fig.13 Analysis of cell lineage differentiation in chimeric monkeys from live births (#10) and abortions (#9) by droplet-based sc / snRNA-seq Figure 6 .

[0167] (A) Left: UMAP visualization showing GFP expression in different cell types identified in PBMC DNBelab C4 data (droplet-based snRNA-seq) from live-born chimeric monkeys + (green) Distribution of cells. Middle: UMAP visualization showing different cell types identified in PBMCs. Cells are colored and labeled by cell type. Right: Bar graph showing GFP expression in different cell types identified in PBMCs + and GFP - The proportion of cells.

[0168] (B) Left: UMAP visualization showing GFP +(Green) Distribution of cells in different cell types identified in the adrenal DNBelabC4 data (droplet-based snRNA-seq) from live-born chimeric monkeys. Middle: UMAP visualization showing different cell types identified in the adrenal gland. Cells are colored and labeled by cell type. Right: Bar graph showing GFP expression in different cell types identified in the adrenal gland + and GFP - The proportion of cells.

[0169] (C) Left: UMAP visualization showing GFP expression in different cell types identified in the DNBelab C4 data (droplet-based snRNA-seq) from the liver of live-born chimeric monkey #10 + Distribution of cells. Middle: UMAP visualization showing the different cell types found in the liver. Cells are colored and labeled by cell type. Right: Bar graph showing GFP in different cell types in the liver -+ and the proportion of GFP cells.

[0170] (D) Left: UMAP visualization showing GFP expression in different cell types identified from DNBelab C4 data (droplet-based snRNA-seq) from the brain of aborted chimeric monkey #9 + Distribution of cells. Middle: UMAP visualization showing different cell types identified in the brain. Cells are colored and labeled by cell type. Right: Bar graph showing GFP + and GFP - The proportion of different cell types in the brain.

[0171] (E) Left: UMAP visualization showing GFP expression in different cell types identified in the cardiac DNBelab C4 data (droplet-based snRNA-seq) + Distribution of cells. Middle: UMAP visualization showing the different cell types found in the heart. Cells are colored and labeled by cell type. Right: Histogram showing GFP + and GFP - The proportion of different cell types in the heart.

[0172] Fig.14 .Testicular and placental tissue mosaicism and analysis of genomic integrity and epigenetic status of ESC-derived cells, related Figure 7 .

[0173] (A) GFP-tagged 4CL Contribution of ESCs in the testes of two chimeric monkeys (#9 and #10). Scale bar, 20 μm.

[0174] (B) GFP-tagged 4CL ESCs contribute to the testes of aborted chimeric monkey #9. GFP antibody was used in combination with VASA (germ cell marker) antibody. Nuclei were labeled with DAPI (blue). Scale bar, 25 μm.

[0175] (C) Testicular GFP of aborted chimeric monkey #9 + Cells as a percentage of total cells and VASAS + Cell ratio. Data are expressed as mean ± SEM.

[0176] (D) GFP-tagged 4CL Contribution of ESCs to the placenta of chimeric monkey #10. Scale bar, 20 μm.

[0177] (E) GFP-tagged 4CL Contribution of ESCs to the placenta of chimeric monkey #10 and wild-type monkeys. GFP antibody was used in combination with MCT4 (a marker for spongiotrophoblast cells) and Proliferin (a marker for trophoblast giant cells). Scale bar, 25 μm.

[0178] (F) FACS analysis of GFP in placentas of #9, #10 chimeric monkeys and wild-type controls + The percentage of nuclei.

[0179] (G) PCR detection of GFP sequences in genomic DNA of placentas from #9, #10 chimeric monkeys and wild type monkeys, with actin as a control.

[0180] (H) UMAP visualization showing the distribution of different cell types in the placenta in chimeric monkey #9 Smart-seq2 data.

[0181] (I) Bubble plots showing the expression frequency and average expression of placental cell-specific marker genes in the chimeric monkey #9 Smart-seq2 data.

[0182] (J) Bar graph showing the percentage of different cell types in the placenta in the chimeric monkey #9 Smart-seq2 data.

[0183] (K)GFP - and GFP + Comparison of DNA methylation levels in different genomic regions in BMCs.

[0184] (L) Pie chart shows GFP - BMCs and GFP + BMCs - Distribution of hypermethylated regions (methylation difference >= 0.2, FDR < 0.05) across the annotated genomic locations. DMR, differentially methylated region.

[0185] (M)GFP +GO analysis of high DMR-enriched genes in BMCs. DETAILED DESCRIPTION

[0186] The inventors have unexpectedly developed a method for preparing chimeric embryos of non-human primates in vitro through extensive and in-depth research, a large number of screenings and attempts, and the chimeric embryos or chimeric non-human primates obtained by the method of the present invention have the characteristics of a high chimerism ratio, and the method of the present invention can significantly increase the contribution rate of immature stem cells, for example, the contribution rate of immature stem cells is as high as 21%-92%. On this basis, the inventors have completed the present invention.

[0187] the term

[0188] Primed pluripotent stem cells (PSC)

[0189] As used herein, "primed pluripotent stem cells (PSCs)" refer to cells that are capable of unlimited proliferation, self-renewal, and multidirectional differentiation in vitro, and have the potential to differentiate into three germ layers in vitro, and their developmental period is similar to that of ectoderm cells after implantation.

[0190] Immature stem cells

[0191] As used herein, "naive stem cells" refer to cells that not only have the characteristics of primed pluripotent stem cells, but also have the ability to form embryonic chimeras, and their developmental stage is similar to that of pre-implantation epiblast cells.

[0192] Mosaic embryos

[0193] As used herein, "chimeric embryo" refers to an embryo that is formed by injecting exogenous cells into the embryo using the embryo injection method so that the cells develop together, that is, an embryo that contains two or more genomes.

[0194] After embryonic stem cells are injected into recipient embryos, through microscopic fluorescence observation and fluorescence staining, obvious GFP-positive signals can be seen in chimeric embryos (such as Figure 2 C, 2E, 2F), all of which indicate the high survival rate of injected chimeric embryos and ESCs. The chimeric embryos injected with 4CL and 5iLAF stem cells were cultured in vitro for 17 days, and it was found that the injected embryonic stem cells could still survive efficiently. Single-cell sequencing proved that monkey embryonic stem cells could develop synchronously with recipient embryonic cells and contribute to different cell lineages of the 17-day monkey embryo ( Figure 3 ).

[0195] Among the 10 monkeys born or aborted, embryonic stem cell chimerism was detected in one monkey that survived and one monkey that aborted. Using a series of rigorous chimera analysis procedures including PCR amplification, microsatellite paternity testing, genomic SNP deep sequencing, flow cytometry detection, green fluorescent protein detection, and immunofluorescence detection, it was found that the contribution of embryonic stem cells in monkeys that survived was as high as about 70%, and the contribution of embryonic stem cells in monkeys that aborted was about 20%. Single-cell transcriptome sequencing also further proved that the injected embryonic stem cells can be synchronously differentiated with the recipient embryonic cells into various different cell lineages of the born individual monkeys ( Figure 5 and Figure 6 ).

[0196] Currently, research on non-human primates is very slow. In previous studies on non-human primates, chimeras were obtained in aborted fetuses and offspring, but the contribution of donor cells in the chimeric tissues was very low, only 0.1-4.5%. The present invention improves the viability of injected ESCs and optimizes the culture system of chimeric embryos to obtain chimeras with a chimerism ratio of up to 90%, paving the way for future transgenic NHPs using gene-edited PSCs.

[0197] Method for culturing primed stem cells or immature stem cells

[0198] The starting cells of the primed stem cells or naive stem cells of the present invention are primed pluripotent stem cells (PSCs), which are cultured under the first culture conditions to obtain primed stem cells or naive stem cells, wherein the first culture conditions include a first culture medium, and the first culture medium is selected from the following group: 4CL, 5iLAF, PXGL, RSeT, LCDM, primed, or a combination thereof.

[0199] In a preferred embodiment, 4CL medium is commercially available (for specific components, see the general method section of the present invention).

[0200] In a preferred embodiment, 5iLAF medium is commercially available (for specific components, see the general method section of the present invention).

[0201] In a preferred embodiment, PXGL medium is commercially available (for specific ingredients, see the general method section of the present invention).

[0202] In a preferred embodiment, RseT culture medium is commercially available (purchased from stem cell company).

[0203] In a preferred embodiment, LCDM medium is commercially available (for specific components, see the general method section of the present invention).

[0204] Methods for culturing chimeric embryos

[0205] The present invention first obtains a non-human primate embryo containing primed stem cells or naive stem cells, and cultures the non-human primate embryo containing primed stem cells or naive stem cells under a second culture condition to obtain a chimeric embryo, wherein the second culture condition includes a mixed culture medium in which a second culture medium and a third culture medium are mixed in a volume ratio of 1:1, the second culture medium is selected from the following group: primed, 4CL, 5iLAF, and the third culture medium includes HECM9.

[0206] Method for preparing chimeric embryos in vitro

[0207] The present invention provides an in vitro method for preparing a chimeric embryo of a non-human primate, comprising the steps of:

[0208] (i) culturing primed pluripotent stem cells (PSCs) under a first culture condition to obtain flat stem cells or naive stem cells, wherein the first culture condition comprises a first culture medium selected from the group consisting of 4CL, 5iLAF, PXGL, RseT, LCDM, primed, or a combination thereof;

[0209] (ii) contacting the flat stem cells (cell types obtained when the first culture medium is primed culture medium) or naive stem cells (cell types obtained when the first culture medium is 4CL, 5iLAF, PXGL, RseT, LCDM culture medium) obtained in step (i) with non-human primate embryos to obtain non-human primate chimeric embryos containing naive stem cells, and culturing the non-human primate chimeric embryos containing naive stem cells under a second culture condition to obtain blastocyst stage chimeric embryos, wherein the second culture condition comprises a mixed culture medium in which a second culture medium and a third culture medium are mixed in a volume ratio of 1:0.5-1.5 (preferably 1:1), the second culture medium is selected from the group consisting of Primed, 4CL, 5iLAF, or a combination thereof, and the third culture medium comprises HECM9.

[0210] Theoretically, the culture medium for chimeric embryos that we optimized is suitable for other flat stem cells or immature stem cells, including the existing 4CL, 5iLAF, PXGL, RseT, LCDM, primed, etc. By mixing the culture medium for embryonic stem cells and the culture medium for embryo culture at a volume ratio of 1:0.5-1.5 (preferably 1:1), the normal development of the embryo can be guaranteed to a certain extent on the basis of maintaining the survival of embryonic stem cells. The present invention discovered for the first time that when the volume ratio of the culture medium for embryonic stem cells and the culture medium for embryo culture is 1:0.5-1.5 (preferably 1:1), the normal development of the embryo can be guaranteed on the basis of maintaining the survival of embryonic stem cells to a large extent, and a higher chimerism ratio can be obtained. 4CL, 5iLAF, PXGL, RseT, LCDM, primed are just examples of specific embryonic stem cell culture media cited in the present invention, and HECM9 is just an example of specific embryo culture medium cited in the present invention. Mammalian pluripotent stem cells have preimplantation embryonic cell-like pluripotency This was demonstrated by complementation of early embryos with homologous cells to produce chimeric animals. Although pluripotency has been well demonstrated in rodents, chimerism is poor in other species, including non-human primates, because donor cells cannot match the developmental state of the host embryo. We investigated various culture conditions for embryonic stem cells and optimized the culture method for chimeric embryos. This method generated one aborted chimeric fetus and one living chimeric monkey with high donor cell contribution. Rigorous characterization demonstrated that in the chimeric monkeys, donor cells efficiently contributed (up to 90%) to various tissues, including gonads and placenta. Our results have implications for primates Research on pluripotency and genetic engineering of non-human primates is of great significance.

[0211] The main advantages of the present invention include:

[0212] (1) The present invention provides for the first time a method for preparing chimeric embryos of non-human primates in vitro. The chimeric embryos or chimeric non-human primates obtained by the method of the present invention have the characteristics of a high chimerism ratio, and the method of the present invention can significantly increase the contribution rate of immature stem cells, for example, the contribution rate of immature stem cells is as high as 21%-92%.

[0213] (2) This invention is the first to systematically test monkey We investigated various culture conditions for embryonic stem cells and optimized the culture method for chimeric embryos. This method generated one aborted chimeric fetus and one living chimeric monkey with high donor cell contribution. Rigorous characterization demonstrated that in the chimeric monkeys, donor cells efficiently contributed (up to 90%) to various tissues, including gonads and placenta. Our results have implications for primates Research on pluripotency and genetic engineering of non-human primates is of great significance.

[0214] (3) The present invention systematically tested the effects of various human PSC culture media on crab-eating macaque ESCs and optimized the injection protocol for early monkey embryos and the in vitro culture of injected embryos. We found that 4CL improved the naive pluripotency of monkeys and enabled monkey donor cells to survive in blastocysts. In addition, we have established a complete set of systematic analysis methods, including GFP-positive cell counting, single nucleotide polymorphism (SNP) analysis of mitochondrial and genomic DNA, and single-cell transcriptome analysis. Using these methods, we demonstrated that monkey ESCs have a high degree of chimerism during delayed in vitro culture and in vivo pregnancy. Donor ESCs showed a high degree of chimerism ranging from 20% to 90% in living chimeric offspring tissues (including gonads and placenta). Our results show that by interacting with homologous Early embryonic complementation of PSCs can generate chimeras with high ESC contribution in NHPs, paving the way for the future generation of transgenic NHPs using gene-edited PSCs.

[0215] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0216] Unless otherwise stated, the materials and reagents in the examples are commercially available products.

[0217] General methods and materials

[0218] Key Resources Table

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226] Experimental model and subject details

[0227] Ethical Statement

[0228] The use and care of animals followed the guidelines of the Animal Advisory Committee of the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences. The ethics application for “Study on pluripotency of non-human primate embryonic stem cells” (#CEBSIT-2020007R01) was approved by the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences. This study was conducted in accordance with the guidelines of the International Society for Stem Cell Research (ISSCR). 75 Euthanasia is considered only after an animal develops life-threatening symptoms such as respiratory failure, persistent bleeding, septic shock, hypothermia, persistent self-mutilation, and infection with pathogenic microorganisms. In this study, after careful consideration, the veterinarian decided to euthanize chimeric monkey #10 due to respiratory failure and hypothermia.

[0229] monkey

[0230] Healthy cynomolgus monkeys (Macaca fascicularis) aged 5 to 12 years were selected for this study. All animals were housed in a sunlit room in the non-human primate facility of the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences. The cynomolgus monkeys used in this study were from Southeast Asia. The monkeys were fed a commercial diet and seasonal fruits. There was no history of any other experiments on the monkeys except for assisted reproduction experiments.

[0231] mouse

[0232] Healthy mice were housed at the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences. Mice were fed a commercial diet and maintained at 22°C-26°C with 12 h light / 12 h dark. The animals had no history of subject (drug or experiment). Teratoma experiments were performed on 6-8 week old NOD / SCID mice. Mouse embryonic fibroblasts (MEFs) were obtained from ICR fetal mice at E13.5.

[0233] Feeder layer and fibroblast culture conditions

[0234] MEFs and human fibroblasts HFFs were cultured in DMEM (Gibco, 11995073) medium supplemented with 15% FBS (BI, 04-001-1ACS), 1mM Glutamax (Gibco, 35050061), 1% penicillin-streptomycin (Gibco, 15140122), 1% nonessential amino acids (Gibco, 11140050), and monkey fibroblasts were cultured in the same medium. All cell lines were mycoplasma negative and stored in an incubator at 37°C and 5% CO2.

[0235] Generation of Cynomolgus Monkey Primed ESCs

[0236] After the zona pellucida was removed in Tyrode's solution (Sigma, T1788), the blastocysts were transferred to HFFs inactivated with mitomycin C (Sigma, M0503-5) and replaced with KOSM medium. KOSM medium consists of DMEM / F12 (Gibco, 10565018) supplemented with 20% KOSR (Gibco, A3181502), 40ng / ml bFGF (Peprotech, 100-18B), 1% nonessential amino acids, 1% Penicillin-Streptomycin, 0.1mMβ-mercaptoethanol (Sigma, M3148), 1mM Glutamax and 10μM ROCK inhibitor Y27632 (Axon, 1683). In general, cells with ESC-like morphology grow out of the ICM within 3-5 days. Then, the cells were digested into small colonies with 2 mg / ml collagenase IV (Invitrogen, 17104019) at 37°C for 5-10 minutes and seeded onto new HFFs. Primed ESCs were passaged every 3-4 days, and the culture medium was changed every day. We obtained 5 primed ESCs lines from 5 monkey blastocysts, and these cell lines were routinely cultured in E8+KOSR medium (mTeSR-E8 medium TM ; STEMCELL Technologies, 05990), 5% KOSR and 2.5 μM IWR (Sigma, I0161, also known as E8+K]. All cell lines were mycoplasma negative and maintained in an incubator at 37°C with 5% CO2.

[0237] monkey Generation of ESCs

[0238] Primed ESCs were digested into single cells at 1000-1500 cells / cm 2 The cells were seeded at a density of 10 μM on the ICR feeder layer in E8+K medium supplemented with 10 μM Y27632 ROCK inhibitor (purchased from Axon, Cat#: 1683). After 24 or 48 hours of seeding, the medium was replaced with different Culture medium.

[0239] 5iLAF

[0240] 5iLAF The generation process of ESCs is similar to that of human PSCs. Briefly, primed ESCs were cultured at 2x10 5 The cells were seeded onto the ICR feeder layer at a density of 10 cells / mL in E8+K medium supplemented with 10 μM Y27632. After 48 hours, the medium was replaced with 5iLAF medium, which consisted of a 1:1 mixture of DMEM / F12 (Gibco, 10565018) and Neurobasal (Gibco, 21103049), supplemented with 1% N2 supplement (Gibco, 17504044), 1% B27 supplement (Gibco, 17502048), 1% nonessential amino acids, 1mM Glutamax, 1% Penicillin-Streptomycin, 0.1mM β-mercaptoethanol, 50μg / ml BSA (Gibco, A1933), 1μM PD0325901 (Axon, 1408), 1μM IM-12 (Selleck, S7566), 0.5μM SB590885 (Selleck, S2220), 1μM PH-4-023 (Selleck, S7565), 10μM Y-27632, 20ng / ml activin A (Peprotech, 120-14E), 8ng / ml bFGF, 20ng / ml human LIF (PeproTech, AF-300-05-100) and 0.5% KOSR. During the next 8-10 days, fresh medium was replaced every day. It is normal for cells to show significant cell death and partial differentiation during transformation. During the process of cell transformation, adding fresh ICR (around day 6) will allow cells to proliferate better. At the beginning, the dome-shaped ESCs proliferate slowly, and significant proliferation is not seen until around P4-P5. Early passages can be passaged at high density to promote cell proliferation. During the passage process, passing the cells through a 40μm cell strainer helps to remove clumping cells and differentiated cells. 5iLAF cells were cultured at 37°C, 5% O2 and 5% CO2.

[0241] R

[0242] Transform and culture using commercial RSeT medium (Stem cell, cat#05975) ESCs, and follow previous studies in humans Briefly, primed ESCs were cultured at 2x10 5 The cells were seeded on ICR at a density of 100 μM in E8+K medium supplemented with 10 μM Y27632. After 24 hours, the medium was changed to RSeT and the medium was changed every day.

[0243] 4CL

[0244] 4CL medium conversion ESCs follow the previous 4CL medium was composed of a 1:1 mixture of advanced DMEM / F12 (Gibco, 12634010) and neurobasal medium, supplemented with 1% N2supplement, 1% B27 supplement, 1% non-essential amino acids, 1mM Glutamax, 1% Penicillin-Streptomycin, 1% sodium-pyruvate, 50μg / ml L-ascorbic acid (Sigma, A4030), 0.2% (v / v) Geltrex (Gibco, A1413302), 20ng / ml activin A, 20ng / ml human LIF, 1μM PD0325901, 5nM TSA (Sigma, V900931), 10nM DZNep (Selleck, S7120) and 5μM IWR-1. The medium was refreshed daily and cells were passaged as single cells every 3-4 days; 5 μM Y-27632 was optionally added during the first 24 h. 4CL cells were cultured at 37°C, 5% O2 and 5% CO2.

[0245] PXGL

[0246] PXGL The conversion and maintenance process of ESCs is similar to that of human PSCs. Briefly, primed ESCs were cultured at 2 × 10 5 The cells were seeded onto the ICR feeder layer at a density of 10 μM Y27632 in E8+K medium. After 24 hours, the medium was changed to PXGL induction medium [N2B27, containing 1 μM PD0325901, 10 ng / ml human LIF and 1 mM VPA (STEMCELL technologies, 72292)], and the medium was changed every day. After 3 days, the cells were changed to PXGL medium (N2B27 supplemented with 1 μM PD0325901, 2 μM XAV939 (Sigma, X3004), 2 μM (Sigma, 365251) and 10 ng / ml human LIF), and the medium was changed every day. Due to massive cell death, poor proliferation and spontaneous differentiation around the 5th or 6th passage, the cells cultured in PXGL were ESCs were passaged at a 1:1 ratio at the beginning of transformation and were not used beyond these passages. PXGL cells were cultured at 37°C with 5% O and 5% CO2.

[0247] LCDM

[0248] The process of converting primed ESCs into expanded potential pluripotent stem cells using LCDM medium is similar to that of human PSCs. LCDM medium consists of a 1:1 mixture of DMEM / F12 and neurobasal medium, 0.5x N2 supplement, 0.5x B27 supplement, 1% non-essential amino acids, 1mM Glutamax, 1% Penicillin-Streptomycin, 1μΜ CHIR99021 (Sigma, SML1046), 5% KOSR, 10ng / ml human LIF, 2μM (S)-(+)-dimethindene maleate (Tocris, 1425) and 2μM minocycline hydrochloride (SantaCruz, sc-203339). PXGL cells were cultured at 37°C, 21% O and 5% CO2.

[0249] Ovulation and oocyte collection

[0250] Healthy female cynomolgus monkeys with regular menstrual cycles were selected for superovulation and oocyte collection. Starting from the third day of the menstrual cycle, 25 IU of recombinant human folliculin was injected intramuscularly twice a day for 7-8 consecutive days. On the 11th day, 1000 IU of human chorionic gonadotropin (hCG) was injected, and 36 hours later, oocytes were extracted from the follicles with a needle of 2-8 mm in diameter. The collected oocytes were cultured in hamster embryo culture medium 9 (HECM-9). The oocytes captured in the middle stage were selected for operation.

[0251] ICSI, culture and embryo transfer of monkey embryos

[0252] For monkey oocystic sperm injection (ICSI), the applicant's previously published method was followed, i.e., a single selected sperm was injected into the oocyst using a voltage-driven micromanipulator, and fertilization was confirmed by the presence of two pronuclei 6 hours later. The embryos were placed in pre-equilibrated HECM-9 and cultured at 37°C, 5% CO2. After the 8-cell stage, the embryos were transferred to HECM-9 supplemented with 5% FBS, and the culture medium was changed every other day until the embryos developed to the blastocyst stage. For embryo transfer, females with synchronized menstrual cycles and ovarian stigmas or fresh corpora lutea were used as surrogate mothers. The embryos (blastocyst stage) were transferred into the fallopian tubes of the surrogate by laparoscopic-assisted minimally invasive surgery.

[0253] Transfection and generation of GFP-labeled ESC cell lines

[0254] Lentiviral particles carrying the GFP gene were generated according to the manufacturer's instructions, and pCDH-CHEF1α-copGFP-P2A-Puro and pMD2.G and psPAX2 plasmids were co-transfected into HEK293T cells using Lipofectamine3000 (Invitrogen, L3000001). Briefly, HEK293T cells were cultured in basal medium (90% DMEM plus 10% FBS), and Lipofectamine 3000 reagent and plasmids were mixed in appropriate proportions in Opti-MEM medium (Gibco, 31985070). Then, the mixture was added to the cells in fresh medium. The medium containing lentivirus was collected twice at 48 hours and 72 hours after transfection. The medium containing lentivirus was centrifuged at the highest speed (12,000 rpm) overnight. TrypLE primed ESCs were digested into single cells and then seeded on a fresh feeder layer on a Geltrex-coated plate at a cell density of 5x10 4 cells / cm 2. 10μM ROCK inhibitor (Y27632) was added to E8+K medium. After 24 hours, the cells were incubated in fresh E8+K medium with lentiviral particles and polybrene for 24 hours. Then, GFP-transduced ESCs were selected with 2μg / ml puromycin, incubated for 24-48 hours, digested, and re-seeded on new HFFs in E8+KOSR medium supplemented with 10μM ROCK. Single cell clones appeared within 3-4 days. GFP-positive ESC clones were then picked, digested, and expanded in E8+K medium.

[0255] Mulberry injection culture

[0256] When fertilized eggs were collected, they were cultured in HECM9+5% FBS medium for 3-4 days at 37°C and 5% CO2. 15-20 monkey ESCs were injected into the morula using a 15μm needle. After injection, the chimeric embryos were cultured in HECM9+5% FBS for 72 hours (Scheme 1); cultured in 4CL medium for 72 hours (Scheme 2); cultured in a 1:1 mixture of 4CL and HECM9 for 24 hours after injection, and then cultured in HECM9 for 48 hours (Scheme 3); cultured in a 1:1 mixed medium of 4CL and HECM9 for 72 hours (Scheme 4); cultured in a 2:1 mixed medium of 4CL and HECM9 for 72 hours (Scheme 5); cultured in a 1:1 primed and HECM9 medium for 72 hours (Scheme 6); cultured in a 1:1 mixed medium of 5iLAF and HECM9 for 72 hours (Scheme 7). During the chimeric embryo period, the medium was supplemented with 5μM Y27632. Morulae were randomly assigned to different culture systems.

[0257] In vitro culture of cynomolgus monkey embryos

[0258] The in vitro culture of cynomolgus monkey embryos was carried out according to the previous protocol for mouse embryo culture, that is, after removing the zona pellucida of the embryos, they were cultured with pre-warmed IVC1 (IVC1,500 500μl / well) at 37°C for 30 minutes and added to a 4-well plate. For the first 3 days, the 4-well plate containing the embryos was not moved to allow all the embryos to adhere to the bottom of the culture dish. After that, IVC1 was replaced with fresh IVC2 medium every 2 days, and monkey embryos were harvested at the designated stages. We measured the development rate of in vitro cultured embryos by detecting embryo attachment (~dpf11), the appearance of obvious ectoderm (~dpf13), and the appearance of the amniotic cavity and yolk sac cavity (dpf15-dpf17). Embryos that did not grow or no ectoderm was observed were considered developmental failures. IVC1 consists of DMEM / F12 supplemented with 20% heat-inactivated serum, as well as 2mM Glutamax, penicillin (25units / ml) / streptomycin (25μg / ml), 1×ITS-X, 8nMβ-estradiol, 200ng / ml progesterone and 25μM N-acetyl-l-cysteine. IVC2 consists of DMEM / F12 supplemented with 30% KOSR, 2mM Glutamax, penicillin (25units / ml) / streptomycin (25μg / ml), 1×ITS-X, 8nMβ-estradiol, 200ng / ml progesterone and 25μM N-acetyl-l-cysteine.

[0259] Teratoma formation

[0260] Monkey ESCs clones were digested into single cells using TrypLE. Cells were suspended with a mixture of 100 μl pre-cooled culture medium and 100 μl Matrigel, approximately 1 million single cells per cell line, and injected subcutaneously into the flank of 6-8 week old NOD / SCID mice. Teratomas are generally formed in 8-12 weeks. Mice were euthanized before the teratoma exceeded 3 cm in diameter. Teratomas were fixed and isolated with 4% paraformaldehyde (PFA), embedded in paraffin, sliced, and histologically characterized with H&E staining to detect the presence of representative tissues of all three germ layers.

[0261] Immunofluorescence staining

[0262] Monkey embryos cultured in vitro were collected, fixed with 4% (v / v) PFA for 20 minutes at room temperature, and then washed with PBS. A portion of the chimeric monkey tissue was fixed and a portion was frozen in a -80°C refrigerator for frozen sections. The fixed embryos or frozen sections were permeabilized with 1% Triton-100, blocked with 3% BSA, and then incubated with primary antibodies at 4°C overnight. After washing at least three times, fluorescently conjugated secondary antibodies and 4', 6-diamino-2-phenylindole (DAPI) were incubated with slides in the dark at room temperature for 2 hours. The following antibodies were used for immunostaining: OCT-3 / 4 (Santa Cruz, sc-5279; Cell Signaling Technology, 2890), NANOG (Abcam, ab173368), GFP (Abcam, ab1218, ab290), KLF17 (ATLASAntibodies, HPA024629), KLF4 (Santa Cruz, sc-166238), TFE3 (Sigma, HPA023881), VASA (Abcam, ab13840), NeuN (Abcam, ab177487), KRT7 (Dako, 20064396), MCT4 (Merck Millipore, ab3314P) and proliferin (Santa Cruz, sc-271891). DAPI was used to stain the cell nucleus. Images were taken using a confocal microscope, and GFP was randomly selected from different tissue fields. + Cells were analyzed (A0331 Olympus FV3000).

[0263] Alkaline phosphatase (AP) staining

[0264] AP staining was performed using an alkaline phosphatase detection kit (Beyotime, C3206) according to the manufacturer's protocol. ESCs were collected at 2×10 4 The cells were seeded at a density of 100 cells in a 24-well plate with a feeder layer and cultured in their respective culture media for 4 days. For AP staining, the cells were washed twice with PBS, fixed with 4% PFA for 10 minutes, and incubated with BCIP / NBT working solution at room temperature for 5-30 minutes. The PBS was washed and the reaction was stopped with ddHO2. The fields of view of different AP-positive clones were randomly selected for analysis under different conditions, with 3 technical replicates each.

[0265] Mitochondrial metabolism assay

[0266] For real-time visualization of mitochondrial imaging, primed or ESCs were incubated in medium containing 50 nM MitoTrackerGreen FM (Cell Signaling Technology) and 100 nM TMRE (Life Technologies) at 37°C for 15 min, and then the staining medium was replaced with the relevant pre-warmed medium and analyzed under a microscope.

[0267] PCR analysis to evaluate chimerism

[0268] The genomic DNA of different tissues of the chimera was isolated using the QIAamp kit (51304, QIAGEN). 20 ng of DNA was taken from each sample each time, and 4 technical replicates were performed. 35 PCR cycles were performed under the conditions of 95°C for 5 minutes, 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds, and then 72°C for 5 minutes. Three independent DNA samples were used for PCR detection for each tissue.

[0269] Real-time quantitative PCR

[0270] Total RNA was isolated from whole culture medium using TRIZOL reagent (Invitrogen) according to the manufacturer's instructions. TM RNA was converted into cDNA using RT reagent Kit (RR047A, TaKaRa). Premix Ex Taq TM The reaction was performed with LightCycler II (RR820A, TaKaRa) and run on a LightCycler 480 Instrument II (Roche). Primers are summarized in Table S6.

[0271] Karyotype analysis

[0272] G-banded karyotyping was performed according to standard protocols. Briefly, colchicine (100 ng / ml) was added to 70-80% ESC culture medium for 1.5 hours, then the medium was removed, the cells were washed with PBS, and the ESCs were digested with TrypLE for 1-2 minutes, centrifuged at 1000 rpm for 5 minutes. Subsequently, 10 ml of hypotonic solution was pre-warmed (37°C), gently mixed with single cells, and placed in a 37°C water bath for 30 minutes. 10 ml of cold fixative was added, stored at room temperature for 10 minutes, and centrifuged at 1000 rpm for 5 minutes. This fixation step was repeated 3 times. Finally, the cell suspension was dropped onto a slide. The slide was then air-dried and stained with Giemsa. The samples were analyzed for G-banded karyotype using a 100x magnifying glass (MetaSystems Ikaros). 20 metaphase spreads randomly selected for each sample were analyzed.

[0273] Short tandem repeat (STR) analysis

[0274] Blood cells and tissue samples were collected from monkeys for DNA extraction. Two microsatellites were selected for analysis (STRs: D6S2741 and D9S9216) for the contribution of ESCs in chimeric monkeys. PCR amplification was performed using locus-specific primers containing a fluorescent dye (FAM). The FAM-labeled STR amplification was diluted and mixed with the internal standard ROX500 and deionized formamide, and electrophoresed on an ABIPRISM 3730 genetic analyzer to obtain raw data. The raw data were analyzed using Gene Marker 2.2.0.

[0275] Single Nucleotide Polymorphism (SNP) Analysis

[0276] For SNP analysis, tissues were collected from monkeys to extract DNA. In general, the target region was amplified from genomic DNA using specific primers in the first round of PCR. The PCR program was 35 cycles of 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 30 seconds, followed by an extension at 72°C for 5 minutes. For chimeric monkeys #9 and #10, mitochondrial DNA (mtDNA, #9 had 2 SNPs and #10 had 3 SNPs) and genomic DNA (gDNA, #9 had 1 SNP and #10 had 3 SNPs) were used. Sanger sequencing of different tissues identified the target region between host embryos and injected ESCs. In the next round of PCR, barcoded primers were used to add forward and reverse barcodes to the ends of the PCR products. Amplicons were purified using the Monarch PCR & DNA Cleanup Kit (NewEngland Biolabs) and quantified using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific). Equal amounts of PCR products were sent to the State Key Laboratory of Rice Biology for sequencing using the Hi-TOM platform (China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou) (https: / / doi.org / 10.1007 / s11427-018-9402-9). Three samples were collected from each tissue of chimeric monkeys #9 and #10 for DNA preparation and PCR amplification.

[0277] RNA-seq data analysis

[0278] RNA-seq was performed with two independent samples. All replicates were successful and no data were excluded. Adapters and low-quality reads were removed using fastp (v0.21.0) (https: / / github.com / OpenGene / fastp) with default options. The trimmed data were aligned to the Macaca_fascicularis_5.0 genome reference using STAR (v2.7.4a) with settings ‘--outSAMtype BAM Unsorted, --quantMode TranscriptomeSAM, --outSAMheader HD\@HD VN:1.4SO:unsorted’. Gene counts were calculated using RSEM (v1.2.18) with setting ‘--paired-end’. After removing genes with zero counts in all samples, DESeq2 (v1.24) in R (v3.6.0) was applied. Differential gene expression analysis was performed between ESC data and primed ESC data. Genes with log2(fold change)>0 and adjusted P value<0.01 were defined as upregulated DEGs. GO enrichment was then performed using ClusterProfiler (v3.6.0), and bar graphs were generated using ggplot2 (v3.3.3). Heatmaps were generated using pheatmap (v1.0.12) in R (v3.6.0) using log 10 (TPM+1) generated.

[0279] Preparation of mononuclear suspension from chimeric monkey tissue

[0280] Cell mononuclear isolation was performed as described in the previous method for human cell mononuclear isolation, that is, the frozen chimeric monkey tissue was minced and transferred to a 1 mL Dounce homogenizer (TIANDZ), which contained 1 mL of pre-cooled homogenization buffer A, which contained 250 mM sucrose (Ambion), 10 mg / ml BSA (Ambion), 5 mM MgCl2 (Ambion), 0.12 U / μl RNasein (Promega, N2115) and 1× complete protease inhibitor cocktail (Roche, 11697498001). The tissue block was incubated with pre-cooled homogenization buffer A in an ice box for 3-5 minutes, and the mixture was pounded 25-50 times with a pestle to make it basically homogenized, and then the mixture was filtered through a 100 μm cell strainer into a 1.5 ml tube (Eppendorf). The efficiency of cell nucleus isolation was checked with trypan blue. After most of the cell nuclei were observed under a microscope, the homogenization was stopped. Afterwards, the mixture was further filtered through a 40 μm filter into a 1.5 ml tube and centrifuged at 500 g for 5 min at 4 °C to pellet the nuclei. The nuclei were then resuspended in PBS with nuclear buffer containing 0.04% BSA for snRNA-seq or Smart-seq2 library construction, FACS sorting, and subsequent DNA extraction.

[0281] FACS

[0282] Chimeric monkey BMC and PBMC were taken and diluted to a certain density (~10^6 cells / ml) with PBS supplemented with 0.1% BSA for flow cytometric analysis. After centrifugation, BMC and PBMC were resuspended in 90% fetal bovine serum supplemented with 10% DMSO and stored in liquid nitrogen. Before FACS, cells or nuclei from different tissues were resuspended in PBS with a buffer containing 0.04% BSA and subjected to flow cytometry. Sorted GFP -+ and GFP cells or nuclei were used for subsequent Smart-seq2, scWGS, DNA extraction, and WGBS analysis.

[0283] Bisulfite whole genome sequencing (WGBS)

[0284] Library construction

[0285] Genomic DNA was extracted from monkey ESCs and libraries were constructed by E-GENE. The Illumina Pair-End protocol was followed with some modifications. In brief, for WGBS, genomic DNA with unmethylated lambda DNA was fragmented using a Bioruptor Pico ultrasonic device. After fragmentation, the purified random fragment DNA was repaired and blunted. The blunt DNA fragments were subsequently 3' adenylated using Klenow fragment (3'-5' exon-) and then ligated to adapters synthesized with 5'-methylcytosine instead of cytosine using T4 DNA ligase. Afterwards, the ZYMO EZ DNA Methylation Gold Kit was used. TM Unmethylated cytosine was converted to uracil according to the manufacturer's instructions. Finally, PCR was performed with 10 amplification cycles. Purified products were analyzed by the Bioanalyzer Analysis System (Agilent, Santa Clara, USA) and quantified by RT-qPCR before analysis with an Illumina Nova sequencer. For low-input DNA whole-genome bisulfite sequencing, genomic DNA was bisulfite converted using the ZYMOEZ DNA Methylation Gold Kit (ZYMO) according to the manufacturer's instructions as described previously. 81 Bisulfite extension was performed to synthesize the first and second strands. The final library was PCR amplified using KAPA HiFi HotStart DNA polymerase (KAPA Biosystems). The final library was analyzed by Agilent Bioanalyzer (Agilent Technologies), quantified by real-time PCR, and then sequenced by Illumina Nova 6000.

[0286] Raw data filtering

[0287] Low-quality bases and adapter sequences were trimmed using trimmomatic. 82 The parameter settings used are "SLIDINGWINDOW:5:15HEADCROP:3AVGQUAL:15LEADING:5TRAILING:5MINLEN:80(low-input:35)".

[0288] Sequence alignment and detection of cytosine methylation levels

[0289] The cleaned reads were mapped back to the reference genome using BSMAP version 2.90 software. 83The parameter settings used were “-v0.08 -g 1-p48”. Methylation ratios were extracted from BSMAP output (SAM) using a Python script (methratio.py) distributed with the BSMAP package. Briefly, methylation levels were calculated based on the percentage of methylated cytosine (mC) in the whole genome, with site methylation level = 100 × [number of methylated cytosine (mC) sequences / total number of valid sequences]. Global average methylation levels were displayed by ViolinPlot using the R software package. The average methylation level of each gene element was calculated using the genomic gff file and visualized using the R software package barplot. Public data were reanalyzed from GSE60166. 38

[0290] Differentially methylated region (DMR) detection

[0291] DMRs were detected in de novo mode in CpG sites with at least 10-fold coverage using the assay. 84 The parameters used were set to "--mincpgs 3-minMethDiff 0.2--mode 1-mtc 2". The detected DMRs were then filtered according to the following criteria: (1) the Q value must be less than 0.05; (2) the methylation level difference must be greater than 0.2; (3) the number of CpGs contained in the DMR must be greater than 5; (4) the length of the DMR must be greater than 50 bp. The relevant elements and genes of DMRs were located by genomic GFP files. Genomic GO annotation was performed using emapper based on the EGGNOG database. GO enrichment analysis of the above-mentioned related hypermethylated genes was then performed using Allenricher (v1.0).

[0292] scRNA-seq sequencing and snRNA-seq library preparation

[0293] Single cell / nucleus libraries were constructed using the DNBelab C4 series single cell library preparation kit (MGI, 1000021082). In brief, single cell / nucleus suspensions were used for droplet encapsulation, emulsion disruption, mRNA capture bead collection, reverse transcription, cDNA amplification, and purification to generate barcode libraries. Indexed sequencing libraries were constructed according to the manufacturer's protocol. Sequencing library concentrations were quantified using the Qubit ssDNA assay kit (Thermo Fisher Scientific, Q10212). The resulting libraries were sequenced using the DIPSEQ T1 sequencer of the China National Gene Bank (CNGB). Monkey ESCs, frozen PBMCs, and BMCs cultured under primed or different naive conditions were subjected to scRNA-seq. Frozen chimeric monkey brain, liver, heart, and adrenal tissues were subjected to snRNA-seq.

[0294] Smart-seq2 library preparation

[0295] Sequencing analysis was performed using Smart-seq2 as described previously for human single cells. Briefly, single cells / nuclei were hand-picked or sorted by FACS into 96-well plates containing lysis buffer, and cell / nucleus lysis, reverse transcription, and cDNA amplification were performed. The resulting cDNA library was further labeled with a unique barcode and sequenced using the BGISEQ-500 sequencer at CNGB. Cells and bone marrow from chimeric IVC embryos injected with 4CL-ESCs on day 18 and nuclei from frozen brain, testis, and placental tissues from live chimeric monkey #10 were sequenced by Smart-seq2.

[0296] scRNA-seq and snRNA-seq data processing

[0297] Raw data processing

[0298] The results of sequencing are in the CNGB database. The raw sequencing reads were filtered and demultiplexed using PISA (v0.2) (https: / / github.com / shiquan / PISA). For DNBelab C4 data of ESC and tissue samples, the reads were aligned to the Macaca_fascicularis_5.0 genome reference using STAR (v2.7.4a)73 and sorted by sambamba (v0.7.0). For scRNA-seq, the reads were aligned to the exons of the mRNA. For snRNA-seq, the reads were aligned to a customized "messenger RNA" reference, which was used to align count reads to introns and exons. Gene expression in snRNA-seq is quantified by including exon and intron reads. Next, a cell / nucleus and gene UMI count matrix was generated by PISA. For Smart-seq2 data, reads were aligned to the Macaca_fascicularis_5.0 genome reference using STAR with settings of "--soloType SmartSeq, --soloUMIdedup Exact, --soloStrand Unranded, and --soloFeatures Gene". The remaining parameters were also applicable to single nucleus Smart-seq2, except that the parameter "--soloFeatures Gene" needed to be changed to "--soloPeatures Gene GeneFull". To identify GFP in chimeric tissues, the STAR indexes used in this paper were generated from macaque FASTA and GTF files with GFP sequences added.

[0299] Quality Control

[0300] The count matrix was processed using the Seurat software package (version 3.1.4). Cells / nuclei with less than 15% mitochondrial reads were retained for the following analyses. For DNBelab C4 data, genes expressed in less than three cells / nuclei were filtered out, and cells / nuclei with less than 500 detected genes and less than 3000 UMIs were excluded. Doublet removal was performed using DoubletFinder with default parameters. For Smart-seq2 data, cells from IVC samples with at least 3000 genes and 1500000 counts and cells from chimeric tissue samples (bone marrow, brain, testis, and placenta) with at least 300000 counts were used for further transcriptome analysis. Reanalysis of GSE74767 31The public data of GSE74767 and GSE130114 were obtained by analyzing the original data according to the literature method. The gene expression matrix of GSE130114 was downloaded from the database. The processing of these two datasets was the same as our Smart-seq2 data.

[0301] DNBelab C4 data analysis

[0302] For the DNBelab C4 data of embryonic stem cells, we first integrated the single-cell data of ESCs with the single-cell data archived in vivo (GSE74767) using Seurat and the FindIntegrationAnchors function. Two objects created from the ESC data and the in vivo embryo data were merged and then normalized with the SCTransform function. 3000 highly variably expressed genes were selected and FindIntegrationAnchors was performed with default parameters. Finally, IntegratedData was applied to integrate the two datasets with default parameters. After the scaling process, principal component analysis (PCA) was performed to select the principal components. Then, UMAP was used for visualization in 2D space. Cells were clustered using Louvain and plots were generated using DimPlot. Correlation analysis was based on the matrix of 3000 highly variable genes in the integration object and the “cor()” function in R (v3.6.2). The correlation matrix was then used as input to pheatmap (v1.0.12) to generate heatmaps. For the DNBelab C4 data of chimeric samples and wild-type monkey testes, count matrices were processed using Seurat, and NormalizeData, FindVariableGenes, and ScaleData functions were used in sequence with default parameters. Dimensionality reduction began with PCA of 3000 significantly variable genes, and the number of principal components selected as input for UMAP visualization and clustering depended on the importance of the embedding. Cells were clustered using Louvain, and a resolution of 0.8 to 2 was used in different samples. Cells with GFP readouts were considered positive, while other cells were considered negative. Plots were generated using DimPlot and DotPlot. Bar graphs were generated using ggplot2 (v3.3.3) in R (v3.6.0). Clusters for each sample were annotated through an extensive literature review and a search for cluster-specific gene expression patterns.

[0303] Smart-seq2 data analysis

[0304] For Smart-seq2 data, cells with one or more GFP reads were considered positive. We integrated GSE74767 (E16 and E17), GSE130114 (dpf16 and dpf17), and our IVC embryo data. The FindIntegrationAnchors function was performed for batch correction as described in the published in vitro and in vivo data of normal monkey embryos (in vivo data: A developmental coordinate of pluripotency among mice, monkeys and humans. In vitro data: In vitro culture of cynomolgus monkey embryos beyond early gastrulation). The integrated data analysis was created by running the SCTransform, FindIntegrationAnchors, and IntegratedData functions in sequence. It is worth noting that some parameters in the above functions need to be adjusted according to the cell numbers in the public data. UMAP visualization using the first 30 principal components showed that the IVC embryo data also closely matched the reported in vivo single-cell data (GSE74767) and other IVC embryo data (GSE130114). The IVC data were annotated using the other two datasets as references. Correlation analysis was performed as described above. IVC embryo data were also individually normalized, scaled, and dimensionally reduced. t-SNE visualization was used to separately visualize GFP + Cells and GFP -Distribution of cells in 2D space. DEGs between different cell types were analyzed by the FindAllMarkers function in Seurat with the settings of “only.pos=TRUE, min.pct=0.25, logfc.threshold=0.25”. Genes with log2(fold change)>0.25 and P value<0.01 were defined as DEGs. DEGs were used for GO enrichment analysis by the clusterProfiler (v3.18.1) R package. Heatmaps were generated by the DoHeatmap function in Seurat. Graphs were generated using DimPlot and FeaturePlot. For Smart-seq2 data of chimeric samples, the count matrix was processed using Seurat in the same way as the DNBelab C4 data of chimeric samples. Graphs were generated using DimPlot and DotPlot. Bar graphs were generated using ggplot2 (v3.3.3) in R (v3.6.0). Cell types were annotated as described above. To integrate the DNBelab C4 data of wild-type monkey testes and the Smart-seq2 data of chimeric testes, batch correction was performed using the FindIntegrationAnchors function as described above. The data of wild-type monkey testes were processed and annotated as described above, with 2000 cells randomly selected for integration.

[0305] Processing of single-cell and whole-genome sequencing (WGS) data

[0306] Single-cell and bulk whole-genome sequencing (WGS) were performed at CNGB using the DNBSEQ-T1 and DNBSEQ-T7 sequencing platforms, respectively. Read length was pair-end 100 (PE100). For each sample, sequencing reads were filtered using the default option fastp (v0.21.0), and the retained reads were then aligned to the Macaca_fascicularis_5.0 reference genome using BWA-MEM. Alignments with low mapping quality (<=30) were screened out, and duplicate sequences were removed using sambamba (v0.7.0). For scWGS analysis, cells that passed the following thresholds were used for downstream analysis: 1) had at least 3x sequence bases; 2) more than 65% of the genomic regions had been covered, and more than 20% of the genomic regions had 5x coverage; 3) more than 60% of the regions had been covered, and the average coverage of each chromosome was more than 2x. Copy number was assessed in 100Kb bins using HMMcopy (v1.42.0). For single-cell WGS data, we also primed P10 cells and Genome-wide association analysis between P10 cells. Logistic regression was used for each bin to test whether the estimated copy number was different between the two groups of cells. There were 28,724 bins between the 28 primed P10 cells and the 20 naive P10 cells. The results are shown in the Quantile-Quantile plot.

[0307] Schematic diagram creation

[0308] The schematic was created using BioRender.com.

[0309] Quantification and statistical analysis of data

[0310] Based on the growth and stable passage of cyESC, we determined Figure 8 Success of primedESC establishment in blastocysts (blastocysts, n=17) in B. Figure 8 Teratoma formation and gene expression (RT-PCR) of cyESCs under different culture conditions in F and 8K, including three biological replicates. Figure 2 GFP in ICM of three injection groups (injected with primed ESC, n=3; injected with 5iLAF ESC, n=3; injected with 4CL ESC, n=4) in F + The cell percentage was analyzed by GraphPad Prism. The data are presented as mean ± SEM. Fig.11 GFP in dpf17 in E + Cell percentage (5iLAF ESC injection, n=3; 4CL ESC injection, n=3). Figure 3 IVC embryonic development in C (for control embryos, dpf7, n = 33; dpf11, n = 24; dpf13, n = 20; dpf15, n = 15; dpf17, n = 15; for 4CL mosaic embryos, dpf7, n = 27; dpf11, n = 18; dpf13, n = 14; dpf15, n = 11; dpf17, n = 11; for 5iLAF mosaic embryos For primed chimeric embryos, dpf7, n = 18; dpf11, n = 12; dpf13, n = 8; dpf15, n = 7; dpf17, n = 7) were determined based on the presence of distinct ectoderm and amniotic and yolk sac cavities. Figure 5We performed three replicate experiments in each SNP to determine the mosaic efficiency in C, 5D, 12C, and 12D. Figure 5 GFP in different tissues (#9 and #10) of chimeric monkeys in K, 5L, 12F, 12J, 12K, 7B and 14C + For cell percentage analysis, more than three random fields were selected, and the error bars represent the SEM of the mean. The differences between the two groups were analyzed by unpaired two-tailed Student's t-test. GraphPad Prism 9 software was used to analyze the data.

[0311] All bioinformatics quantitative and statistical analyses were performed using R and python computing environments and packages. Spearman rank correlation coefficients were used Figure 1 Artifact correlation analysis of scRNA-seq data in D and 11H. Figure 3 P values ​​were calculated for differential expression between cell types in I and 7K using the Wilcoxon rank sum test and adjusted for multiple testing using the Bonferroni correction. Fig. 9 Differential expression between monkey embryonic stem cells in A was calculated using the Wald test and adjusted for multiple testing using the Benjamini-Hochberg correction. Figure 3 When performing GO analysis in J, 9A, and 14M, P values ​​were calculated using the hypergeometric test and adjusted for multiple testing using the Benjamini-Hochberg correction. Quantile-quantile plots (QQ plots) were used Fig. 9 Genome-wide copy number variation analysis in J. DMRs were assessed by hypergeometric test and then using Fig.14 Multiple testing adjustment was performed with the Beniamin-Hochberg correction in L. More details can be found in the relevant section of the Methods Details.

[0312] Example 1: Obtaining monkey ICM-like pluripotent stem cells using human PSC culture protocol

[0313] We first established 9 primed cynomolgus monkey ESC lines from 17 E7 (embryonic day 7) blastocysts. These monkey ESC cells showed a flat morphology similar to that of human PSCs ( Figure 8 A). The establishment efficiency is about 53% ( Figure 8 B) Then, we selected four types of people In addition to the LCDM culture system, we also added a PSC culture system with strong expansion stem cells and differentiation capacity. In each culture condition, 5 primed monkey ESC lines (2 male and 3 female) were used to convert to In addition to RSeT being in primed and The intermediate form ( Figure 1 A), other culture conditions all showed dome-shaped clones within 2 to 4 generations (similar to mouse All five monkey ESC lines were successfully transformed into stable cultured cells under the conditions of 4CL, RSeT, and LCDM, whereas only two and three ESC lines could be transformed using 5iLAF and PXGL, respectively ( Figure 8 C). The ESC lines obtained in 4CL, RSeT and LCDM showed stable clonal morphology after more than 20 passages. In PXGL, 3 cell lines could be successfully transformed, but due to massive cell death and differentiation, the transformed cells lost the dome-like clonal morphology after 4 passages. For 5iLAF, only 2 female ESC lines could be successfully transformed, but after 10 passages, the cell morphology was between primed-like and -like intermediate form ( Figure 8 D) All stably transformed monkey ESC cell lines had higher survival rates and cloning efficiencies after single-cell isolation than primed-like ESCs ( Figure 8 E and Figure 8 F).

[0314] Teratoma assay was used to detect primed and Differentiation ability of ESCs. In all culture systems tested, monkey ESCs were able to form teratomas efficiently (75%-100%), and derivatives of the three germ layers were found in these teratomas ( Figure 8 G and 8H). It has been reported that ESCs are primed and Compared with primed ESCs, all transformed ESCs showed increased signals of tetramethylrhodamine ethyl ester (TMRE) staining, indicating that their oxidative phosphorylation activity was significantly increased ( Figure 8 I). In addition, ESCs cultured in 4CL, PXGL, and 5iLAF showed The nuclear localization of PSC marker TFE3 was increased. TFE3 was mainly localized in the cytoplasm in primed ESCs and ESCs cultured in RSeT or LCDM ( Figure 8 J).

[0315] Quantitative RT-PCR (RT-qPCR) analysis showed that the expression levels of classical pluripotency genes in all transformed ESCs were comparable to those in primed ESCs. The expression levels of pluripotency genes were higher than those of primed ESCs to varying degrees, and the highest expression levels were found in cells cultured in 5iLAF and 4CL ( Figure 8 K). Immunostaining for KLF17, SOX2, and NANOG further confirmed these findings ( Figure 1 B). RNA sequencing (RNA-seq) detected down-regulation of multiple primed ESC genes in all transformed ESCs, and down-regulation in 4CL and 5iLAF ESCs. Activation of the pluripotency network is more efficient ( Figure 1 C). GO analysis showed that genes related to stem cell population maintenance and DNA modification were enriched in 4CL and 5iLAF ESCs, while genes related to axon guidance and neural differentiation were enriched in PXGL ESCs ( Fig. 9 A) These results suggest that 4CL and 5iLAF ESCs are in a more stable state, while PXGL ESCs are in an unstable state, which also reflects the specific differences in the same culture medium between different species. We then detected the developmental state of primed ESCs and ESCs cultured in 4CL, 5iLAF and PXGL by droplet-based single-cell RNA sequencing (scRNA-seq). At the same time, the data were compared with existing cynomolgus macaque early embryonic development datasets. Correlation analysis and nonlinear dimensionality reduction algorithm (UMAP) representation showed that 4CL and 5iLAF ESCs are closer to the ICM and pre-implantation epiblast, primed ESCs are closer to the late post-implantation epiblast, and PXGL ESCs exist in both clusters ( Figure 1 D and 1E).

[0316] Since DNA demethylation is an epigenetic mechanism, primed ESCs are transformed into In the pluripotent state, we found that ESCs cultured in RSeT, PXGL, and LCDM had high DNA methylation levels (83-86%), which was similar to the methylation levels of primed ESCs. 4CL ESCs had low methylation levels (72%), and 5iLAF ESCs had the lowest methylation levels (52%) ( Figure 1 F and Fig. 9 B). 4CL and 5iLAF ESCs show imprinted gene DNA methylation closer to ICM ( Fig. 9 C). We also provide 4CL Different cell generations during the transformation process were sequenced for methylation and it was found that all genomic elements (including DNA demethylation was observed in pluripotency loci (KLF17, DPPA3, and DNMT3L3). Figure 1 G and Fig. 9 D-9G). In addition, demethylation dynamics were also detected in the promoter regions of imprinted genes during transformation ( Fig. 9 H). Karyotype analysis of ESCs cultured in RSeT, LCDM, and 4CL medium for more than 15 generations showed normal chromosome numbers ( Figure 1 H), indicating that genomic integrity was maintained. PXGL ESCs showed normal karyotype within a limited number of passages, but we did not follow their karyotype in subsequent passages due to the difficulty in maintaining the culture. Notably, all 5iLAF ESC lines acquired abnormal karyotypes after 8 passages, which is consistent with previous studies in humans. To further investigate the genomic integrity of 4CL ESCs, we performed single-cell whole-genome sequencing (scWGS). After the same passage number (P10), although both cell lines contained cells presenting copy number variations (CNVs) compared to the reference genome ( Fig. 9 I), but we did not observe obvious differences between 4CL ESCs and primed ESCs, which is consistent with previous findings in cultured human PSCs. We further analyzed the genome-wide copy number variation in the two cases by association analysis, and we did not find any significant differences between primed and 4CL The genomic regions with CNVs that were significantly different between ESCs ( Fig. 9 J).

[0317] In conclusion, our data suggest that 4CL ESCs exhibit more balanced genome-wide DNA demethylation compared with monkey ESCs cultured in other media, High expression of pluripotency genes and genome stability.

[0318] Example 2 Optimization of the culture protocol for producing chimeric blastocysts using monkey ESCs

[0319] We focused on 4CL ESCs, optimized the injection method for early monkey embryos, and improved ESC survival. ESCs and injected into morula-stage monkey embryos ( Figure 2 A and Fig.10 A). For each morula, approximately 15 GFP-labeled 4CL ESCs were injected within 5-10 generations ( Fig.10B). When the injected morulae were cultured in monkey embryo medium HECM9 for 72 hours (Scheme 1), we observed that the blastocysts developed normally, but the GFP signal was weak, indicating that few donor cells survived, which is similar to previous reports. When the injected morulae were cultured in 4CL for 72 hours (Scheme 2), we observed strong GFP signals in most embryos, but no normally developed blastocysts were obtained. After culturing with a 1:1 mixture of 4CL and HECM9 for 24 hours and then culturing in HECM9 for 48 hours (Scheme 3), GFP signals were detected in most embryos and normal blastocysts were obtained. However, in this case, the GFP signal in each blastocyst was limited. Therefore, we cultured the injected embryos in a 1:1 mixture of 4CL and HECM9 for 72 hours (Scheme 4). This greatly enhanced the GFP signal without affecting the development of the blastocyst. Further modification of the 4CL / HECM9 ratio to 2:1 (Scheme 5) resulted in defects in blastocyst formation. We concluded that protocol 4 (ESC medium mixed with HECM9 at a ratio of 1:1 for 72 h) was a good balance between ESC survival and blastocyst development ( Figure 2 B-2D and Fig.10 C).

[0320] Next, we explored the effects of morula injection of monkey ESCs produced in different culture media on embryonic development according to the culture conditions of protocol 4. GFP-labeled monkey primed ESCs and 5iLAF ESCs were injected into morulae according to the modified protocol, which we refer to as protocol 6 (primed ESCs) and protocol 7 (5iLAF ESCs). We found that in both groups of injections, the injected morulae showed a similar blastocyst development rate as 4CL ESCs. Similarly, in all three groups of injections, GFP + There was no significant difference in the proportion of blastocysts ( Figure 2 C,2D and Fig.10 D, 10E). Immunostaining results of chimeric blastocysts showed that 22%-48% of GFP and OCT4 double positive cells were located in the ICM in blastocysts injected with these three ESCs. Figure 2 E, 2F and Fig.10 F). Consistent with previous studies, we also observed 14-23% GFP signals in the trophectoderm (TE) of chimeric blastocysts injected with the three ESCs ( Figure 2 D and Fig.10 G), indicating that monkey ESCs contribute to both embryonic and extraembryonic lineages.

[0321] Example 3 Contribution of monkey ESCs to chimeric embryos during extended in vitro culture

[0322] Next, we used the in vitro delayed culture (IVC) system to test the developmental capacity of injected ESCs in embryos. This experiment selected three ESCs (primed, 4CL and 5iLAF) produced GFP under the optimized culture schemes 4, 6 and 7 respectively. + Chimeric embryos. Around 10 days after fertilization (dpf), most chimeric embryos begin to attach to the culture dish. During 11-13 days after fertilization, the embryonic trophectoderm cells proliferate rapidly, while the ICM remains a dense cell mass. On days 15-17, the embryonic disc-like structure clearly appears ( Figure 3 A, 3B and Fig.11 Importantly, the in vitro development efficiency of embryos in the three groups injected with ESCs was comparable to that of the control group (no ESC injection) (39-45%, IVC embryos with embryonic disc structures), indicating that ESC injection did not reduce the efficiency of in vitro development of embryos ( Figure 3 C). We also observed that at dpf 17, 3 / 7, 8 / 11, and 5 / 7 embryos in the primed, 4CL, and 5iLAF ESC-injected groups, respectively, showed clear GFP signals ( Figure 3 D) In ​​primed ESC-injected embryos, the GFP signal increases very slowly with the extension of culture time, so the 3 GFP + Embryos at dpf17 all showed low fluorescence intensity ( Fig.11 A and 11C). In contrast, in the 4CL and 5iLAF injection groups, the GFP signal increased significantly with the extension of culture time ( Figure 3 A, Fig.11 B and 11C). By single-cell isolation of dpf17 embryos, 44% and 22% of GFP-positive cells were found in 4CL and 5iLAF ESC-injected embryos, respectively ( Fig.11 D,11E).

[0323] It is noteworthy that we observed a large amount of GFP in the chimeric embryos obtained from the 4CL ESC injection group on day 17. + cell( Figure 3 A). We then performed Smart-seq2 analysis. A total of 265 cells passed quality control and were further analyzed. We used Seurat to integrate these data with published normal in vivo and in vitro monkey embryo datasets. This integrated analysis identified 6 clusters (ectoderm, early primordial germ cells, gastrula cells, visceral / yolk sac endoderm, extraembryonic mesenchyme, and trophectoderm cells) ( Figure 3 E, 3F, and 11F). It is noteworthy that GFP-positive cells in chimeric embryos are annotated to the same cell types as those in normal in vitro cultured embryos ( Figure 3G and 11G). Gene expression correlation analysis further confirmed their high similarity ( Fig.11 H). GFP-positive cells made great contributions to EPI (68 / 74, 92%), Gast1 (2 / 2, 100%), and TE (25 / 54, 46%). Figure 3 H). Further analysis showed that there were specific differentially expressed cell types (DEGs) in cells of different lineages in IVC embryos ( Figure 3 I). GO analysis results showed that EPI was enriched with genes involved in mRNA decomposition and metabolism, translation initiation, and stem cell population maintenance, while VE / YE was enriched with genes involved in cell morphogenesis, differentiation, and establishment or maintenance of cell polarity. EXMC and TE were enriched with genes involved in extracellular matrix structure organization, extracellular matrix organization, and uterine embryonic development ( Figure 3 J).

[0324] Therefore, using an in vitro culture system, we demonstrated that monkey ESCs have the ability to generate embryos with a higher proportion of chimeric embryos than primed ESCs. Although the extended in vitro culture system only partially mimics the environment of natural embryonic development, in both tested Among ESCs, 4CL ESCs showed a higher chimerism rate. In addition, the chimerism of donor ESCs in IVC embryos recapitulated key events of early embryogenesis.

[0325] Example 4 Producing a high proportion of chimeric monkeys by syngeneic monkey ESCs

[0326] Subsequently, we transplanted the chimeric monkey embryos into surrogate mothers in pursuit of the production of live chimeric monkeys. In terms of embryo transplantation, GFP-labeled 4CL ESCs (2 female and 1 male cell lines) were injected into morula embryos and cultured in vitro to the blastocyst stage using the optimized scheme 4. A total of 91 blastocysts were obtained from 206 injected morula embryos. Among them, 74 blastocysts with clear GFP signals were transplanted into 40 surrogates, and 12 surrogates successfully became pregnant. Among these 12 pregnancies, 4 aborted fetuses and 6 full-term live-born offspring were obtained. PCR detected the GFP sequence in the genomic DNA of an aborted male fetus #9 and a live-born male fetus #10, as well as chimeric blastocysts with strong GFP signals corresponding to the two fetuses ( Figure 4A-4C). It is noteworthy that in our chimeric experiments, even fetuses without GFP-positive cell contribution had a high miscarriage rate. This suggests that although normal blastocyst rates were observed in this protocol, the HECM9 and 4CL mixed medium may affect embryonic development, and apoptosis of injected ESCs may also have a negative impact on normal embryonic development. Strikingly, live monkey baby #10 showed strong GFP signals throughout the body, including fingers, tail, and eyes, indicating a high level of contribution from ESC-derived cells ( Figure 4 D). Short tandem repeat (STR) analysis further confirmed the contribution of ESCs in these two monkeys ( Figure 4 E, 4F). After surviving for 10 days, chimeric monkey No. 10 developed respiratory failure, hypothermia and other health deterioration, and was euthanized by a veterinarian for detailed analysis. The results are summarized in Figure 4 G.

[0327] The chimerism ratio was assessed using a series of rigorous methods. Various tissues from the two chimeric monkeys that had been frozen previously were used for PCR detection of GFP sequences. Clear GFP sequences were found in almost all tissues of the two monkeys ( Figure 5 A and Fig.12 A). To quantify the chimerism ratio of injected ESCs in these tissues, we used deep sequencing technology (deep-seq) to select SNPs in mitochondrial DNA (mtDNA) and genomic DNA (gDNA) between host embryos and injected ESCs. The ESC / host SNP ratio was then used to calculate the donor ESC contribution ratio in various tissues of chimeric monkeys. For live-born monkey #10, 3 SNPs located in mtDNA were identified, and deep sequencing showed that the contribution of ESCs ranged from 21% to 92%, with an average chimerism rate of 67% in 26 tissues ( Figure 5 B, 5C). Analysis of three additional SNPs located on chromosome X showed that the average ESC contribution of monkey #10 was 63% ( Figure 5 B, 5D). For aborted monkey #9, deep sequencing of 2 SNPs from mtDNA and 1 SNP from chromosome X revealed an average ESC contribution of 15% and 18%, respectively ( Fig.12 B-12D). Overall, the consistency of mtDNA and gDNA SNP analysis results demonstrates the reliability of our quantification.

[0328] We also further examined the contribution of ESCs by GFP fluorescence imaging. We detected GFP-positive cells in the bone marrow cells (BMCs) of the two chimeric monkeys and in the peripheral blood mononuclear cells (PBMCs) of chimeric monkey #10 ( Figure 5 E and Fig.12E) FACS analysis showing GFP expression in PBMC and BMC of chimeric monkey #10 + The cell ratios were 88% and 78%, respectively. The counts of chimeric monkey #9 showed that GFP + The cell ratio is 20% ( Figure 5 F and Fig.12 F). In the other 9 tissues of monkey #10 (brain, heart, kidney, liver, lung, ileum, jejunum, rectum and amnion) and 4 tissues of chimeric monkey #9 (brain, heart, kidney and liver) ( Figure 5 G-5I and Fig.12 Strong GFP fluorescence signals were also observed in G and H. Co-staining with GFP and NeuN antibodies further confirmed the contribution of ESCs to the brains of these two chimeric monkeys ( Figure 5 J and Fig.12 I). The quantitative GFP ratios in these tissues were evaluated by immunostaining and showed general agreement with the SNP quantification results ( Figure 5 K,5L and Fig.12 J, 12K), further confirming the high-level contribution of ESCs in these chimeric monkeys.

[0329] Example 5 Functional integration of embryonic stem cells in chimeric monkeys

[0330] We performed transcriptome analysis using solid tissues and blood-related cells from two chimeric monkeys. For chimeric monkey #10, we first performed Smart-seq2 sequencing on cryopreserved BMCs. A total of 161 cells passed quality control and were further analyzed. Of the sequenced BMCs, 79% (127 / 161) were GFP + , which is consistent with the results of SNP and FACS analysis ( Figure 6 A). We annotated the sequenced cells based on gene expression and identified 10 cell types, including monocytes, natural killer T cells, and erythrocytes ( Figure 6 B, 6C). GFP + The proportion of cells among these annotated cell types was high, ranging between 53-100%, with two cell lines derived entirely from injected ESCs (granulocyte-monocyte progenitors and natural killer T cells) ( Figure 6 D).

[0331] Given that Smart-seq2 can only capture a limited number of cells, we increased the number of cells sequenced by droplet-based scRNA-seq. We obtained 3011 BMC cells in chimeric monkey #10 and annotated 16 cell types ( Figure 6E, 6F). Using this higher throughput approach enabled more lineages to be successfully identified, including pre-B cells, mature B cells, and neutrophils. The percentage of GFP-positive cells from the donor was lower in this sequencing method than in Smart-seq2 (32%), which may be due to the smaller number of specific molecular labels (UMIs) captured by droplet scRNA-seq compared to Smart-seq2. Cell lineage annotation was performed for GFP-positive cells and GFP-negative cells. Interestingly, there were more red blood cells in GFP-positive cells, while there were more mononuclear cells in GFP-negative cells, suggesting that there was a deviation in differentiation direction during the development of the chimeric embryo ( Figure 6 G).

[0332] We applied the same strategy to the brain tissue of chimeric monkey #10 frozen in liquid nitrogen. Using the extracted nuclei, we sequenced 170 and 9450 nuclei using Smart-seq2 and droplet-based single-nucleus RNA sequencing (snRNA-seq), respectively. According to the Smart-seq2 results, the injected ESCs contributed 91% of the brain cells ( Figure 6 H), which is consistent with the SNP analysis. + Brain cells include 93% excitatory neurons, 91% inhibitory neurons, 77% microglia, and 100% neural progenitor cells ( Figure 6 IK). SnRNA-seq further demonstrated that the injected donor cells also contributed other brain cells, such as astrocytes, oligodendrocytes, and VIP + 、SST + and PVALB + Inhibitory neurons ( Figure 6 In addition, we performed droplet-based sc / snRNA-seq sequencing on three additional tissues (PBMCs, adrenal glands, and liver) from chimeric monkey #10 and two tissues (brain and heart) from chimeric monkey #9. Multiple cell types were annotated in PBMCs (12), adrenal glands (7), liver (5), brain (10), and heart (6). Fig.13 A- Fig.13 E) In both chimeric monkeys, most cell types were abundantly GFP positive.

[0333] Our results showed that GFP from donor ESCs + The cells in the monkey chimeras had functions similar to those of the host cells.

[0334] Example 6 Contribution of ESCs to Chimeric Monkey Germ Cells and Placenta

[0335] Germ cell differentiation is different from other tissue differentiation in that it involves extensive DNA demethylation and remethylation. The placenta originates from the trophectoderm, an extraembryonic tissue. The contribution and differentiation of PSCs to these two tissues are often used to evaluate their developmental potential. Therefore, we studied the injected monkeys Whether ESCs contribute to the testes and placenta of two chimeric monkeys.

[0336] For the testes, sequencing of mtDNA and gDNA SNPs revealed that they contributed 6% and 9% of ESCs, respectively, in chimeric monkey #9 and 36% and 41%, respectively, in chimeric monkey #10 (see Figure 5 C, 5D and Fig.12 C, 12D). In addition, GFP signals can be clearly detected in the testis cryosections of the two chimeric monkeys ( Fig.14 A). Co-localization of the germ cell-specific marker VASA and GFP signals on the same cell confirms the in vivo differentiation of donor ESCs into germ cells ( Figure 7 A and Fig.14 B) In the testis of chimeric monkey #10, GFP + Cells in all cells and in all VASA + The average percentages were 58% and 62% in monkey #1 and 11% and 28% in monkey #9 ( Figure 7 B and Fig.14 C). Consistent with previous mouse studies, female ESCs can differentiate into spermatogonia in male chimeric monkeys, indicating that the fate of germ cells is influenced by the testicular environment. We also performed scRNA-seq analysis on testicular cells from chimeric monkey #10 and testicular cells from age-matched wild-type monkeys. For chimeric monkey #10, a total of 256 cells passed quality control and were further analyzed. Notably, the GFP expression in chimeric monkeys + Cells and GFP - The cells include interstitial cells, peritubular myoid cells, supporting cells, endothelial cells, proliferating cells and spermatogonia. + The proportion ranged from 42% (supporting cells) to 90% (peritubular myoid cells). Comprehensive cluster analysis showed that the GFP + and GFP - These six major lineages of cells can be clustered together with the major lineages of control monkeys ( Figure 7 C-7F). We further analyzed GFP in supporting cells + GFP - Cell population, XY GFP - Sertoli cells highly express genes of testicular Sertoli cells (SOX9, CIT- ED1, DMRT1 and SERPINA5), while XX GFP + Sertoli cells highly express genes of ovarian granulosa cells (CYP19A1, WNT6, AMHR2 and CRHBP) Figure 7 G) These results are consistent with previous findings, indicating that the Y chromosome plays a major role in this process and that the ability of XX ESCs to differentiate into testicular Sertoli cells is therefore limited.

[0337] For the placenta, by directly detecting GFP fluorescence, GFP signals were clearly detected in the cryosections of chimeric monkey #10 ( Fig.14 D). In the placenta of chimeric monkey #10, immunofluorescence analysis also detected signals such as GFP, KRT7 (a marker of villous trophoblast cells), MCT4 (a marker of cavernous trophoblast cells), and trophoblast giant cell markers ( Figure 7 H, Fig.14 E). We then performed FACS analysis on the percentage of GFP-positive nuclei in the placenta and observed that GFP was expressed in the placenta tissues of wild-type placenta and chimeric monkeys #9 and #10, respectively. + The nuclear ratio is 0%, 6% and 20% ( Fig.14 F) GFP expression in the placenta of chimeric monkeys #9 and #10 by FACS + PCR was performed on the nuclei to confirm the integration of GFP ( Fig.14 G). We also analyzed the GFP expression in the placenta of chimeric monkey #9 by FACS sorting. + The nuclei were sequenced by Smart-seq2. A total of 288 GFP + The nuclei passed the quality control, including 145 villous cytotrophoblast cells, 77 macrophages, and 66 stromal cells ( Fig.14 H-14J).

[0338] These findings provide convincing evidence that monkeys ESCs have the ability to form germ cells and placental tissue in vivo. The former has great practical significance for genetic engineering, while the latter proves that 4CL Developmental potential of monkey ESCs.

[0339] Example 7 Epigenetic state and genomic integrity of ESC-derived cells in chimeric monkeys

[0340] To gain insight into the causes of low frequency chimerism, miscarriage in chimeric monkey #9, and compromised health in live-born chimeric monkey #10, we investigated the DNA methylation status and genomic integrity of donor ESC-derived cells. Abnormal DNA methylation levels are known to cause developmental impairment in chimeric mice, so we chose DNA methylation to investigate epigenetic status. At the time of euthanasia of monkey #10, we also collected the monkey's ear tips and isolated fibroblasts, which were expanded and cryopreserved. ESC-derived GFP was sorted by FACS + and host embryo-derived GFP - We used low-input WGBS to analyze GFP + BMCs and GFP - BMCs, interestingly, GFP + The DNA methylation level of BMCs (~80%) is higher than that of GFP - BMCs (~72%) Figure 7 I,7J). This hypermethylation is distributed in different genomic regions ( Fig.14 K) In GFP + BMCs and GFP - A total of 18,462 differentially hypermethylated regions (hyperDMRs) were identified between BMCs, 9% of which were located in promoter regions and 870 showed hypomethylation (hypoDMRs) ( Fig.14 L). GO analysis showed that the genes corresponding to these hyperDMRs were enriched in development-related biological processes, such as anatomy, morphogenesis, and phylogeny ( Fig.14 M). However, no enrichment of genes related to BMC hematopoiesis was observed. We further selected 100 genes highly expressed in BMCs and tested their expression in GFP. + BMCs and GFP - Methylation levels and gene expression in BMCs. We found that GFP + BMCs and GFP - There were no significant differences in methylation levels among BMCs. These findings are consistent with single-cell transcriptomic analysis of GFP + BMCs and GFP - The gene expression levels of BMCs were very similar and consistent ( Figure 7 K). We also analyzed the DNA methylation levels of classical imprinted genes and observed that GFP + The DNA methylation levels of the promoter regions of imprinted genes in BMCs were slightly higher. + BMCs and GFP -Gene expression levels were similar between BMCs, suggesting that 4CL ESCs-generated monkey chimeras show no obvious loss of genomic imprinted genes ( Figure 7 L) To investigate the genomic integrity of chimeric monkey donor ESC-derived cells, we used GFP + BMCs and GFP - BMCs, GFP + Fibroblasts and GFP - Whole genome sequencing (WGS) was performed on four types of fibroblasts. We found that GFP + BMCs and GFP + Ear fibroblasts and GFP - Cells have almost no genomic abnormalities compared to Figure 7 M).

[0341] Although we cannot conclude that the different DNA methylation levels are the cause of the miscarriages and poor health in the chimeric monkeys, the possibility exists and this is consistent with our observations in monkeys. The DNA methylation level of ESCs was higher than that of ICMs. + BMCs and GFP + The overall number of abnormalities in fibroblasts was relatively low, suggesting that cells with higher fitness may have been selected during development.

[0342] discuss

[0343] In this study, we describe the generation of living chimeric monkeys and High contribution of ESCs (up to 90% in some tissues). By combining transgenic GFP reporter genes, STR and SNP analysis, and two types of single-cell transcriptome analysis, we extensively verified the high level of chimerism in various tissues in chimeric monkeys. We found that in sharp contrast to the low proportion of chimerism in all other fetuses, the contribution of donor cells in the two monkey chimeras obtained was very high, and the underlying mechanism to explain this phenomenon may be factors such as cell competition and induction of apoptosis in the host embryo. Our method to promote the survival of ESCs in early embryos may require a relatively high percentage of ESCs to achieve ultimate survival. In this case, only maintaining a high proportion of ESCs alive at early developmental stages can contribute to the formation of highly chimeric tissues. Through competition from host cells, ESCs with low survival rates may be more easily excluded during organogenesis. Factors affecting whether donor cells can survive in early embryos may be related to the state of the cultured host embryo or the heterogeneity of ESCs. Using single-cell transcriptome analysis to understand the cellular dynamics of chimeric embryos at various early stages during in vivo development can provide clues to this issue.

[0344] Since it was recognized that human PSCs cultured under conventional (primed) conditions share characteristics with postimplantation-like mouse epiblast stem cells, several groups have reported converting primed human PSCs into mouse-like epiblast stem cells by using culture media containing different combinations of growth factors and small molecule inhibitors of signaling pathways or epigenetic modulators. PSCs or expanded / expanded pluripotent stem cells. In this study, we systematically investigated the characteristics of monkey ESCs grown in various media previously used to culture human PSCs. We chose 4CL ESCs to assess isogenic monkey chimerism in vivo because they show High expression levels of pluripotency genes without inducing extensive DNA demethylation while maintaining genomic stability. However, we cannot exclude that If monkey ESCs cultured under normal karyotype and high expression of The possibility of obtaining similar results is also possible with the use of pluripotency genes. Our optimized embryo injection method and embryo culture protocol, as well as the 4CL medium method, also improve the chimerism of donor cells in IVC embryos. In addition to providing a test of the developmental potential of monkey ESCs, the IVC model can also be used for mechanistic studies of cell fate progression using wild-type or genetically modified ESCs, such as through barcoding and lineage tracing.

[0345] The generation of living chimeric monkeys from homologous PSCs provides a powerful method for producing monkey models with complex genetic modifications for both basic and translational research. The recent development of gene editing technologies and their application in fertilized eggs has made it relatively easy and efficient to achieve precise gene targeting in embryos of multiple species, including macaques. However, more complex gene editing, such as large fragment insertions or replacements and multi-site gene targeting remain difficult. Previously reported cloning by somatic cell nuclear transfer provides a possible approach, but the efficiency remains low. Therefore, the use of gene editing The reproducible generation of germline chimeric monkeys from PSCs would represent a major advance in the field, and our findings pave the way toward achieving this goal.

[0346] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for preparing a chimeric embryo of a non-human primate in vitro, comprising the steps of: (i) culturing primed pluripotent stem cells (PSCs) under a first culture condition to obtain primed stem cells or naive stem cells, wherein: The first culture condition comprises a first culture medium, wherein the first culture medium is selected from the group consisting of 4CL, 5iLAF, PXGL, RseT, LCDM, primed, or a combination thereof; (ii) contacting the flat stem cells or naive stem cells obtained in step (i) with a non-human primate embryo to obtain a non-human primate chimeric embryo containing the flat stem cells or naive stem cells, and culturing the non-human primate chimeric embryo containing the flat stem cells or naive stem cells under a second culture condition to obtain a blastocyst stage chimeric embryo, wherein the second culture condition comprises a mixed culture medium in which a second culture medium and a third culture medium are mixed in a volume ratio of 1:0.5-1.5, wherein the second culture medium is selected from the group consisting of Primed, 4CL, 5iLAF, or a combination thereof, and the third culture medium comprises HECM9.

2. The method according to claim 1, characterized in that The first culture condition is a culture condition suitable for differentiation of flat stem cells or immature stem cells.

3. The method according to claim 1, characterized in that The first culture medium is selected from the group consisting of Primed, 4CL, 5iLAF, or a combination thereof.

4. The method according to claim 1, characterized in that The pluripotent stem cells are derived from embryonic stem cells of non-human primates.

5. The method according to claim 1, characterized in that The naive stem cells include naive embryonic stem cells.

6. The method according to claim 1, characterized in that The second culture condition includes a mixed culture medium in which the second culture medium and the third culture medium are mixed in a volume ratio of 1:

1.

7. The method according to claim 1, characterized in that The embryos or chimeric embryos include morula stage embryos.

8. The method according to claim 1, characterized in that The chimeric embryos have a high chimerism ratio.

9. A culture medium composition for producing chimeric embryos, characterized in that include: (a) a first culture medium for culturing and obtaining primed stem cells or naive stem cells, selected from the group consisting of 4CL, 5iLAF, PXGL, RseT, LCDM, primed, or a combination thereof; (b) A mixed culture medium for obtaining chimeric embryos, the mixed culture medium comprising a second culture medium and a third culture medium, the volume ratio of the second culture medium to the third culture medium in the mixed culture medium is 1:0.5-1.5, the second culture medium is selected from the following group: Primed, 4CL, 5iLA, or a combination thereof; the third culture medium comprises HECM9.

10. Use of the combination of culture media according to claim 9, characterized in that: Non-human primates for the preparation of chimeras.

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