Use of bcl2 in regulating the pluripotent state of human embryonic stem cells and / or activation of 8-cell like cells

Abstract

The application provides application of BCL2 in regulating pluripotency state of human embryonic stem cells and / or activation of 8-cell-like cells, and belongs to the technical field of genetic engineering. By up-regulating BCL2 expression in human embryonic stem cells, the application can effectively promote conversion of human embryonic stem cells from a naive state to a ground state, stably maintain pluripotency state of human embryonic stem cells, efficiently induce human embryonic stem cells to transform into 8-cell embryo-like cells, effectively maintain long-term stable self-renewal and omnipotency molecular characteristics of the 8-cell embryo-like cells, so that the 8-cell embryo-like cells can simultaneously contribute to an inner cell mass and a trophoblast in vivo, and overcome defects such as low induction efficiency of 8-cell embryo-like cells, easy apoptosis and differentiation in long-term culture, unstable maintenance of omnipotency, and limited lineage differentiation ability in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of BCL2 in regulating the pluripotency state of human embryonic stem cells and / or 8-cell-like cell activation. Background Technology

[0002] Human embryonic stem cells (HSCs) possess unlimited self-renewal capacity and multipotent differentiation potential, making them a core in vitro research model for studying early human embryonic development, cell fate regulation, regenerative medicine, and disease mechanisms. Based on developmental regulatory characteristics and molecular phenotypic differences, HSCs are mainly classified into two pluripotent states: the primordial state and the primitive state. Primordial HSCs have limited differentiation potential and their lineage differentiation is biased towards embryonic tissues; primitive HSCs more closely resemble the characteristics of early pre-implantation embryonic cells, exhibiting lower DNA methylation levels and a broader pluripotency regulatory network, making them an important cell model for simulating early human embryonic development in vitro. Therefore, exploring the key molecules regulating the pluripotency transition of HSCs and achieving efficient and stable conversion from the primordial state is of significant research value for elucidating the regulatory mechanisms of stem cell pluripotency.

[0003] Human early embryonic development is a highly precise and ordered biological process. The eight-cell stage is a critical window for zygotic genome activation. During this stage, cells possess totipotency and can differentiate bidirectionally to form embryonic tissues and extra-embryonic trophoblast tissues, which are essential for maintaining normal embryo implantation and development. However, due to the scarcity of human embryo samples, ethical restrictions, and difficulties in obtaining experimental materials, directly using natural early human embryos for mechanistic research presents significant limitations. Eight-cell embryo-like cells can be obtained through in vitro reprogramming of human embryonic stem cells. These cells can stably mimic the transcriptional characteristics, zygotic genome activation characteristics, and developmental potential of human eight-cell embryos, making them an ideal alternative model for studying human zygotic genome activation, early lineage differentiation, and totipotency regulation.

[0004] However, existing in vitro induction systems still have significant drawbacks: under conventional induction conditions, the induction efficiency of eight-cell embryo-like cells is limited, and these cells are prone to differentiation and apoptosis during long-term culture, making it difficult to achieve long-term stable self-renewal. Simultaneously, the pluripotency of eight-cell embryo-like cells induced from wild-type embryonic stem cells is unstable, and their bidirectional lineage differentiation capacity in vivo is weak, making it difficult for them to stably participate in the construction of the inner cell mass and trophoblast lineage of chimeric embryos, greatly limiting the application and in-depth research of early embryonic development models. Furthermore, the key regulatory genes and molecular mechanisms driving human embryonic stem cells back to the eight-cell early developmental stage, maintaining pluripotency, and enhancing bidirectional differentiation potential are still not fully elucidated. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide the application of BCL2 in regulating the pluripotency state of human embryonic stem cells and / or activating 8-cell-like cells. This overcomes the problems of low conversion efficiency from primordial to primitive state, unstable maintenance of pluripotency, limited induction efficiency of 8-cell embryonic-like cells, easy apoptosis and differentiation during long-term culture, difficulty in maintaining pluripotency characteristics stably, and insufficient bidirectional lineage differentiation ability in existing technologies. This provides a new strategy for efficiently obtaining and stably culturing primitive human embryonic stem cells and 8-cell embryonic-like cells.

[0006] The objective of this invention is achieved through the following technical solution: This invention provides the application of reagents that increase BCL2 gene expression levels and / or increase BCL2 protein levels in the preparation of reagents or kits for regulating the pluripotency state of human embryonic stem cells.

[0007] Preferably, the amino acid sequence of BCL2 is shown in SEQ ID NO.1.

[0008] Preferably, the human embryonic stem cells include primordial human embryonic stem cells and / or primitive human embryonic stem cells.

[0009] Preferably, the regulation of the pluripotency state of human embryonic stem cells includes at least one of the following: (1) Promotes the transformation of human embryonic stem cells from the primordial state to the primitive state; (2) Maintaining the pluripotency of human embryonic stem cells; (3) Promote the transformation of primitive human embryonic stem cells into 8-cell embryo-like cells.

[0010] This invention provides a method for in vitro enhancement of the pluripotency of human embryonic stem cells or in vitro induction of human embryonic stem cells to transform into cells of the early embryonic stage, comprising: Upregulates the expression level of the BCL2 gene and / or the protein level of BCL2 in human embryonic stem cells.

[0011] This invention provides the application of reagents that increase BCL2 gene expression levels and / or increase BCL2 protein levels in the preparation of reagents or kits for inducing the transformation of human embryonic stem cells into 8-cell embryo-like cells.

[0012] Preferably, the application includes at least one of the following: (I) Promotes the transformation of human embryonic stem cells into 8-cell embryo-like cells; (II) Maintaining the long-term stable self-renewal of 8-cell embryo-like cells; (III) Maintain the totipotency of 8-cell embryo-like cells; (IV) Enhance the bidirectional lineage differentiation potential of 8-cell embryo-like cells.

[0013] This invention provides a method for in vitro induction of human embryonic stem cell transformation into 8-cell embryo-like cells, comprising: Upregulate the expression level of the BCL2 gene and / or upregulate the BCL2 protein level in the human embryonic stem cells.

[0014] The present invention provides a human 8-cell embryonic-like cell, which is obtained by increasing the expression level of the BCL2 gene and / or the BCL2 protein level in human embryonic stem cells.

[0015] The present invention provides a primer set for identifying whether the pluripotency of human embryonic stem cells is enhanced, characterized in that it includes: hBCL2-F as described in SEQ ID NO.4 and hBCL2-R as described in SEQ ID NO.5.

[0016] The beneficial effects of this invention are: This invention provides the application of reagents that increase BCL2 gene expression and / or BCL2 protein levels in the preparation of reagents or kits for regulating the pluripotency of human embryonic stem cells. This invention clarifies for the first time the role of BCL2 in the regulation of human embryonic stem cell pluripotency and the induction of early embryonic cells. By upregulating BCL2 expression, it can effectively promote the conversion of human embryonic stem cells from the primordial state to the primitive state, stably maintain the pluripotency of human embryonic stem cells, and efficiently induce the transformation of human embryonic stem cells into 8-cell embryo-like cells. It effectively maintains the long-term stable self-renewal and pluripotency molecular characteristics of 8-cell embryo-like cells, significantly enhances the differentiation potential of 8-cell embryo-like cells, enabling them to participate in the construction of chimeric embryos in vivo and simultaneously contribute to the inner cell mass and trophoblast. This overcomes the problems of low induction efficiency of 8-cell embryo-like cells, easy apoptosis and differentiation during long-term culture, unstable maintenance of pluripotency, and limited lineage differentiation ability in existing technologies. This invention verifies the role of BCL2 in regulating the pluripotency of human embryonic stem cells and promoting the acquisition of 8-cell embryo-like cells. It provides a valuable cell model for studying the human zygote genome activation (ZGA)-like process under in vitro conditions, provides a new technical means for studying early embryonic development, and provides good theoretical support for research on human early embryonic development and stem cell-derived embryonic models. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 To generate stable BCL2 Schematic diagram of the piggyBac transposon system overexpressing human initial state ESC; Figure 2 for BCL2Representative morphologies of overexpressed hESCs and WT hESCs, scale bar: 100 μm; Figure 3 for BCL2 Overexpression of hESCs and WT hESCs BCL2 Figure 1 shows the qPCR analysis results of pluripotent genes; error bars represent mean ± standard deviation (n = 3); p-values ​​were calculated using a two-tailed t-test, ** indicates p < 0.01, *** indicates p < 0.001, n = 3 biological replications; Figure 4 for BCL2 Immunoblot analysis of BCL2 protein in overexpressed hESCs and WT hESCs; Figure 5 A schematic diagram of the experimental design for converting initial-state ESCs into pristine-state ESCs using 4CL culture medium; Figure 6 Representative morphological diagrams of WT and BCL2_OE ESCs in 4CL medium; scale bar, 100 micrometers; Figure 7 To investigate the WT and BCL2_OE ESC core pluripotent genes and Na in 4CL medium qPCR analysis of the ve marker gene; error bars represent mean ± standard deviation (n = 3); p-values ​​were calculated using a two-tailed t-test, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and so on; n = 3 biological replications; Figure 8 For WT and BCL2 Western blot analysis of OCT4, NANOG, and SOX2 protein levels in OE ESC; Figure 9 A schematic diagram of the experimental design for inducing the transformation of primitive human ESCs into 8C-like cells (8CLCs) using e4CL culture medium; Figure 10 After 6 days of e4CL induction, WT and BCL2 Representative image of the primitive human ESC in OE; scale bar, 100 micrometers; Figure 11 For WT and BCL2 Figure _Immunochromatographic analysis of OE embryonic stem cells after 6 days of induction in e4CL medium; C represents the results of immunostaining after 6 days of e4CL induction. BCL2 Immunostaining images of key pluripotency proteins OCT4, SOX2, and NANOG in OE ESCs; scale bar, 50 μm; D is the immunostaining image of key pluripotency proteins OCT4, SOX2, and NANOG in WT ESCs after 6 days of e4CL induction; scale bar, 50 μm; E is the immunostaining image of key pluripotency proteins OCT4, SOX2, and NANOG in WT ESCs after e4CL induction. BCL2 Immunostaining images of TPRX1 and ZSCAN4 in _OE ESCs; scale bar, 50 μm; F is an immunostaining image of TPRX1 and ZSCAN4 in WT ESCs after e4CL induction; scale bar, 50 μm; Figure 12 Key 8CLC-related genes TPRX1, TRIM43, LEUTX, ZSCAN4, RFPL4A, H3. X / Y In WTESCs and BCL2 Relative RNA expression levels in OE ESCs; error bars represent mean ± standard deviation (n = 3); p-values ​​were calculated using a two-tailed t-test, n = 3 biological replications; Figure 13 Western blot analysis results for the expression of TPRX1, DUX, ZSCAN4, DPPA5 and NELFA proteins; Figure 14 For e4CL conditions, WT and BCL2 Morphological images and immunostaining images of core pluripotent proteins in long-term culture of OE ESCs; Top image shows morphology, scale bar 100 μm; Bottom image shows immunostaining images of core pluripotent proteins OCT4, SOX2, and NANOG, scale bar 50 μm. Figure 15 After 18 days of e4CL culture, WT and BCL2 Relative expression levels of core 8C-like genes in _OE ESC; error bars represent mean ± standard deviation (n = 3); p-values ​​were calculated using a two-tailed t-test, n = 3 biological replications; Figure 16 For WT and BCL2 Western blot validation images of the selected 8C-like markers TPRX1, ZSCAN4, DUX, NELFA, and TP53 from _OE ESC; Figure 17 For in WT and BCL2 Immunostaining verification diagram of 8C-like markers TPRX1 and ZSCAN4 in OE ESC; scale bar, 50 micrometers; Figure 18 for BCL2 Immunostaining image of eight-cell embryos injected with OE ESCs; A represents eight-cell embryos injected with mCherry-labeled... BCL2 Representative images of chimeric blastocysts immunostained with OE ESCs after 52 hours of in vitro culture, using OCT4 and GATA3 antibodies; scale bar, 50 μm; B indicates quantification. BCL2 Contribution of OE ESCs to ICM and TE lineages in chimeric embryos (n=35); Figure 19Immunostaining image of eight-cell embryos injected with WT ESCs; representative image of chimeric blastocysts immunostained with OCT4 and CDX2 antibodies after eight-cell embryos were injected with mCherry-labeled e4CL_WT ESCs and subsequently cultured in vitro for 52 hours; cell nuclei stained with DAPI; scale bar, 50 μm. Detailed Implementation

[0019] This invention provides the application of reagents for increasing BCL2 gene expression levels and / or increasing BCL2 protein levels in the preparation of reagents or kits for regulating the pluripotency state of human embryonic stem cells. In this invention, the amino acid sequence of BCL2 is shown in SEQ ID NO.1; encoding... BCL2 The mRNA sequence is shown in SEQ ID NO.2; encoding BCL2 The CDS sequence is shown in SEQ ID NO.3.

[0020] As an optional embodiment of the present invention, the human embryonic stem cells include primordial human embryonic stem cells and / or primitive human embryonic stem cells. As an optional embodiment of the present invention, the regulation of the pluripotency state of human embryonic stem cells includes at least one of the following: (1) promoting the conversion of human embryonic stem cells from the primordial state to the primitive state; (2) maintaining the pluripotency state of human embryonic stem cells; (3) promoting the conversion of primitive human embryonic stem cells to 8-cell embryo-like cells. The results of the embodiments of the present invention show that by increasing the expression level of the BCL2 gene and the BCL2 protein level in human embryonic stem cells, the conversion of primordial human embryonic stem cells (primed hESCs) to primitive human embryonic stem cells (na) can be effectively promoted. BCL2 overexpression significantly accelerated the transformation of cell morphology from a flat, loose primordial state to a dense, three-dimensional primitive "dome-shaped" clone in the 4CL-induced culture system. Simultaneously, the expression levels of core pluripotency markers such as OCT4, SOX2, and NANOG were significantly upregulated, along with primitive-state-specific genes. TFCP2L1, DNMT3L, KLF4The expression of BCL2 gene and the level of BCL2 protein are also significantly activated, resulting in higher pluripotency. The results of this invention through examples show that increasing BCL2 gene expression and BCL2 protein levels can stably maintain the pluripotency characteristics of human embryonic stem cells, especially primitive human embryonic stem cells, during long-term in vitro culture. BCL2 overexpression can effectively inhibit apoptosis and stress damage under in vitro culture conditions, maintain stable cell self-renewal capacity, and prevent spontaneous differentiation or dedifferentiation; simultaneously, through continuous high expression of the core regulatory network of pluripotency, it ensures that cells maintain homogeneity and stability during passage and long-term culture. The results of this invention through examples show that upregulating BCL2 expression can further promote the efficient transformation of primitive human embryonic stem cells into 8-cell embryo-like cells (8CLCs). BCL2 overexpression can significantly promote the induction of 8-cell embryo-like cells and promote… DUX, TPRX1, ZSCAN4, NELFA, H3.X / Y The stable and high expression of the core characteristic genes of 8CLC enables the induced 8-cell embryo-like cells to possess typical 8-cell embryonic transcriptional characteristics and pluripotent molecular markers, thereby significantly improving the induction efficiency and obtaining a more stable 8-cell embryo-like cell population.

[0021] This invention provides a method for in vitro enhancement of the pluripotency of human embryonic stem cells (HECs) or in vitro induction of HECs into early embryonic stage cells, comprising: upregulating the expression level of the BCL2 gene and / or upregulating the BCL2 protein level in HECs. By upregulating the gene transcription and protein translation levels of BCL2 in HECs, this invention can stably regulate the pluripotency state of HECs, promoting efficient conversion of primordial HECs into primitive HECs and maintaining pluripotency homeostasis. It can also drive further reprogramming of primitive HECs to achieve targeted conversion into 8-cell embryo-like cells in the early embryonic stage, effectively activating the unique transcriptional characteristics and developmental potential of early embryos. This invention does not specifically limit the upregulation method; any technical solution that can significantly increase the intracellular BCL2 gene expression level and / or BCL2 protein level using conventional molecular biology techniques falls within the scope of protection of this invention. As an optional embodiment of this invention, the upregulation method can be to introduce an overexpression vector containing the BCL2 gene into HECs, and continuously achieve BCL2 gene overexpression by utilizing the stable expression of the vector within the HECs. As an optional embodiment of the present invention, the overexpression vector can be constructed using the piggyBac transposon system, which can achieve stable integration and long-term expression of the target gene in stem cells. The present invention does not impose any special limitations on the construction method of the BCL2 overexpression vector; any recombinant vector capable of stably driving efficient expression of the BCL2 gene in human cells can be constructed using conventional cloning, vector construction, and element assembly methods in the art.

[0022] This invention provides the application of reagents that increase BCL2 gene expression levels and / or BCL2 protein levels in the preparation of reagents or kits for inducing the transformation of human embryonic stem cells into 8-cell embryo-like cells. As an optional embodiment of this invention, the application includes at least one of the following: (I) promoting the transformation of human embryonic stem cells into 8-cell embryo-like cells; (II) maintaining the long-term stable self-renewal of 8-cell embryo-like cells; (III) maintaining the totipotency of 8-cell embryo-like cells; (IV) enhancing the bidirectional lineage differentiation potential of 8-cell embryo-like cells. The results of this invention, through its embodiments, demonstrate that increasing the expression level of the BCL2 gene and the level of the BCL2 protein can directionally induce the transformation of primordial human embryonic stem cells into 8-cell embryo-like cells, activate the specific transcriptional characteristics of 8-cell embryos, and improve the induction efficiency of 8-cell embryo-like cells; maintain the long-term stable self-renewal capacity of 8-cell embryo-like cells under long-term e4CL culture conditions in vitro, inhibit environmental stress, passage damage, and apoptosis in vitro, reduce spontaneous cell differentiation, and ensure morphological stability and proliferation capacity during continuous cell passage; stably maintain the totipotency molecular characteristics of 8-cell embryo-like cells, continuously and highly express core totipotency markers such as TPRX1, ZSCAN4, DUX4, and LEUTX, and stabilize the gene expression profile unique to early embryonic cells; enhance the bidirectional lineage differentiation potential of 8-cell embryo-like cells, enabling them to possess in vivo chimeric development capabilities, and simultaneously differentiate and integrate into the inner cell mass and trophoblast of chimeric embryos, breaking through the lineage differentiation limitations of traditional human embryonic stem cells and fully utilizing the developmental potential of early embryo-like cells. The results of the embodiments of the present invention show that BCL2 overexpression induces human ESCs to acquire the characteristics of 8-cell embryo-like cells and enables them to have extended pluripotency, which can simultaneously contribute to the ICM and TE lineages of chimeric blastocysts.

[0023] The present invention provides a method for in vitro induction of human embryonic stem cells into 8-cell embryo-like cells, comprising: upregulating the expression level of the BCL2 gene and / or upregulating the BCL2 protein level in the human embryonic stem cells.

[0024] This invention provides a human 8-cell embryo-like cell, obtained by increasing the expression level of the BCL2 gene and / or the BCL2 protein level in human embryonic stem cells. The cell sustainably and highly expresses one or more of the 8-cell embryo-specific markers TPRX1, ZSCAN4, DUX, NELFA, and H3.X / Y, stably maintaining the transcriptional characteristics specific to the 8-cell embryonic stage. Simultaneously, the cell possesses excellent in vivo developmental and differentiation potential. After microinjection into mouse 8-cell stage embryos, it can effectively participate in the construction of chimeric embryos, simultaneously differentiating and integrating into both the inner cell mass and trophoblast of the chimeric embryo, overcoming the limitation of traditional human embryonic stem cells being confined to embryonic lineage differentiation. This cell can stably persist in chimeric tissues without significant differentiation abnormalities, exhibiting significantly superior bidirectional chimerism compared to wild-type control cells, possessing both stable pluripotency and broadened differentiation potential. As an optional embodiment of this invention, the cell may also stably express mCherry fluorescent markers, facilitating cell tracking, positive screening, and chimerism assay localization. The cells provided by this invention can be widely used in research on early human embryonic development mechanisms, lineage differentiation regulation, and in vitro embryonic model construction.

[0025] This invention provides a primer set for identifying whether the pluripotency of human embryonic stem cells is enhanced, comprising: hBCL2-F as described in SEQ ID NO.4 and hBCL2-R as described in SEQ ID NO.5. Using this primer set, real-time quantitative PCR of human embryonic stem cell cDNA can determine the BCL2 gene expression level. A significant increase in BCL2 gene expression indicates enhanced pluripotency of human embryonic stem cells.

[0026] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0027] The human embryonic cell line used in this invention is the H9 human embryonic cell line, which originated from Professor Jie Wei of Tsinghua University.

[0028] Primed hESCs were cultured using mTeSR1 or E8 culture systems.

[0029] Human primed embryonic stem cell line, H9 used in this invention, was routinely cultured on Matrigel (Corning, 354230) plates using mTeSR1 medium (Stemcell Technologies, 85850) or E8 medium (Thermo Fisher, A1517001), following the manufacturer's instructions. Typically, ESCs were passaged every 3-4 days, with the medium changed daily. Before passage, cells were washed once with DPBS (Thermo Fisher, 14200075), then treated with 0.5 mM EDTA (Thermo Fisher, 15575020) for 5 minutes. Digestion was then stopped with a stop solution, and cells were passaged as single cells. Cells were cultured in an incubator at 37°C and 5% CO2. All cell lines were confirmed to be mycoplasma-negative.

[0030] Termination solution formulation: 90% DMEM / F12 (Gibco, 11320-033); 10% Fetal Bovine Serum (Gibco, 10099-141); 1X Penicillin-Streptomycin (Gibco, 15140122).

[0031] The primer sequences involved in the following technical solutions are shown in Tables 1 and 2.

[0032] Table 1 Primer Information

[0033] Table 2 Primer Information II

[0034] Example 1 1. BCL2 (human) has a Gene ID (Entrez ID) of 596 in NCBI. BCL2 chromosome location: 18q21.33. BCL2 For detailed mRNA information, please refer to NCBI Reference Sequence: NM_000633.3. For detailed amino acid sequence information of BCL2, please refer to NCBI Reference Sequence: NP_000624.2.

[0035] The amino acid sequence of BCL2 is shown in SEQ ID NO.1, specifically: MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPHPAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLGHK。

[0036] The mRNA sequence encoding BCL2 is shown in SEQ ID NO.2, specifically:

[0037] coding BCL2 The CDS sequence is shown in SEQ ID NO.3, specifically as follows: .

[0038] 2. BCL2 OE cell line construction process.

[0039] Stable [process] was generated using the piggyBac transposable subsystem under the control of the CAGGS promoter. BCL2 Overexpression of human initial state ESCs ( BCL2 _OE ESC), mCherry as a fluorescent reporter (e.g. Figure 1 ).

[0040] 1) Construction of overexpression vector system: Construction of a PiggyBac-based induction system BCL2 The overexpression vector, namely the PB-CAG-BCL2-mCherry expression plasmid, was constructed using the PiggyBac system. BCL2 Gene overexpression plasmids were used to achieve stable integration and induced expression of the vector. For specific methods, please refer to the relevant literature [Zhao L, Gao X, Zheng Y, et al.Establishment of bovine expanded potential stem cells[J]. Proceedings of the National Academy of Sciences, 2021, 118(15): e2018505118.].

[0041] The specific steps are as follows: (1) In order to construct the overexpression recombinant plasmid, targeting BCL2 Gene-specific primers (upstream primer, as shown in SEQ ID NO.38, specifically: TCATTTTGGCAAAGAATTCATGGCGCACGCTGGGAGAACA; downstream primer, as shown in SEQ ID NO.39, specifically: TTGACAGGAGCGACAATTTTACTAGACTTGTGGCCCAGATAGGCAC) were designed to amplify from the hESCscDNA template. BCL2 Target gene CDS sequence. First, total RNA was extracted from well-primed hESCs, and cDNA was obtained by reverse transcription as amplification. BCL2 The DNA template of the gene. Obtained through amplification. BCL2 The gene CDS product showed a clear target band at 750 bp, and this fragment was recovered via gel electrophoresis. To obtain the PiggyBac vector backbone for subsequent ligation experiments, the vector was double-digested with EcoRI and BamHI restriction endonucleases. The digestion reaction was performed at 37℃ for 30 min in a PCR instrument. The vector size was then detected by agarose gel electrophoresis. A band of the expected size was detected at 5000–8000 bp. The linearized vector was confirmed to meet the requirements of the standard ligation reaction through gel recovery, purification, and concentration detection. The amplified... BCL2The target gene fragment and the linearized vector were ligated using T4 DNA ligase. The ligation product was heat-shocked, transformed into competent cells, plated, and incubated overnight at 37°C. Single colonies were observed on LB agar plates after 12–16 h. Ten to fifteen single colonies were randomly selected and placed in ampicillin-containing LB broth for amplification at 37°C and 250 rpm for 3 h. Colony PCR was used to verify the inserted fragment. The results showed that all colonies exhibited specific amplification bands consistent with the expected size, indicating successful recombination of the target gene and vector backbone. To further confirm the ligation of the target fragment and vector, the bacterial solutions corresponding to these positive colonies were sent to a sequencing company for DNA sequencing. The recombinant plasmid... BCL2 The sequencing results of the coding region were completely consistent with the known sequences in the GenBank database, and no base mutations or deletions were detected, indicating that the recombinant plasmid was successfully constructed. Subsequently, colonies with correct sequencing results were selected for amplification culture. 20 μL of positive bacterial culture was inoculated into 45 mL of LB liquid medium containing ampicillin and cultured at 37°C with shaking at 250 rpm for 12-16 h. The plasmid was then extracted using a kit to obtain the recombinant plasmid, which was subsequently named PB-CAG-BCL2-mCherry. The extracted plasmid was used for subsequent experiments.

[0042] (2) Cell transfection: Recombinant plasmids were co-introduced into human primed embryonic stem cells (hESCs) using liposome transfection reagent to establish cells. BCL2 Overexpression cell lines were established by co-introducing PB-CAG-BCL2-mCherry (2 μg), PB-EF1α-transposase plasmid (2 μg), and PB-EF1α-rTTA plasmid (2 μg) into human primed embryonic stem cells using liposome transfection reagent. BCL2 Overexpression cell lines. Stable integration of the exogenous gene into the genome was achieved using the piggyBac transposon system. Transfection was performed using the Lipofectamine Stem Reagent kit. After transfection, cells were selected using puromycin (3 μg / mL) for 24–48 hours to remove untransfected cells. Surviving cells were then seeded into 96-well plates for single-cell clonal culture using mTesR medium. The ROCK inhibitor Y-27632 (10 μM) was added to promote single-cell survival. Y-27632 was removed after 24 hours of culture, and the medium was changed daily. Wells forming single clones were selected for amplification culture.

[0043] (3) Culture and passage of hESCs: Before passage, the culture medium needs to be preheated. After thawing, replace the medium with fresh medium 24 hours later and remove any dead cells that have not adhered. Generally, hESCs are passaged every 3-4 days, and the medium is replaced daily. Observe the clones with clear edges and close arrangement. If there are many cell fragments, gently rinse with DPBS 1-2 times before replacing the medium. When the clone density reaches 70%-80%, discard the old medium, avoid touching the cell layer at the bottom of the well plate, gently rinse once with DPBS, add 200 µL of TrypLE for digestion, and let it stand in the cell culture incubator for about 3 minutes until about 60% of the adhered cells are in suspension under a microscope. Add 800 µL of stop solution to stop the digestion, and gently pipette (5-10 times) until a single cell or small cell cluster suspension is formed. Collect the suspension in a 1.5 mL centrifuge tube and centrifuge at 1300 rpm for 3 minutes. Discard the supernatant and add an appropriate volume of mTeSR TM 1. Resuspend the cell pellet in culture medium, discard excess Matrigel from the well plate, and seed the cells into Matrigel-pretreated culture dishes at a ratio of 1:3 to 1:6. Gently shake the culture dishes until the cells are uniformly observed under a microscope, then transfer them to a cell culture incubator for further culture and observe the cells the next day.

[0044] 4. To BCL2 Fluorescence microscopy was performed on overexpressed hESCs and WT hESCs. See details below. Figure 2 Where WT stands for WT hESCs. BCL2 _OE is BCL2 Overexpression of hESCs; Left image: 38th generation wild-type (WT) human embryonic stem cells; Right image: 14th generation BCL2 Overexpression ( BCL2 Human embryonic stem cells (OE). From Figure 2 From this, we can conclude BCL2 Ectopic expression did not alter the key morphological characteristics of the human initial state ESC.

[0045] 5. To BCL2 RT-qPCR was performed on overexpressed hESCs and WT hESCs to detect the expression levels of related genes. Primer sequences are detailed in Table 1.

[0046] RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, 74104). cDNA was obtained via reverse transcription using a Reverse Transcription System (Qiagen, 205311). Real-time quantitative PCR reactions were performed in duplicate using the KAPASYBR FAST qPCR kit (KAPA Biosystems, KK4601), and each experiment was repeated at least three times independently, with similar results. GAPDH Expression levels were used as internal controls. All samples were analyzed using the LightCycler®96 Instrument (Roche Molecular Systems). Relative expression levels were calculated using the 2-ΔΔCt method.

[0047] The specific steps for extracting RNA and reversing cDNA are as follows: Total RNA was extracted from cells using the RNasy Plus Mini kit. To prevent RNA degradation during extraction, the high-speed centrifuge was pre-cooled to 4°C, the work surface and pipettes were wiped with RNase inhibitor spray, enzyme-free pipette tips were used, masks were worn throughout the process, and ice was prepared (the entire procedure was performed on ice). The extraction method is as follows: ① Add 350 µL buffer RLT to a 1.5 mL sample tube (cell pellet) for lysis, mix well and let stand for 10 min; ② Add an equal volume of 70% ethanol and mix well; ③ Transfer the obtained 700 µL liquid to a 2 mL centrifuge column and centrifuge at 9000 rpm for 1 min; ④ Discard the liquid, add 700 µL of buffer RW1, and centrifuge at 9000 rpm for 1 min; ⑤ Discard the liquid, add 500 µL of buffer RPE, and centrifuge at 9000 rpm for 1 min; ⑥ Discard the liquid, add 500 µL of buffer RPE, and centrifuge at 9000 rpm for 2 min; ⑦ Discard the liquid, place the centrifuge column on a new 1.5 mL collection tube, add 30-50 µL of enzyme-free water dropwise, and centrifuge at 9000 rpm for 1 min. The liquid in the collection tube is the cell RNA. ⑧ After determining the concentration, prepare for reverse cDNA processing, or store in an ultra-low temperature freezer at -80℃ for later use.

[0048] Template cDNA Acquisition The steps for obtaining cDNA using the QuantiTect® Reverse Transcription Kit are as follows: First, determine the RNA loading amount based on the concentration, and proceed in two steps. The first step is the removal of genomic DNA. Add 1 µg RNA, 2 µL gDNA wipeout buffer (7×), and enzyme-free water to a final volume of 14 µL. Place the mixture in a PCR instrument and incubate at 42°C for 2 min. Immediately after the reaction, incubate on ice for 5 min. The second step is the reverse transcription reaction. Add 4 µL Quantiscript RT Buffer (5×), 1 µL Quantiscript Reverse Transcriptase, 1 µL RT Primer Mix, and x µL RNA to obtain template cDNA. If not used immediately, it can be stored at -20°C for later use.

[0049] BCL2 Overexpression of hESCs and WT hESCs BCL2 , OCT4 , SOX2 and NANOG The relative expression level, such as Figure 3 As shown. By Figure 3 The results showed that: BCL2 Overexpression only leads to BCL2 Pluripotency-related genes in _OE ESCs include the upregulation of OCT4, NANOG, and SOX2.

[0050] 6. To BCL2 Western blot analysis was performed on the BCL2 protein levels in overexpressed hESCs and WT hESCs, and the results are as follows: Figure 4 As shown.

[0051] Protein extraction: 1) The entire protein extraction process is carried out on ice, and the centrifuge is pre-cooled to 4°C. (2) When the cell confluence reaches 80%, digestion is performed. After digestion is terminated, centrifuge at 1300 rpm for 3 min, discard the supernatant, add 1 mL of DPBS to resuspend, and centrifuge again. (3) The lysis buffer should be protected from light and prepared fresh. Prepare the lysis buffer according to the ratio of lysis buffer: phosphatase inhibitor: protease inhibitor = 100: 10: 1. After mixing, resuspend the cell pellet with the prepared lysis buffer, mix well, and place at -20℃ or on ice with shaking for 20 min. (4) Centrifuge the suspension from step four at 4°C and 12,000 rpm for 10 min and collect the supernatant. (5) Concentration measurement: Take 15 μL from the total supernatant, dilute the protein supernatant with the lysis buffer at a ratio of 1:4, and then use Pierce to measure the concentration. TM According to the instructions, prepare the A:B mixture in a ratio of 50:1, using solution A:solution B. Add the mixture three times for each sample to a 96W cell culture dish (protected from light) and incubate at 37°C for 30 minutes. Use a microplate reader to test the concentration.

[0052] (6) Cooking the protein: Dilute the remaining supernatant with 6× loading buffer at a ratio of 1:5, then heat in boiling water at 100°C for 5 minutes, cool to room temperature and store in a -80°C refrigerator for later use.

[0053] 2) Western Blot: (1) Preparation: Separating and stacking gels were prepared, with the gel concentration selected based on the molecular weight of the target protein. In this chapter, a 10% gel with a thickness of 1.5 mm was used. The gel preparation system was prepared according to the one-step PAGE gel preparation kit (10%) (separating gel: 4 mL lower gel solution, 4 mL lower gel buffer, 80 μL modified coagulant; stacking gel: 1 mL upper gel solution, 1 mL upper gel buffer, 20 μL modified coagulant). β-ACTIN was used as the internal control protein in all Western blotting experiments in this study.

[0054] (2) Sample loading: First, add 1× running buffer to the electrophoresis tank, following the principle of higher outside and lower inside to ensure the stability of the subsequent electrophoresis process. Select an appropriate electrophoresis program according to the molecular weight of the target protein. In this experiment, the program used was set to run at 60 V for 30 min to allow the sample to enter the separating gel smoothly, and then adjust the voltage to 90 V and continue electrophoresis for 2 h to ensure that the protein is fully separated; (3) Transfer treatment: The transfer method used was wet transfer. Pretreatment: Immerse the PVDF membrane in methanol for 15 seconds to activate it, then place it in distilled water and swirl until stopped, then soak for 10 minutes. Place the sponge, gel, and pretreated PVDF membrane together in 1× transfer buffer and oscillate for 30 minutes. Place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge sequentially on the negative electrode plate of the transfer tank. After each layer, gently roll it with a glass rod or similar tool to carefully remove air bubbles, avoiding residual air bubbles that could lead to uneven protein transfer or even failure. Select 180 V voltage and 300 mA current, and continue transfer for 50 minutes to ensure efficient and complete protein transfer to the PVDF membrane.

[0055] (4) Blocking: Prepare a 5% blocking solution (dissolve 2 g of skim milk powder in 40 mL of 1× TBST), shake until the milk powder is completely dissolved to form a uniform blocking solution. After the transfer is completed, immediately immerse the PVDF membrane in the blocking solution and continue to block it on a shaker at room temperature for 1 h to block the non-specific binding sites on the membrane. (5) After the sealing is completed, wash the membrane with 1× TBST on a shaker for 5 minutes each time, and wash three times to remove unbound impurities and residual sealing solution. (6) Incubation of primary antibody: Prepare primary antibody according to the species of antibody used and the recommended dilution ratio. Then, according to the molecular weight of the target protein, cut the PVDF membrane into appropriate strips, put the cut membrane strips into the prepared primary antibody, ensure that the membrane strips are completely immersed, and place them in a 4°C refrigerator. It is best to incubate them overnight on a shaker with slow shaking to allow the primary antibody to fully bind to the target protein. (7) Washing primary antibody: Wash the membrane with 1× TBST on a shaker for 10 min each time, three times, to completely wash away unbound primary antibody and avoid non-specific signals in subsequent experiments; (8) Incubation of secondary antibody: Prepare secondary antibody according to the species and dilution ratio of the antibody, put it into the corresponding secondary antibody, and shake on a shaker at room temperature for 1 h; (9) Washing secondary antibody: Wash the membrane with 1× TBST on a shaker for 10 min each time, three times, to remove unbound secondary antibody and reduce background interference; (10) According to Pierce TM The instructions for the ECL Western Blotting Substrate kit require that the chromogenic solution be prepared in a dark environment. After preparation, the chromogenic solution is evenly added to the PVDF membrane. Then, the protein bands on the membrane are detected and observed using a chemiluminescence imaging system or traditional X-ray exposure, with the exposure time selected according to the actual situation. The expression of the target protein is analyzed based on the presence, intensity, and position of the bands.

[0056] Based on the above analysis, it can be concluded that this experiment successfully constructed a BCL2-overexpressing human primordial embryonic stem cell line with stable genetic background and good pluripotency. This provides a cellular tool and experimental basis for subsequent research on the function of BCL2 in human embryonic stem cell 8-cell-like transformation and extended pluripotency regulation.

[0057] Example 2 Human embryonic stem cells (hESCs), possessing unlimited proliferative capacity and multipotent differentiation potential, have become an important cellular resource for regenerative medicine and developmental biology research. The pluripotent states of hESCs are generally classified into two types: primordial and primitive. Primitive hESCs, due to their developmental characteristics being closer to the inner cell mass of the preimplantation blastocyst in humans, exhibit higher levels of pluripotent gene expression and lower DNA methylation levels, providing a good in vitro research model for studying early human development and cell reprogramming.

[0058] Explore BCL2 Functions and roles exhibited by human embryonic stem cells during the transition from the initial state to the primitive state.

[0059] 1. Using the established 4CL medium, human primordial states were cultured... BCL2 _OE ESC transforms into the original state (e.g.) Figure 5 ).

[0060] To generate Na using 4CL culture medium ve hESCs, first put WT primed hESCs and BCL2 OE-primed hESCs were washed once with DPBS, digested with TrypLE, and distilled at 1000–1500 cells / cm². 2 Cells were seeded at a density of [insert density here] on Matrigel-pretreated culture plates and cultured in mTeSRTM1 medium. After 24 hours, the medium was replaced with 4CL medium. The 4CL medium was changed daily, and cells were passaged every 3-4 days. Cells were cultured in an incubator at 37°C and 5% CO2.

[0061] The 4CL medium consisted of a 1:1 mixture of Neurobasal medium (Gibco, 21103049) and Advanced DMEM / F12 (Gibco, 12634010), with the addition of 0.5% N2 additive (Gibco, 17502048), 1% B27 additive (Gibco, 17504044), 1% Glutamax (Gibco, 35050061), 1% MEM NEAA (Gibco, 11140050), 100 U penicillin / streptomycin (Gibco, 15140122), 1 mM sodium pyruvate (Gibco, 11360070), 0.1 mM β-mercaptoethanol (Gibco, 21985023), 10 nM DZNep (Selleck, S7120), and 5 nM... TSA (Sigma, T8552), 1 μM PD0325901 (Miltenyi biotec, 130-103-923), 5 μM IWR-1 (Selleckchem, S7086), 20 ng / mL human LIF (Peprotech, 300-05), 20 ng / mL ACTIVIN A (Peprotech, 338-AC), 50 μg / mL L-ascorbic acid (Sigma, 49752), and 0.2% (v / v) matrix gel.

[0062] WT and BCL2 _OE hESCs from primed to na VE conversion was detected 25 days after induction.

[0063] The 4CL culture method is described in the reference [Mazid, Md Abdul, et al. Rolling back humanpluripotent stem cells to an eight-cell embryo-like stage. Nature, 605.7909(2022): 315-324.].

[0064] 2. In BCL2 Embryonic stem cells overexpressing hESCs and WT hESCs were observed under a fluorescence microscope on days 7 and 25 of culture in 4CL medium. The results are detailed below. Figure 6 .Depend on Figure 6 Therefore, compared to WT ESC, BCL2 _OE ESCs are morphologically closer to the original "dome-shaped" clone.

[0065] 3. Considering that morphological similarity is insufficient to conclude that cells have acquired primitive pluripotent status, further gene expression analysis was performed. BCL2 On day 25 of culture of overexpressing hESCs and WT hESCs in 4CL medium, the expression levels of the pluripotency markers were quantified by RT-qPCR, using the same method as in Example 1. Primer sequences are detailed in Table 1. Results are detailed below. Figure 7 .Depend on Figure 7 We can obtain, BCL2 _OE ESCs in core pluripotent genes ( OCT4, NANOOG, and MYC ) and primitive state marker genes ( TFCP2L1, DNMT3L and KLF4 The expression level of ) was significantly higher than that of WT ESCs.

[0066] 4. For those cultured for 25 days BCL2 Immunofluorescence staining and Western blotting were performed on overexpressed hESCs and WT hESCs, and the results are as follows: Figure 8 .show BCL2 The levels of OCT4, NANOG, and SOX2 were significantly higher in _OE ESCs.

[0067] In summary, BCL2 Promoting the transition of human embryonic stem cells (hESCs) from their primordial to primitive state demonstrates the role and application of BCL2 in regulating hESC pluripotency, specifically driving the transformation of hESCs into early embryonic stem cell stages. This provides new insights for efficiently obtaining stable primitive-state hESCs. This research not only deepens our understanding of the self-renewal network of primitive-state hESCs but also provides cellular resources for establishing stem cell-based regenerative medicine research models. This study is significant for a deeper understanding of the molecular mechanisms of BCL2 in stem cell fate determination and provides a theoretical basis for developing novel stem cell therapy strategies.

[0068] Example 3 BCL2 Promote the transformation of human embryonic stem cell 8C-like cells Early embryonic development is a complex biological process, with the 8-cell stage playing a crucial role as a critical period for zygotic genome activation. During this stage, embryonic cells are considered to possess totipotency and exhibit unique transcriptional characteristics, laying the molecular foundation for subsequent cell differentiation and normal embryonic development.

[0069] Efficiently activating and stably maintaining the 8CLC characteristics has become a major challenge for scientists. Zygote genome activation (ZGA) occurs in the eight-cell stage of human embryos. As a key event in early development, understanding the molecular mechanisms regulating ZGA is crucial for human reproductive health. However, due to limitations in embryo availability and ethical considerations, obtaining a sufficient number of human embryos for research remains challenging. Therefore, embryonic stem cells have become an ideal model system for in vitro studies of early embryonic development.

[0070] Explore BCL2 Does overexpression promote human na? ve ESC (Na 8CLC induction in primitive human embryonic stem cells.

[0071] 1. In order to be in na Inducing 8CLCs in ve hESCs, first WT and BCL2 _OE na ve hESCs were washed once with DPBS, digested into single cells with TrypLE, and then sputtered at 2000-3000 cells / cm². 2 Cells were seeded at a density of 4% on Matrigel-pretreated culture plates and cultured in 4CL medium for 24 hours, then the medium was replaced with e4CL. The e4CL medium was changed daily, and cells were passaged every 3-4 days. Cells were cultured in an incubator at 37°C and 5% CO2.

[0072] The testing period was the 6th day induced by the above method.

[0073] The induction method is described in the reference [Mazid, Md Abdul, et al. Rolling back humanpluripotent stem cells to an eight-cell embryo-like stage. Nature, 605.7909(2022): 315-324.], such as... Figure 9 As shown.

[0074] WT and 2.6 days after e4CL induction BCL2 Representative images of the primitive human ESC (Extreme Human Pathogen) are as follows: Figure 10 As shown.

[0075] 3.WT and BCL2 Immunostaining analysis results of OE embryonic stem cells induced for 6 days in e4CL medium are as follows: Figure 11 As shown.

[0076] Immunofluorescence staining experimental method: (1) Add 100 μL of Matrigel to an 8-well cell culture dish and place it in a cell culture incubator for 45 min. Seed an appropriate amount of cells and culture them to a suitable density. (2) Rinse each well of the 8-well cell culture dish with 500 μL of DPBS to remove cell impurities and dead cells; (3) Add 500 μL of 4% paraformaldehyde (PFA) solution to each well, and then place the cells at room temperature for 30 min to fix them in a stable state for subsequent operations.

[0077] (4) Discard the fixative and wash the cells with DPBS to completely remove any residual fixative components (if staining experiments are not performed, 500 μL of DPBS can be added to the fixed sample and stored at 4°C for later use). (5) Permeabilize cells with IF buffer for 30 min (IF buffer preparation method: 50 mL DPBS + 0.1% Triton X-100 + 1% BSA); (6) Discard the IF buffer, add the diluted primary antibody, and incubate overnight at 4°C; (7) Discard the primary antibody and rinse three times with IF buffer, 10 min each time; (8) Add the corresponding secondary antibody and incubate at room temperature for 1 h to enable the secondary antibody to specifically recognize and bind to the primary antibody; (9) Discard the secondary antibody and rinse three times with IF buffer, 10 min each time; (10) Use DAPI to stain cell nuclei for about 5 minutes; (11) Discard the DAPI staining solution and wash the cells with DPBS for 5 min; (12) Cover with a glass slide, seal with nail polish, take a picture using a laser confocal microscope or temporarily store at -20℃.

[0078] 4. RT-qPCR detection of WT and BCL2 The relative expression levels of pluripotency and 8CLCs-related genes in OE embryonic stem cells after 6 days of induction in e4CL medium were determined. Primer information is detailed in Table 2. The results are as follows: Figure 12 As shown.

[0079] 5. Western blot analysis results of TPRX1, DUX, ZSCAN4, DPPA5, and NELFA protein expression are as follows: Figure 13 As shown.

[0080] 6. Results Analysis To investigate whether BCL2 overexpression also promotes human nausea and vomiting. ve ESC (Na 8CLC induction in primordial human embryonic stem cells (BCL2_OE) was performed. Immunostaining analysis showed that 6-day e4CL induction significantly increased the expression levels of key pluripotency proteins (OCT4, SOX2, and NANOG) in BCL2_OE and WT ESCs. BCL2 overexpression led to a significant upregulation of key 8CLC-related genes (TPRX1, TRIM43, LEUTX, ZSCAN4, RFPL4A, and H3X / Y) compared to WT ESCs. Consistent with this transcriptional signature, the protein levels of key markers in BCL2_OE ESCs were also significantly upregulated, more significantly than in the control group. The upregulation of these genes was directly verified at the protein level by immunostaining (TPRX1, ZSCAN4) and Western blotting (TPRX1, DUX, ZSCAN4, DPPA5, NELFA). In summary, overexpression of these genes indicates that 8CLC induction significantly increases the expression levels of key pluripotency proteins (OCT4, SOX2, and NANOG) in BCL2_OE and WT ESCs. BCL2 It can efficiently induce the generation of 8-cell embryo-like cells, overcoming the problem of difficulty in efficiently obtaining primitive embryonic stem cells and 8-cell embryo-like cells in existing technologies, and providing new technical means for studying the mechanism of early human embryonic development, establishing in vitro development models, and obtaining seed cells with higher developmental potential.

[0081] Example 4 BCL2 Enabling 8C-like cells to self-renew for extended periods under specific conditions Under in vitro culture conditions, factors such as serum, oxidative stress, and mechanical damage caused by passage culture have a significant impact on the in vitro self-renewal and pluripotency maintenance of human embryonic stem cells. Given that the sustained activation and long-term maintenance of the pluripotency of 8CLCs remains challenging even under specific conditions, it is crucial to develop a more stable and long-term maintained 8C-like human embryonic stem cell model.

[0082] Explore BCL2 Can overexpression promote long-term amplification and stabilize the e4CL-induced 8C-like state? 1. The experimental methods, cell culture, and cell culture medium were the same as in Example 3. The cells from Example 3 were cultured for a long period of time, and the detection days after the long-term culture were 18 days and 25 days.

[0083] 2. Under e4CL conditions, WT and BCL2 Morphological characteristics of long-term cultured OE ESCs, such as Figure 14 The above figure shows the results. The results indicate that after prolonged culture under e4CL conditions, WT ESCs cannot pass beyond 23 days (6 generations), and ES-like clones are almost undetectable. In contrast, BCL2 _OE ESCs maintained a strong self-renewal capability and maintained an ES-like population for 10 generations.

[0084] 3. Based on the above findings, cells from day 18 were selected for subsequent comparative analysis. After prolonged e4CL culture (day 18), WT and... BCL2 Immunostaining was performed on the core pluripotency proteins (OCT4, SOX2, NANOG) of OE ESCs, and the results are as follows: Figure 14 The image below is shown.

[0085] 4.18 days after e4CL culture, WT and BCL2 The relative expression levels of the core 8C-like genes in _OE ESC are detailed in Table 2, with primer information provided. The results are as follows: Figure 15 As shown.

[0086] 5. For WT and BCL2 The 8C-like markers (TPRX1, ZSCAN4, DUX, NELFA, TP53) selected in the _OE ESC were validated by Western blotting, and the results are as follows: Figure 16 As shown.

[0087] 6. In WT and BCL2 Immunostaining verification of 8C-like markers (TPRX1, ZSCAN4) in OE ESC, the results are as follows: Figure 17 As shown.

[0088] BCL2 Under prolonged e4CL conditions, compared to WT ESCs, _OEs not only maintained high protein levels of OCT4, SOX2, and NANOG, but also exhibited significant transcriptional upregulation of most core 8C-like genes, including KLF17, TPRX1 TRIM43, DUX4, LEUTX, ZSCAN4, RFPL4A and H3X / Y .

[0089] BCL2 Under prolonged e4CL conditions, compared to WT ESCs, _OEs not only maintained high protein levels of OCT4, SOX2, and NANOG, but also exhibited significant transcriptional upregulation of most core 8C-like genes, including KLF17, TPRX1 TRIM43, DUX4, LEUTX, ZSCAN4, RFPL4A and H3X / Y We verified the increased protein levels of these 8C-like gene subsets using Western blotting (TPRX1, ZSCAN4, DUX, NELFA, TP53) and immunostaining (TPRX1, ZSCAN4), and the results were consistent with their transcriptomic profiles.

[0090] BCL2 overexpression not only enhances the pluripotency of human primordial embryonic stem cells (ESCs), but more importantly, it contributes to the long-term and stable maintenance of human 8CLCs. Conversely, wild-type ESCs induced over a long period exhibited progressive apoptosis. These results suggest that the anti-apoptotic function of BCL2 supports the continuous self-renewal of 8CLCs. Overall, the previously unknown role of BCL2 in regulating pluripotency and promoting the acquisition of 8C-like states provides a valuable cell model for studying human zygote genome activation (ZGA)-like processes under in vitro conditions, offering a new technological approach for studying early embryonic development.

[0091] Example 5 BCL2-induced human 8-cell embryo-like cells possess extended pluripotency. By culturing e4CL BCL2 _OE ESC was injected into 8-cell stage embryos to assess their in vivo developmental potential.

[0092] 1. Human-mouse chimera experiment Mice were housed at the Laboratory Animal Center of Inner Mongolia University, China. All mouse handling and experiments were conducted in accordance with the relevant guidelines of the Institutional Animal Care and Use Committee (IACUC) of Inner Mongolia University (License No.: SYXK(Mongolia)2020-0006) and were performed humanely.

[0093] Eight-cell stage embryos were obtained by flushing the oviducts of female ICR mice.

[0094] The WT or WT cultured in e4CL medium in the above examples BCL2 After digestion and dispersion, 15 cells were taken from each OE ESC and microinjected into 8-cell stage mouse embryos.

[0095] Following microinjection, 8-cell stage embryos were cultured for 52 hours at 37°C and 5% CO2 in G-2TM PLUS (Vitrolife, 10132). The resulting chimeric blastocysts were then fixed for immunofluorescence staining analysis.

[0096] 2. Immunofluorescence staining: Immunostaining was performed using OCT4, GATA3, and CDX2 antibodies. Cell nuclei were counterstained with DAPI. Immunostaining was performed three independent experiments. The number of embryos contributing to the inner cell mass (ICM) and those contributing to both the inner cell mass (ICM) and trophoblast (TE) was counted.

[0097] Immunofluorescence staining results as follows Figures 18-19 As shown. Figure 18 yes BCL2 Immunostaining image of eight-cell embryos injected with OE ESCs. Figure 19 This is an immunostaining image of an eight-cell embryo injected with WTESCs. The purpose of this experiment was to determine whether the injected WTESCs cells could develop to the ICM and TE stages of the embryo. OCT4 is a marker used to detect ICM. Figure 18 and Figure 19 Both are used for detection. GATA3 and CDX2 are markers used to detect TE. Figure 18 The test used was GATA3. Figure 19 The test used CDX2, although it uses a different detection marker, it represents the expression status of TE cells.

[0098] By culturing e4CL BCL2 _OE ESC was injected into 8-cell stage embryos to assess their in vivo developmental potential. After 48 hours of culture and immunostaining analysis, it was observed that... BCL2 Overexpressing cells significantly contributed to both the inner cell mass (65.7%, 23 / 35) and trophoblast (31.4%, 11 / 35) of chimeric embryos. In contrast, WT ESCs contributed very little to the trophoblast (3.3%, 1 / 30). These results suggest that BCL2 possesses extended differentiation potential, enabling it to contribute to both the embryonic and extraembryonic lineages under specific culture conditions.

[0099] In summary, BCL2-overexpressing 8C-like cells exhibit expanded pluripotency and can contribute to the inner cell mass and trophoblast of mouse embryos, while 8C-like cells transformed from wild-type embryonic stem cells cannot contribute to the trophoblast. This indicates that BCL2 overexpression can significantly improve the expanded pluripotency of human embryonic stem cells, providing a good theoretical support for research on early human embryonic development and stem cell-derived embryonic models.

[0100] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of reagents that increase BCL2 gene expression and / or BCL2 protein levels in the preparation of reagents or kits for regulating the pluripotency of human embryonic stem cells.

2. The application according to claim 1, characterized in that, The amino acid sequence of BCL2 is shown in SEQ ID NO.

1.

3. The application according to claim 1 or 2, characterized in that, The human embryonic stem cells include primordial human embryonic stem cells and / or primitive human embryonic stem cells.

4. The application according to claim 1, characterized in that, The regulation of the pluripotency state of human embryonic stem cells includes at least one of the following: (1) Promotes the transformation of human embryonic stem cells from the primordial state to the primitive state; (2) Maintaining the pluripotency of human embryonic stem cells; (3) Promote the transformation of primitive human embryonic stem cells into 8-cell embryo-like cells.

5. A method for enhancing the pluripotency of human embryonic stem cells in vitro or inducing the transformation of human embryonic stem cells into early embryonic stage cells in vitro, characterized in that, include: Upregulates the expression level of the BCL2 gene and / or the protein level of BCL2 in human embryonic stem cells.

6. Application of reagents that increase BCL2 gene expression levels and / or increase BCL2 protein levels in the preparation of reagents or kits for inducing the transformation of human embryonic stem cells into 8-cell embryo-like cells.

7. The application according to claim 6, characterized in that, The application includes at least one of the following: (I) Promotes the transformation of human embryonic stem cells into 8-cell embryo-like cells; (II) Maintaining the long-term stable self-renewal of 8-cell embryo-like cells; (III) Maintain the totipotency of 8-cell embryo-like cells; (IV) Enhance the bidirectional lineage differentiation potential of 8-cell embryo-like cells.

8. A method for inducing the transformation of human embryonic stem cells into 8-cell embryo-like cells in vitro, characterized in that, include: Upregulate the expression level of the BCL2 gene and / or upregulate the BCL2 protein level in the human embryonic stem cells.

9. A human 8-cell embryonic-like cell, characterized in that, The cells were obtained by increasing the expression level of the BCL2 gene and / or the level of the BCL2 protein in human embryonic stem cells.

10. A primer set for identifying whether the pluripotency of human embryonic stem cells is enhanced, characterized in that, include: hBCL2-F as described in SEQ ID NO.4 and hBCL2-R as shown in SEQ ID NO.5.

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