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

Abstract

The application provides a method for regulating the pluripotency state of human embryonic stem cells and / or activating 8-cell-like cells NELFA The application belongs to the technical field of genetic engineering and provides a method for regulating the pluripotency state of human embryonic stem cells and / or activating 8-cell-like cells NELFA The application provides a method for regulating the pluripotency state of human embryonic stem cells. NELFA The application successfully discloses and verifies the role of the key positive regulator in driving the conversion of human embryonic stem cells into early embryonic stage cells, and provides a new technical means for efficiently obtaining primitive stem cells and 8-cell embryo-like cells, and then obtaining seed cells with expanded pluripotency.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving NELFA Application in regulating the pluripotency of human embryonic stem cells and / or activating 8-cell-like cells. Background Technology

[0002] Human embryonic stem cells (hESCs) are derived from the inner cell mass of the blastocyst. They possess the potential for self-renewal and multipotent differentiation, and have significant applications in developmental biology research, disease model construction, drug screening, and regenerative medicine. Currently, it is generally believed that human embryonic stem cells exist in two distinct pluripotent states: the primitive state (na...) and the na... Pluripotency is categorized into two states: primitive (e.g., primordial state) and primed state. Primitive state stem cells resemble the pre-implantation ectoderm of the embryo and possess the highest developmental potential; primed state stem cells resemble the post-implantation ectoderm, developing later than primitive state and exhibiting relatively lower pluripotency. These two states of stem cells differ significantly in morphology, clonogenic capacity, signaling pathway dependence, epigenetic characteristics, and metabolic patterns. Therefore, exploring the maintenance mechanisms of different pluripotency states and the molecular regulatory networks of their interconversion is crucial for understanding early human embryonic development and optimizing stem cell culture systems.

[0003] Mammalian embryonic development begins with a fertilized egg and then undergoes continuous lineage differentiation. In early human embryonic development, the 8-cell stage is a crucial turning point—zygotic genome activation (ZGA) initiates at this stage, marking the transfer of developmental control from maternal factors to the embryo's own genome, and the formal commencement of a new individual developmental program. Cells at the 8-cell stage possess unique molecular characteristics and developmental potential; however, due to the scarcity of human embryonic material and ethical constraints, research on the regulatory mechanisms of this stage has long faced bottlenecks.

[0004] In recent years, with the advancement of in vitro stem cell culture technology, researchers have developed various induction systems that enable pluripotent stem cells to be reprogrammed to earlier developmental states in vitro. For example, through specific culture medium combinations, primordial human embryonic stem cells can be induced to a primitive state; further research has shown that primitive or primordial cells can be transformed into 8-cell embryo-like cells (8CLCs) under specific culture conditions. These cells are similar to 8-cell embryos in vivo in terms of transcriptomic characteristics, epigenetic modifications, and developmental potential. 8CLCs not only express zygotic genome activation-related characteristic genes but also possess bidirectional differentiation potential, capable of contributing to both the inner cell mass and extraembryonic trophoblast tissue.

[0005] Although the establishment of the aforementioned induction system provides an important tool for studying early human embryonic development, little is known about the key molecular mechanisms regulating the transformation of pluripotent stem cells into early embryonic cells. Existing research has revealed some regulatory factors involved in this process, such as... OTX2 Reported as a suppressor that limits 8CLCs-induced inhibition, knockout OTX2 It can significantly promote the generation of 8CLCs; other studies have found that the deubiquitinating enzyme USP7 limits the extraembryonic developmental potential of human embryonic stem cells by regulating mTOR activity; metabolic remodeling also plays an important role in the induction of 8CLCs. However, the positive regulatory factors driving the transformation of human embryonic stem cells from the primordial state to the primitive state, and then to 8-cell embryo-like cells, still need to be discovered.

[0006] Therefore, screening and identifying key molecules that can promote the transformation of human embryonic stem cells into earlier embryonic stages and revealing their regulatory mechanisms are of great scientific significance and application value for a deeper understanding of early human developmental events, optimization of in vitro induction systems, and acquisition of seed cells with higher developmental potential. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide... NELFA Its application in regulating the pluripotency state of human embryonic stem cells, particularly providing a method for regulating... NELFA The method of expressing or regulating the activity of human embryonic stem cells to induce the transformation of human embryonic stem cells into early embryonic stage cells can overcome the problem of difficulty in efficiently obtaining primitive embryonic stem cells and 8-cell embryo-like cells in existing technologies, and provide new technical means for studying the development mechanism of human early embryos, establishing in vitro development models, and obtaining seed cells with higher developmental potential.

[0008] The objective of this invention is achieved through the following technical solution: This invention provides NELFAApplication in regulating the pluripotency of human embryonic stem cells.

[0009] Preferably, the regulation includes promoting the conversion of human embryonic stem cells from the primordial state to the primitive state.

[0010] Preferably, the regulation includes: overexpression NELFA To promote the acquisition of primitive pluripotency in human embryonic stem cells; or to inhibit NELFA It is expressed in order to inhibit the conversion of human embryonic stem cells to the primitive state.

[0011] Preferably, obtaining primitive pluripotency includes increasing the expression level of primitive pluripotency-related factors in cells and / or making cells exhibit primitive-specific morphology.

[0012] This invention provides overexpression NELFA Application in the preparation of reagents or kits for inducing the transformation of human embryonic stem cells into 8-cell embryo-like cells.

[0013] Preferably, the human embryonic stem cells include primordial human embryonic stem cells and / or primitive human embryonic stem cells.

[0014] Preferably, the 8-cell embryo-like cells have cellular characteristics of the early embryonic development stage.

[0015] This invention provides overexpression NELFA Application in enhancing cell expansion pluripotency; the enhancement of cell expansion pluripotency includes enhancing the expansion pluripotency of 8-cell embryo-like cells.

[0016] This invention provides a method for in vitro induction of human embryonic stem cells to transform into early embryonic stage cells, comprising increasing the concentration of certain components in the human embryonic stem cells. NELFA The expression or activity of.

[0017] This invention provides an 8-cell embryo-like cell, which is overexpressed by human embryonic stem cells in vitro. NELFA It was prepared.

[0018] The beneficial effects of this invention are: This invention provides NELFA Application in regulating the pluripotency state of human embryonic stem cells. This invention successfully reveals and verifies... NELFA As a key positive regulator, it plays a role in driving the transformation of human embryonic stem cells into early embryonic cells, providing a new technical means for efficiently obtaining primitive stem cells and 8-cell embryo-like cells, and then obtaining seed cells with expanded pluripotency.

[0019] This invention utilizes overexpression NELFA This significantly improved the efficiency of human embryonic stem cell transformation to the primitive state. Experimental results showed that stem cells cultured in 4CL medium… NELFA Overexpression of embryonic stem cells resulted in the emergence of dome-shaped clones significantly earlier than in the wild-type control group, and these clones were able to be cultured for extended periods and maintain this typical primitive morphology. Compared to wild-type cells, NELFA Overexpressing cells significantly upregulated pluripotency markers on day 9. SOX2 , OCT4 , TFCP2L1 Expression levels, and key primitive genes DPPA3 , DPPA5 and TFCP2L1 The expression of these proteins was also significantly higher than that of wild-type cells. Western blot analysis further confirmed that the protein levels of OCT4, NANOG, and SOX2 were slightly upregulated, fully demonstrating that... NELFA It can efficiently promote the transformation of nascent cells to the primitive state.

[0020] Based on this, the present invention further confirms overexpression NELFA It can efficiently induce the generation of 8-cell embryo-like cells. Cultured in e4CL medium. NELFA The number of dome-shaped colonies in the overexpressed embryonic stem cells was significantly greater than that in the wild-type control group; DPPA3 Upregulated expression of core characteristic transcripts in 8-cell embryo-like cells DUX4 , TRIM43 and H3.X / Y Significant upregulation was observed, with immunostaining results showing a significant increase in the number of TPRX1 and ZSCAN4 positive cells. Western blot analysis also confirmed a significant upregulation of ZSCAN4, DUX, and TPRX1 protein levels. These experimental data fully demonstrate that... NELFA It can efficiently activate the generation of human 8-cell embryo-like cells.

[0021] This invention further confirms that, through overexpression NELFA The obtained 8-cell embryo-like cells exhibited typical extended pluripotency. Chimera experiments showed that... NELFA The induced cells were able to contribute to different germ layer lineages simultaneously in chimeric blastocysts. Among the recovered embryos, OCT4-positive inner cell mass cells accounted for 64.5% and CDX2-positive trophoblast cells accounted for 15.2%. This proportion was significantly higher than that of the wild-type control group, demonstrating that these cells have the developmental potential to differentiate into both the embryonic inner cell mass and extraembryonic trophoblast, i.e., they have typical extended pluripotency characteristics.

[0022] Furthermore, the present invention has confirmed through inhibition experiments that... NELFA in primed-to-na The necessity of ve transformation. After inhibiting the NELFA promoter in 4CL medium, even under induction culture conditions, the core primitive state pluripotency-related genes... NANOG , DPPA3 and DPPA5 The expression level of [the substance] was still significantly lower than that of the control group, indicating that [the substance] was significantly lower than that of the control group. NELFA Although not an essential gene in primordial embryonic stem cells, it is crucial for the transition from primordial to primitive state, a discovery that deepens our understanding of the molecular mechanisms of pluripotency state transition.

[0023] In summary, this invention regulates... NELFA The expression or activity of this technology can efficiently drive human embryonic stem cells to transform sequentially into primitive and 8-cell embryo-like cells, and obtain early embryonic stage cells with expanded pluripotency. This provides new technical means and important tools for studying the developmental mechanism of early human embryos, establishing in vitro developmental models, exploring the mechanism of placental-related diseases, and obtaining seed cells with high developmental potential. Attached Figure Description

[0024] 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.

[0025] Picture 1 Dox-induced type NELFA Construction of human embryonic stem cell overexpression lines and verification of pluripotency-related molecular expression; a) shows the construction of piggyBAC vector-based lines from primordial human embryonic stem cells. NELFA Experimental design for overexpression cells; b) Representative bright-field and fluorescence images of human-primed ESCs; Left: Wild-type (WT), passage number 15; Right: Induced by doxycycline (Dox) NELFA Cell morphology and fluorescence changes 72 hours after activation; scale bar is 50 μm; c represents RT-qPCR detection of wild-type and c-type cells under E8 culture conditions. NELFA Overexpression cells NELFA and pluripotency genes OCT4 , SOX2 and NANOG The relative expression level; error bars represent mean ± standard deviation (n=3); p-values ​​were calculated using a two-tailed t-test, p<0.05, n=3 biological replicates; d represents Western blot detection. NELFA Expression of NELFA protein in _OE hESCs, with β-actin as a loading control; Right figure: Relative expression level of NELFA protein calculated after normalization to β-actin; Error bars represent mean ± standard deviation (n=3); P value was calculated using a two-tailed t-test, p<0.05, n=3 biological replicates; Picture 2 For primed to na Experimental design diagram for VE conversion; Picture 3 Wild type and NELFA Changes in cell state of overexpressing embryonic stem cells cultured in 4CL medium on days 7 and 18; scale bar: 50 μm; Picture 4 To convert from primed to na in 4CL medium During the VE conversion process, wild type and NELFA Percentage of primitive colonies of overexpressing cells at day 7 and day 18; error bars represent mean ± standard deviation (n=3); p-values ​​were calculated using a two-tailed t-test, p<0.05, n=3 biological replicates; Picture 5 For RT-qPCR detection of wild-type and wild-type under different culture conditions NELFA The relative expression levels of pluripotency-related genes in overexpressed embryonic stem cells; error bars represent mean ± standard deviation (n=3); p-values ​​were calculated using a two-tailed t-test, p<0.05, n=3 biological replicates; Picture 6 For RT-qPCR assays, WT and NELFA Pluripotency genes in OE ESCs SOX2 , OCT4 , TFCP2L1 The relative expression level; the error bar represents the mean ± standard deviation (n=3); the p-value was calculated using a two-tailed t-test, p<0.05, n=3 biological replicates; Picture 7 The results of Western blot analysis are shown below; Left panel: identification of OCT4, SOX2, and NANOG protein levels by Western blot; Right panel: 12 relative OCT4, SOX2, and NANOG levels calculated based on β-actin normalization; Error bars represent mean ± standard deviation (n=3); P-values ​​were calculated using a two-tailed t-test, p<0.05, n=3 biological replicates; Picture 8 For WT and NELFA Immunofluorescence staining results of pluripotency markers OCT4, NANOG and KLF4 in _OE hESCs; cell nuclei stained with DAPI; scale bar at 50 μm; error bars represent mean ± standard deviation (n=3). Picture 9 Interference with CRISPRi system NELFA Flowchart and results of embryonic stem cell expression preparation; a) Schematic diagram of the inducible CRISPR interference (CRISPRi) system used for promoter inhibition; b) Bright-field and fluorescence images showing the interference in the CRISPRi system. NELFADuring expression, the state of embryonic stem cells induced by Dox and TMP for 3 days; scale bar is 50 μm; c represents the different treatment groups detected by RT-qPCR. NELFA The relative expression level; the error bar represents the mean ± standard deviation (n=3); the p-value was calculated using a two-tailed t-test, p<0.05, n=3 biological replicates; d represents the protein level of NELFA in different treatment groups as identified by Western Blot; Picture 10 To enable human embryonic stem cells to transition from primed to na under the inhibition of the NELFA promoter in the CRISPRi system. Morphological changes on days 3 and 6 during VE conversion and relative expression levels of pluripotency genes in different types of embryonic stem cells three days after induction; e represents the conversion of human embryonic stem cells from primed to na under the condition of NELFA promoter inhibition in the CRISPRi system. Morphological changes on days 3 and 6 during ve conversion; scale bar: 50 μm; f: relative expression level of pluripotency gene of different types of embryonic stem cells cultured under different conditions after three days of induction, detected by RT-qPCR; error bar: mean ± standard deviation (n=3); p-value was calculated by two-tailed t-test, p<0.05, n=3 biological replicates; Picture 11 To knock down NELFA Human embryonic stem cells in primed to na Quantitative and morphological graphs of survival and proliferation during ve conversion; g is a bar chart of live cell counts; error bars represent mean ± standard deviation (n=3); p-values ​​were calculated using a two-tailed t-test, p<0.05, n=3 biological replicates. NELFA Morphological evolution of stem cells after knockdown on days 1, 3, and 7; scale bar: 50 μm; Picture 12 for NELFA Overexpression promotes human na a) Experimental procedure and phenotypic verification diagram for the conversion of VE-state embryonic stem cells into 8-cell-like cells; b) Schematic diagram of 8C-like cell induction procedure; c) WT and NELFA Morphological changes of OE embryonic stem cells after 5 days of induction in e4CL medium; scale bar: 50 μm. Picture 13 for NELFA Overexpression regulation of human na Molecular and cellular validation diagrams of VE-state embryonic stem cell pluripotency and 8-cell-like cell activation; d and e show the relative expression levels of pluripotency and 8-CLCs-related genes detected by RT-qPCR after 7 days of induction in e4CL medium; error bars represent mean ± standard deviation (n=3); p-values ​​were calculated using a two-tailed t-test, p<0.05, n=3 biological replicates; f shows the detection of WT and... NELFA Protein levels of TPRX1 and ZSCAN4 in OE embryonic stem cells 7 days after induction in e4CL medium; cell nuclei stained with DAPI; immunofluorescence staining was performed in three independent experiments; scale bar: 30 μm; Picture 14 for NELFA Molecular validation and quantitative analysis of overexpression-induced expression of 8CLCs-related proteins; g is for Western blotting detection of WT and NELFA Expression levels of 8CLCs-related proteins in OE embryonic stem cells; h and i represent the quantitative analysis of the percentage of TPRX1 and ZSCAN4 positive cells based on f immunofluorescence staining results; error bars represent mean ± standard deviation (n=3); p-values ​​were calculated using a two-tailed t-test, p<0.05, n=3 biological replicates; j represents the relative 8C-like cell (8CLCs)-related protein levels after standardization with β-actin as an internal reference (correlation with left figure g); error bars represent mean ± standard deviation (n=3); p-values ​​were calculated using a two-tailed t-test, p<0.05, n=3 biological replicates; Picture 15 For verification by immunofluorescence staining NELFA Overexpressed 8CLCs were chimerically distributed in the trophoblast (CDX2) and inner cell mass (OCT4) in early embryos; scale bar: 50 μm; Picture 16 The figure shows the results of quantitative statistical analysis of the chimerism rate between the TE and ICM regions in chimeric embryos; Picture 17 Verification diagram of the ability of wild-type 8CLCs to chimerize into the embryonic trophoblast (TE) and inner cell mass (ICM); scale bar is 50 μm. Detailed Implementation

[0026] This invention provides NELFA Application in regulating the pluripotency of human embryonic stem cells.

[0027] In an optional embodiment of the present invention, the regulation includes promoting the conversion of human embryonic stem cells from a primordial state to a primitive state. In an optional embodiment of the present invention, the human embryonic stem cells include primed human embryonic stem cells. In an optional embodiment of the present invention, the regulation is achieved by overexpression... NELFATo promote the acquisition of primitive pluripotency in human embryonic stem cells; or to inhibit NELFA Expression is used to inhibit the conversion of human embryonic stem cells to a primitive state. As an optional embodiment of the invention, the regulation can also be achieved through overexpression. NELFA Improve the pluripotency of human embryonic stem cells; inhibit NELFA The expression of factors that reduce the pluripotency of human embryonic stem cells is reduced. In this invention, obtaining primitive pluripotency includes increasing the expression levels of primitive pluripotency-related factors in cells and / or causing cells to exhibit primitive-specific morphology.

[0028] This invention utilizes overexpression NELFA This significantly improved the efficiency of human embryonic stem cell transformation to the primitive state. Experimental results showed that... NELFA Overexpression can be significantly upregulated SOX2 and NANOG mRNA levels. This was determined by culturing in 4CL medium. NELFA Overexpression of embryonic stem cells resulted in the appearance of dome-shaped clonal patterns significantly earlier than in the wild-type control group. NELFA Overexpression promotes na Ve-like colony formation efficiency and long-term maintenance The VE pluripotency status was significantly superior to that of wild-type cells in both aspects. Compared with wild-type cells, NELFA Overexpressing cells significantly upregulated pluripotency markers on day 9. SOX2 , OCT4 , TFCP2L1 Expression levels, and key primitive genes DPPA3 , DPPA5 and TFCP2L1 The expression of OCT4 was also significantly higher than that of wild-type cells, and Western blot analysis further confirmed that the protein levels of OCT4, NANOG and SOX2 were significantly upregulated.

[0029] This invention further confirms through inhibition experiments that, after inhibiting the NELFA promoter in 4CL medium, even under induced culture conditions, the core primitive state pluripotency-related genes are preserved. NANOG , DPPA3 and DPPA5 The expression level of [the substance] was significantly lower than that of the control group, and it inhibited [the activity]. NELFA This can lead to a significant decrease in cell survival / proliferation capacity.

[0030] This invention provides overexpression NELFAApplication 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 the 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 invention, the 8-cell embryo-like cells have cellular characteristics of an early embryonic developmental stage. As an optional embodiment of the invention, the cellular characteristics include a significantly increased number of dome-shaped colonies; DPPA3, DUX4 , TRIM43 and H3.X / Y Expression was upregulated; the number of TPRX1 and ZSCAN4 positive cells increased significantly; the protein levels of ZSCAN4, DUX and TPRX1 were upregulated.

[0031] The results of the embodiments of this invention show that overexpression NELFA It can efficiently induce the generation of 8-cell embryo-like cells. Cultured in e4CL medium. NELFA The number of dome-shaped colonies in the overexpressed embryonic stem cells was significantly greater than that in the wild-type control group; DPPA3 Upregulated expression of core characteristic transcripts in 8-cell embryo-like cells DUX4 , TRIM43 and H3.X / Y Significant upregulation was observed, with immunostaining showing a significant increase in the number of TPRX1 and ZSCAN4 positive cells. Western blot analysis also confirmed a significant upregulation of ZSCAN4, DUX, and TPRX1 protein levels, fully demonstrating... NELFA It can efficiently activate the generation of human 8-cell embryo-like cells.

[0032] This invention provides overexpression NELFA Application in enhancing cell expansion pluripotency. As an optional embodiment of the present invention, enhancing cell expansion pluripotency includes enhancing the expansion pluripotency of 8-cell embryo-like cells; the cell expansion pluripotency includes the ability of the cells to simultaneously contribute to different germ layer lineages in a chimeric embryo.

[0033] The results of the embodiments of the present invention show that, through overexpression NELFA The obtained 8-cell embryo-like cells exhibited typical extended pluripotency. Chimeric experiments showed that overexpression... NELFA The induced cells were able to contribute to different germ layer lineages simultaneously in chimeric blastocysts. Among the recovered embryos, OCT4-positive inner cell mass cells accounted for 64.5% and CDX2-positive trophoblast cells accounted for 15.2%. This proportion was significantly higher than that of the wild-type control group, demonstrating that these cells have the developmental potential to differentiate into both the embryonic inner cell mass and extraembryonic trophoblast, i.e., they have typical extended pluripotency characteristics.

[0034] This invention provides a method for in vitro induction of human embryonic stem cells to transform into early embryonic stage cells, comprising increasing the concentration of certain components in the human embryonic stem cells. NELFA The expression or activity of [certain substances]. This invention aims to improve the expression or activity of [certain substances] in human embryonic stem cells. NELFA There are no particular limitations on the method of expression; any conventional method in the art can be used. As an optional embodiment of the present invention, the method of improving the concentration of [specific components] in human embryonic stem cells... NELFA The expression method can be constructed through the PiggyBac-TET on / off system. NELFA Gene overexpression plasmid. The present invention does not specifically limit the preparation method of the overexpression plasmid; any conventional preparation method in the art can be used. The present invention improves the expression of genes in human embryonic stem cells. NELFA There are no particular limitations on the method of activation; any conventional method in the field may be used.

[0035] This invention provides an 8-cell embryo-like cell, which is overexpressed by human embryonic stem cells in vitro. NELFA The 8-cell embryo-like cells described in this invention possess extended pluripotency, capable of simultaneously contributing to different germ layer lineages in chimeric embryos. Specifically, in chimeric blastocysts, they can simultaneously contribute to both the inner cell mass (ICM) and extraembryonic trophoblast (TE) lineages. Chimeric experiments of this invention show that these cells express OCT4-positive inner cell mass markers (64.5% of recovered embryos) and CDX2-positive trophoblast markers (15.2% of recovered embryos) in chimeric blastocysts, and the chimeric proportions are significantly higher than those in the wild-type control group, demonstrating their developmental potential for bidirectional differentiation into both intraembryonic and extraembryonic lineages.

[0036] 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.

[0037] See the reference [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.] for the piggyBac Tet-on induction plasmid.

[0038] The RT-qPCR primers involved in the following technical solutions are shown in Table 1 below.

[0039] Table 1 RT-qPCR primers

[0040] Table 2 sgRNA Information

[0041] The H9 human embryonic cell line used in this invention was obtained from Professor Jie Wei of Tsinghua University.

[0042] Primed hESCs were cultured using the E8 culture system.

[0043] 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. ESCs were typically passaged every 3-4 days, with the medium changed daily. Before passage, cells were washed once with DPBS (Thermo Fisher, 14200075) and then treated with 0.5 mM EDTA (Thermo Fisher, 15575020) for 5 minutes. The EDTA was then removed, 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.

[0044] Example 1 1. NELFA (human) has a Gene ID (Entrez ID) of 7469 in NCBI. NELFA chromosome location: chr4p16.3. NELFA For detailed mRNA information, please refer to NCBI Reference Sequence: NM_005663.5. For detailed amino acid sequence information of NELFA, please refer to NCBI Reference Sequence: NP_005654.4.

[0045] The amino acid sequence of NELFA is shown in SEQ ID NO.31, specifically: MASMRESDTGLWLHNKLGATDELWAPPSIASLLTAAVIDNIRLCFHGLSSAVKLKLLLGTLHLPRRTVDEMKGALMEIIQLASLDSDPWVLMVADILKSFPDTGSLNLELEEQNPNVQDILGELREKVGECEASAMLPLECQYLNKNALTTLAGPLTPPVKHFQLKRKPKSATLRAELLQKSTETAQQLKRSAGVPFHAKGRGLLRKMDTTTPLKGIPKQAPFRSPTAPSVFSPTGNRTPIPPSRTLLRKERGVKLLDISELDMVGAGREAKRRRKTLDAEVVEKPAKEETVVENATPDYAAGLVSTQKLGSLNNEPALPSTSYLPSTPSVVPASSYIPSSETPPAPSSREASRPPEEPSAPSPTLPAQFKQRAPMYNSGLSPATPTPAAPTSPLTPTTPPAVAPTTQTPPVAMVAPQTQAPAQQQPKKNLSLTREQMFAAQEMFKTANKVTRPEKALILGFMAGSRENPCQEQGDVIQIKLSEHTEDLPKADGQGSTTMLVDTVFEMNYATGQWTRFKKYKPMTNVS。

[0046]

[0047] The CDS sequence of NELFA is shown in SEQ ID NO.33, specifically as follows:

[0048] 2. NELFA OE cell line construction process NELFA For details on the construction process and detection methods of the OE cell line, please refer to [link / reference needed]. Picture 1 'a' in 'a'.

[0049] 1) Construction of overexpression vector system: Construction of Tet-On induction system NELFA The overexpression vector, namely the Tet-on-TRE-NELFA-EGFP expression plasmid, was constructed using the PiggyBac-TET on / off system. NELFA Gene overexpression plasmids were used to achieve stable integration and induced expression of the vector. Specific methods are described in 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.]. The specific steps are as follows: (1) Construct a Tet-on inducible overexpression system based on the piggyBac transposon vector, and then... NELFA The CDS sequence of the gene was cloned downstream of the TRE promoter. Specifically, using cDNA containing the full-length CDS of NELFA as a template, the target fragment was obtained by PCR amplification (EcoRI and BamHI restriction sites were pre-introduced at both ends of the primers to ensure correct reading frames). The NELFA CDS fragment and the vector plasmid were then treated with EcoRI and BamHI double digestion, respectively. After recovery and purification, the fragment was directionally cloned into the multiple cloning site region of the vector using T4 DNA ligase. This site is located downstream of the TRE promoter, enabling... NELFA The expression of the NELFA fragment is completely regulated by the Tet-On system. The ligation product was transformed into competent E. coli DH5α, and colony PCR and Sanger sequencing confirmed that the NELFA fragment was free of mutations and inserted in the correct direction.

[0050] (2) Simultaneously ligate the EGFP reporter gene, which uses... Not I and BamH I enzymes are inserted into the target gene ( NELFAFollowing the F2A expression cassette, an F-2A linker is used in the middle to indicate the expression status. Specifically: an EGFP fusion fragment containing the F2A peptide coding sequence is designed (with BamHI and NotI restriction sites introduced at both ends to ensure contiguity with the NELFA CDS reading frame); the fusion fragment and the aforementioned intermediate vector are double-digested with BamHI and NotI, purified, and then ligated to form the F2A-EGFP expression cassette in series with the NELFA CDS reading frame. NELFA Downstream of CDS, an integrated "NELFA-F2A-EGFP" expression unit is formed; sequencing verification confirms that the F2A peptide sequence is mutation-free and the element linkage is correct, ensuring that subsequent expression can be achieved through F2A autocleavage. NELFA Simultaneous expression of EGFP, with EGFP used for visual indication. NELFA The induced expression state.

[0051] The EGFP reporter gene is an enhanced green fluorescent protein (EGFP) derived from a mutant of green fluorescent protein (GFP) from Aequorea victoria. Its amino acid sequence is referenced from UniProt database accession number P42212 and includes mutations such as F64L and S65T.

[0052] The obtained recombinant plasmid was identified, and all elements were confirmed to be normal. The recombinant overexpression plasmid was finally obtained. NELFA Gene overexpression plasmids (e.g.) Picture 1 (as shown in a), it can also be simply referred to as a recombinant plasmid, or as a Tet-on-TRE-NELFA-EGFP plasmid.

[0053] (3) This system relies on rTTA (reverse tetracycline-controlled transactivator) to induce doxycycline (Dox) expression.

[0054] 2) Cell transfection: Recombinant plasmids were co-introduced into human primed embryonic stem cells (primed hESCs) using liposome transfection reagent to establish... NELFA Overexpression cell lines.

[0055] Tet-on-TRE-NELFA-EGFP plasmid (2 μg), PB-EF1α-transposase plasmid (2 μg), and PB-EF1α-rTTA plasmid were co-introduced into human primed embryonic stem cells using liposome transfection reagent to establish... NELFAOverexpression 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, following the kit's instructions.

[0056] 3) Positive cell selection and single-cell establishment: After transfection, puromycin (3 μg / mL) was used for selection for 24–48 hours to remove untransfected cells. Subsequently, surviving cells were seeded into 96-well plates for single-cell clonal culture under the following conditions: (1) Use mTesR / E8 medium. Add ROCK inhibitor Y-27632 (10 μM) to promote single cell survival.

[0057] (2) Remove Y-27632 after 24 hours of culture and change the culture medium daily. Select wells that form a single clone for amplification culture.

[0058] 4) Identification of positive clones Identification of the expanded monoclonal cells: Sanger sequencing was used to confirm the exogenous origin. NELFA Proper integration of expression boxes and sequence integrity.

[0059] 5) NELFA Induction of overexpression After obtaining a stable cell line, induction was performed by adding doxycycline (Dox) to the culture medium. NELFA The mechanism of action is as follows: (1) Dox activates rTTA; (2) rTTA binds to the TRE promoter; (3) NELFA-EGFP transcriptional expression is initiated; (4) EGFP signal can be used to monitor the overexpression status in real time.

[0060] 3. Regarding NELFA Fluorescence microscopy was performed on overexpressed hESCs and WT hESCs. See details below. Picture 1 b in the text. Where WT stands for WT hESCs. NELFA _OE is NELFA Overexpression of hESCs.

[0061] Depend on Picture 1 As shown in b, normal, untreated hESCs typically have clear and smooth colony edges, tightly packed cells, a high nucleus-to-cytoplasm ratio, large and prominent nuclei, and relatively little cytoplasm. The colonies as a whole present a flattened clonal appearance and exhibit a certain degree of gloss and three-dimensionality under a microscope. Based on the bright-field image of the cells, it can be observed that... NELFA After overexpression, the two did not show any obvious difference in morphology.

[0062] 4. To NELFART-qPCR was performed on overexpressed hESCs and WT hESCs to detect the expression levels of related genes. Primer sequences are detailed in Table 1.

[0063] 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 biological 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.

[0064] NELFA Overexpression of hESCs and WT hESCs NELFA , OCT4 , SOX2 and NANOG The relative expression level, such as Picture 1 As shown in c in the figure. At the mRNA level, WT and NELFA In _OE's hESCs NELFA Expression levels were compared. The results showed that, compared to WT hESCs, NELFA In _OE NELFA Gene expression levels were significantly elevated. Analysis of core pluripotency genes revealed that in hESCs, NELFA Transient overexpression of pluripotent genes on mRNA OCT4 and NANOG The expression of [certain substances] was unaffected or only slightly affected. However, compared to wild-type (WT) embryonic stem cells, NELFA Overexpression in embryonic stem cells SOX2 The mRNA level was significantly upregulated.

[0065] 5. The overexpression effect was detected at the protein level, for WT and NELFA Western blot analysis was performed on cells with _OE, with β-ACTIN used as an internal control protein.

[0066] The Western Blot experimental method is as follows: (1) Extraction and concentration determination of total cell protein Sufficient cell volume is required for cell protein extraction. First, transfer cells cultured in 24-well plates to 6-well plates and culture for several days until the cell density reaches the target level (approximately two wells of a 6-well plate, with confluence exceeding 90%). Wash cells three times with DPBS to remove dead cells, digest and centrifuge to obtain cell pellet. Mix the pellets from both wells and add pre-prepared cell lysis buffer, lysing on ice for 20 min. Then, centrifuge at 12000 rpm for 10 min in a pre-chilled centrifuge at 4°C. Carefully aspirate the supernatant (avoiding aspiration of the pellet) and transfer it to a new centrifuge tube. Take 15 μL of the supernatant and dilute it 1:5 with Lysis as the protein concentration assay sample; repeat the assay three times for each sample. Add 6× Loading Buffer to the remaining supernatant proportionally, dilute the 6× Loading Buffer to 1×, boil in water for 5 min to denature the proteins, and then store at -20°C for later use.

[0067] For protein concentration determination, solution A and solution B were prepared at a ratio of 1:50. The test area was marked with a marker pen in a 96-well plate. 200 μL of the prepared BCA working solution and 25 μL of the test sample were added to each well. The plate was placed in a constant temperature incubator and incubated at 37°C in the dark for 30 min. Finally, the protein concentration was determined using a microplate reader.

[0068] Cell lysis buffer preparation: Lysis: phosphatase inhibitor; protease inhibitor = 100:10:1. Generally, 250 μL of lysis buffer is needed for one six-well plate.

[0069] (2) Western Blot detection The experimental steps are as follows: Calculate the average value based on the protein concentration measured in the previous step, and then calculate the loading amount based on a concentration of 20 μg.

[0070] Gel preparation: Prepare 10% gels according to the instructions. The separating gel preparation method is: 4 mL lower gel solution, 4 mL upper gel buffer, and 80 μL modified electrophoresis reagent; the stacking gel preparation method is: 1 mL upper gel solution, 1 mL upper gel buffer, and 20 μL modified electrophoresis reagent.

[0071] Remove the gel plate from the gel casting rack, wipe the liquid off the outer surface of the glass plate with paper, and then attach it to the gel holder with electrodes. Next, pour 1×Running Buffer into the electrophoresis tank.

[0072] Sample loading: Pull the comb vertically upwards, then add the protein sample to the SDS-PAGE gel wells according to the calculated protein concentration. Perform electrophoresis using the following program: 60 V, 30 min; 90 V, 1.5 h. After electrophoresis, remove the glass plate from the electrode holder and mark the front and back sides immediately. Cut a PVDF film to the same size as the gel, activate it in methanol for 15 s, and then place it in distilled water for 10 min. After activation, immerse it, the gel, and a sponge in 1× transfer buffer and shake for 20 min.

[0073] Place the sponge-gel-PVDF membrane-sponge in the center of the transfer clamp and clamp tightly. Transfer program: 110 V, 300 mA, 50 min, start transfer.

[0074] Blocking: After the transfer is complete, remove the transfer clamp and place the PVDF membrane in the pre-prepared blocking solution on a shaker for 1 hour. The blocking solution consists of 0.2 g of skim milk powder and 40 mL of 1×TBST.

[0075] After sealing is complete, discard the sealing solution, add an appropriate amount of 1×TBST to wash the membrane, repeat 3 times, 10 min each time.

[0076] Cut the desired protein band from the corresponding position according to the Maker band size, place it in the diluted primary antibody, and incubate overnight at 4°C.

[0077] Wash three times with 1×TBST, 10 min each time. After washing, place in the prepared secondary antibody and incubate at room temperature for 60 min. After secondary antibody incubation, wash three more times with 1×TBST, 10 min each time.

[0078] Development solution preparation: The development solution was prepared using Pierce ECL Western blot substrate at a 1:1 ratio to visualize the protein signal, and the protein was detected using the e-BLOT contact non-destructive quantitative WB imaging system.

[0079] For WT and NELFA Western blot analysis was performed on OE cells, with β-ACTIN used as an internal control protein. The results showed... NELFA The level of NELFA protein was significantly increased in hESCs of _OE ( Picture 1 The results in (d) were consistent with those of RT-qPCR, indicating that we successfully obtained... NELFA Overexpression cell lines.

[0080] Example 2 In order to obtain na Ve cells, selected from those in the primed state. NELFA_OE hESCs, use 4CL na VE culture medium to induce NA ve state transition.

[0081] 1. people na For details on embryonic stem cell culture (VE), please refer to [link / reference]. Picture 2 .

[0082] To generate 4CL na VE embryonic stem cells, respectively WT and NELFA Overexpression ( NELFA Embryonic stem cells (OE) were washed once with DPBS, digested into a single-cell suspension using TrypLE™ Express (Gibco, 12563029), and concentrated at 1,000-1,500 cells / cm³. 2 The culture medium was inoculated at a density of 10 µM Y-27632 on a matrix gel-coated culture plate using mTeSR 1 medium supplemented with 10 µM Y-27632. After 24 hours, the culture medium was replaced with 4CL medium. The composition of 4CL medium was: Neurobasal medium (Gibco, 21103049) and Advanced DMEM / F12 (Gibco, 12634010) mixed in a 1:1 ratio, 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), and 10 nM DZNep (Selleck, The medium consisted of 5 μM S7120, 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) Matrigel. The medium was changed daily to 4 ml, and cells were passaged as single cells every 3–4 days. Cells were cultured at 37°C in a 5% CO2 incubator.

[0083] During the 4CL medium culture process, no dox was added to WT hESCs; NELFA Overexpressing hESCs, separate processing groups are set up with and without adding dox.

[0084] 2. In NELFA Embryonic stem cells overexpressing hESCs and WT hESCs cultured for 7 days and 18 days, respectively, were observed under a fluorescence microscope. See details below. Picture 3 In 4CL medium, from primed to na During the VE conversion process, wild type and NELFA The percentage of primitive-like colonies in overexpressing cells at days 7 and 18 is as follows: Picture 4 As shown.

[0085] Depend on Pictures 3~4 It can be seen that in 4CL culture medium, we observed that whether NELFA Both _OE hESCs and WT hESCs underwent significant morphological changes. Bright-field and fluorescence images show that wild-type and NELFA Changes in cell state of overexpressing embryonic stem cells cultured in 4CL medium on days 7 and 18. Compared with WT hESCs, NELFA The dome-shaped cloning of _OEhESCs appeared significantly earlier and could be cultured for a long time and maintain this morphology. Moreover, this phenotype was maintained even on day 18.

[0086] 3. During the transition from initiation to initialization in 4CL medium, on day 18 of culture... NELFA The expression levels of overexpressed hESCs and WT hESC 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. Picture 5 RT-qPCR detection of wild-type and... NELFA The relative expression levels of pluripotency-related genes overexpressed in embryonic stem cells at 6 and 9 days of culture are as follows: Picture 6 As shown.

[0087] Pluripotency-related genes and core pluripotency genes were analyzed by RT-qPCR. OCT4 , NANOG , SOX2 and core na ve markers DPPA3 , DPPA5 , TFCP2L1 , KLF17 The key na VE gene DPPA3 , DPPA5 and TFCP2L1Cultured in 4CL medium NELFA The expression level in overexpressed embryonic stem cells was significantly higher than that in WT embryonic stem cells cultured in 4CL medium.

[0088] 4. During the transition from initiation to initialization in 4CL medium, on day 18 of culture... NELFA Overexpression of hESCs and WT hESCs was performed using immunofluorescence staining and Western blotting.

[0089] The immunofluorescence staining procedure is as follows: (1) After digesting the cells into single cells using trypsin, the cells were cultured in an eight-well plate at a certain ratio using a cell counting method.

[0090] (2) When the cell confluence reaches a suitable density, fix the cells with 4% PFA (cell tissue fixative) at room temperature for 30 min.

[0091] (3) Discard PFA and wash with 500 μL DPBS for 5 min. If the experiment is not to be performed immediately, add 500 μL DPBS and store in a 4℃ refrigerator. Keep the cells moist during this period.

[0092] (4) Discard DPBS, add 500 μL IF Buffer, and permeate for 30 min.

[0093] (5) Add 200 μL of primary antibody and incubate overnight at 4°C. (The primary antibody should be diluted with IF Buffer according to the specified ratio).

[0094] (6) Add 200 μL of the corresponding secondary antibody and incubate at room temperature in the dark for 1 h.

[0095] (7) Discard the secondary antibody, add 500 μL IF Buffer, wash 3 times after 5 min.

[0096] (8) Add 200 μL of DAPI and incubate for 5 min.

[0097] (9) Discard DAPI, add 500 μL IF Buffer, wash 3 times for 5 min. Remove the mold, add an appropriate amount of glycerol, and seal the slide with a coverslip. Finally, wrap the slide with aluminum foil to keep it in a light-proof state to prevent fluorescence quenching. Mark and record the information for later review. Image the slide using a Nikon AX upright laser confocal microscope.

[0098] Immunostaining results showed that wild-type and... NELFA In embryonic stem cells overexpressing OCT4, NANOG, and KLF4 proteins, the detected levels were comparable. Picture 8Western blot analysis showed that, compared with wild-type embryonic stem cells cultured in 4CL medium, embryonic stem cells cultured in 4CL medium... NELFA The protein levels of OCT4, NANOG, and SOX2 were slightly upregulated in embryonic stem cells overexpressing OCT4. Picture 7 Based on the above analysis, we can conclude that... NELFA Promotes the transformation of human embryonic stem cells from primed to na The transformation of the ve state was used to prepare the... NELFA _OE na VE embryonic stem cells.

[0099] Example 3 NELFA Inhibition hinders the process of human embryonic stem cells to primed na Transition of ve state In order to investigate NELFA From primed to na The functional role of ve conversion process was investigated, and an inducible CRISPR interference (CRISPRi) plasmid system based on piggyBAC was constructed. Picture 9 (a)

[0100] Transgenic KRAB-dCas9-DHFR fusion protein is controlled by a doxycycline (Dox) inducible promoter. In the absence of Dox and its stabilizer—the ligand trimethoprim (TMP) for the DHFR domain—KRAB-dCas9-DHFR fusion protein produced by any leakage activity of the inducible promoter is degraded by the proteasome due to the action of its DHFR degradation domain. Simultaneous addition of Dox and TMP induces potent mRNA expression of the KRAB-dCas9 CRISPRi element and stabilizes its protein, respectively. After stable integration of the inducible CRISPRi transgene and the sgRNA-encoding plasmid targeting the promoter into the genome of primordial human embryonic stem cells, cells were converted to a primitive state in the presence of Dox and TMP, and gene expression was detected on days 4–6 post-treatment. IRES, Internal Ribosome Entry Site.

[0101] 1. Construction of CRISPRi expression vector (The construction of CRISPRi expression vector is described in reference [Tang WWC, Castillo-Venzor A, Gruhn WH, et al. Sequential enhancer state remodelling defines human germline competence and specification[J]. Nature Cell Biology,2022, 24(4): 448-460.).

[0102] Using the CRISPRa plasmid as a backbone vector, nucleic acid fragments encoding the KRAB-dCas9-ecDHFR fusion protein gene and the IRES-EGFP reporter gene were inserted downstream of the TRE3G inducible promoter, and the CRISPRi expression vector was constructed using a one-step cloning method. The CRISPRi expression vector has the following characteristics: (1) KRAB-dCas9 protein is strictly regulated by the doxycycline (Dox) inducible promoter; (2) The fused ecDHFR protein domain serves as a degradation tag, enabling the KRAB-dCas9 protein to degrade under stabilizer-free conditions; (3) IRES-EGFP allows for fluorescence visualization monitoring of CRISPRi system expression.

[0103] 2. CRISPRi sgRNA design and screening targeting NELFA against NELFA Four specific sgRNAs were designed from the gene promoter region and the sequence near the transcription start site (TSS), and three non-targeting scramble sgRNAs were designed as negative controls. Detailed information on the sgRNAs is shown in Table 2.

[0104] The above sgRNA sequences were digested with the Eppendorf restriction endonuclease and then cloned into the piggyBac-U6-sgRNA-EF1α-Neo expression vector to obtain the NELFA-targeting sgRNA expression vector. This vector uses the piggyBac transposon as its backbone and contains an sgRNA expression element driven by the U6 promoter and a neomycin resistance selection marker driven by the EF1α promoter. It has two Eppendorf restriction sites at its multiple cloning site, enabling targeted insertion of the sgRNA fragment.

[0105] To ensure gene repression efficiency, four sgRNAs were cloned into sgRNA expression vectors containing neomycin resistance genes, and then co-transfected into human embryonic stem cells with CRISPRi vector and piggyBac transposase vector, respectively. Detection was performed by quantitative PCR and Western blotting after transfection. NELFA Efficiency of gene expression inhibition.

[0106] 3. Construction and induction of stable cell lines The following plasmids were co-transferred into human primed embryonic stem cells (hESCs): (1) CRISPRi expression vector based on piggyBac transposon system (containing puromycin resistance gene) (CRISPRi expression vector constructed above). (2) sgRNA expression vector (containing neomycin resistance gene) (NELFA-targeted sgRNA expression vector); (3) Highly active piggyBac transposase expression vector (refer to [Tang WWC, Castillo-Venzor A, Gruhn WH, et al. Sequential enhancer state remodelling defines human germline competence and specification[J]. Nature Cell Biology, 2022, 24(4):448-460.]). Transfection was performed using the Lonza 4D-Nucleofector system via electroporation. Cell lines stably integrating the CRISPRi system and sgRNA were selected 7–10 days post-transfection using a combination of puromycin and neomycin to obtain stable cell lines.

[0107] 4. Induction and CRISPRi system NELFA Gene suppression Human primordial embryonic stem cells (hESCs) stably integrated with the CRISPRi system and sgRNA were seeded in mTeSR medium. After obtaining a stable cell line, the medium was replaced with 4CL medium supplemented with doxycycline (Dox, 1 μg / mL) and trimethoprim (TMP, 10 μM) for continuous induction treatment for 3 days.

[0108] (1) Dox induces KRAB-dCas9 transcriptional expression; (2) TMP stabilizes the ecDHFR domain, preventing degradation of the fusion protein; (3) KRAB-dCas9 targets under the guidance of sgRNA NELFA Gene promoter regions, inhibiting their transcriptional activity; (4) EGFP expression is used to monitor the activation status of the system.

[0109] A control group (scramble sgRNA group) without Dox and TMP was set up simultaneously.

[0110] During the induction process, embryonic stem cells were subjected to fluorescence visualization detection, RT-qPCR and Western blot analysis, including (1) RT-qPCR detection. NELFA mRNA expression level; (2) Western blot detection of NELFA protein expression level. The results showed that a stable human primordial embryonic stem cell line carrying sgRNA and EGFP reporter gene Dox-induced CRISPRi transgene was established ( Figure 9 (b), and confirmed by RT-qPCR and Western blot analysis, respectively. NELFA Knockdown of mRNA and protein expression levels ( Figure 9 (c~d in the text).

[0111] 5. Human embryonic stem cells from primed to na Morphological changes on days 3 and 6 during VE conversion, such as Figure 10 As shown in e, RT-qPCR was used to detect the relative expression levels of pluripotency genes in different types of embryonic stem cells cultured under different conditions three days after induction. Figure 10 As shown in f, in e, CRISPRi of NELFA promoter corresponds to the results of human primordial embryonic stem cells integrating the CRISPRi system and sgRNA; WT corresponds to the results of untreated human primordial embryonic stem cells; in f, mTeSR_WT: the results of unconverted primed embryonic stem cells; 4CL_WT: the results of normally primed embryonic stem cells converted to NELFA. wild-type cells converted from ve; 4CL_CRISPRi_ NELFA (w / o Dox+TMP): Human primordial embryonic stem cells with integrated CRISPRi system and sgRNA cultured in a medium without the addition of Dox+TMP; 4CL_CRISPRi_ NELFA (with Dox+TMP): Human primordial embryonic stem cells with integrated CRISPRi system and sgRNA cultured with Dox+TMP added to the culture medium.

[0112] In 4CL medium, inhibiting the NELFA promoter did not induce the transition of human embryonic stem cells from the primordial state to the primitive state; however, WT embryonic stem cells successfully completed the transition from primordial to naïve state. ve conversion ( Figure 10 (e). Under Dox induction, despite CRISPRi-mediated NELFA promoter repression under 4CL conditions, some core primitive pluripotency-related genes, such as NANOG , DPPA3 and DPPA5 The expression level was still significantly lower than that of embryonic stem cells cultured in 4CL medium. Figure 10 (f)

[0113] 6. Cell proliferation and survival experimental procedure Experimental grouping and initial inoculation: 4CL_WT: Wild-type hESCs, cultured in 4CL medium; 4CL_CRISPRi of NELFA (w / o Dox+Tmp): hESCs stably integrated with the NELFA-targeting CRISPRi system were cultured in 4CL medium without the addition of Dox and TMP; 4CL_CRISPRi of NELFA (with Dox+Tmp): Stable integration NELFA hESCs targeting the CRISPRi system were cultured in 4CL medium and induced with the addition of Dox and TMP.

[0114] Initial seeding: On day 0, the three groups of cells were seeded at the same density (3 × 10⁻⁶). 4 Inoculate into petri dishes and incubate at 37°C in a 5% CO2 incubator.

[0115] On day 3 of culture, viable cell counts were performed on the three groups of cells, and the number of viable cells in each group was recorded. After counting on day 3, the three groups of cells were seeded again at the same density into new culture dishes and cultured under the original conditions until day 7. The results are as follows: Figure 11 g and h in the text.

[0116] The results showed that there was no significant difference in the number of viable cells between the 4CL_WT group and the uninduced group; however, the number of viable cells in the induced knockdown group was significantly different. NELFA The number of viable cells decreased significantly, and the number of cells inhibiting the NELFA promoter was much smaller than that of wild-type cells, indicating that... NELFA Inhibition can lead to impaired cell survival or proliferation.

[0117] In summary, NELFA It is not essential in primed embryonic stem cells, but it is necessary for primed to na The transformation process of ve is crucial; in this process, the expression at its basal level is necessary for maintaining the self-renewal of primitive pluripotency.

[0118] Example 4 NELFAActivation of 8-cell embryonic-like cells in human embryonic stem cells 1. e4CL culture system Human 8CLCs were generated using the e4CL method. For details, please refer to [link to documentation]. Figure 12 In b, WT and NELFA _OE na VE embryonic stem cells (i.e., those cultured in Example 2) NELFA _OE na Embryonic stem cells (VE) are digested into single cells at a concentration of 2,000-3,000 cells / cm³. 2 Cells were seeded at a density of [insert density here] on Matrigel-coated culture plates and cultured in 4CL medium. After 24 hours, the medium was replaced with e4CL medium. The composition of e4CL is the same as 4CL, except that the concentrations of DZNep (50 nM) and TSA (20 nM) are higher. The e4CL medium was changed daily until day 6. Cells were cultured in an incubator at 37°C and 5% CO2.

[0119] 2. Regarding NELFA In the _OE group, doxycycline (Dox) was added to e4CL medium to induce... NELFA Overexpression; WT group synchronous processing, without adding Dox.

[0120] On day 5 of culture, the embryonic stem cells were subjected to fluorescence visualization detection, and the results were as follows: Figure 12 c in the text.

[0121] Human 8CLCs were induced in e4CL medium, and the results showed that those cultured in e4CL medium... NELFA In overexpressing embryonic stem cells, the number of dome-shaped colonies was greater than that of wild-type embryonic stem cells cultured in e4CL medium.

[0122] 3. RT-qPCR detection of WT and NELFA The relative expression levels of pluripotency and 8CLCs-related genes in OE embryonic stem cells were determined 7 days after induction in e4CL medium. The results are as follows: Figure 13 d~e in. Immunofluorescence staining detection of WT and NELFA The protein levels of TPRX1 and ZSCAN4 in OE embryonic stem cells were measured 7 days after induction in e4CL medium. The results are as follows: Figure 13 f in Western Blot detection of WT and NELFA Expression levels of proteins related to 8CLCs of OE embryonic stem cells were examined, along with WT and NELFA OE embryonic stem cells in na The expression levels of related proteins in embryonic stem cells were as follows: Figure 14 As shown.

[0123] Compared to WT embryonic stem cells cultured in e4CL medium, those cultured in e4CL medium... NELFA Overexpression of pluripotency-related genes in embryonic stem cells (including OCT4 and SOX2 The level of expression for ) has slightly decreased, while DPPA3 The expression level was then increased ( Figure 13 (d). Importantly, compared to wild-type embryonic stem cells cultured in e4CL medium, those cultured in e4CL medium... NELFA Overexpression of 8CLCs-related transcripts in embryonic stem cells DUX4 , TRIM43 and H3.X / Y Significantly upward ( Figure 13 (e). Immunostaining results showed that, compared with WT embryonic stem cells cultured in e4CL medium, those cultured in e4CL medium... NELFA The number of TPRX1 and ZSCAN4 positive cells increased in overexpressing embryonic stem cells. Figure 13 f and Figure 14 We also quantified the levels of proteins related to 8CLCs using Western blot analysis. The results showed that, compared to WT embryonic stem cells cultured in e4CL medium, those cultured in e4CL medium... NELFA Protein levels of ZSCAN4, DUX, and TPRX1 were significantly upregulated in embryonic stem cells overexpressing ZSCAN4, DUX, and TPRX1. Figure 14 The above results clearly indicate that... NELFA It can activate human 8CLCs.

[0124] Example 5 NELFA Induced human 8-cell embryo-like cells have expanded pluripotency 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.

[0125] Eight-cell stage embryos were obtained by flushing the oviducts of ICR female mice.

[0126] Green fluorescent protein (EGFP) labeled, cultured in e4CL medium NELFAWild-type embryonic stem cells overexpressing embryonic stem cells and labeled with green fluorescent protein (EGFP) cultured in e4CL medium were digested and dispersed separately, and then extracted from cells cultured under e4CL conditions. NELFA Overexpression of embryonic stem cells ( NELFA Fifteen cells were taken from _OE ESCs and wild-type embryonic stem cells (WT) and injected into 8-cell stage mouse embryos, respectively.

[0127] After microinjection, 8-cell stage embryos were placed in KSOM medium (Millipore, MR-101) and cultured at 37°C and 5% CO2 for 48–60 h. Subsequently, the resulting chimeric blastocysts were fixed for immunofluorescence staining analysis.

[0128] Immunostaining was performed using OCT4 and CDX2 antibodies. Cell nuclei were counterstained with DAPI. Three independent immunostaining experiments were conducted, and the results are as follows: Figures 15-17 As shown. WT and WT cultured in e4CL medium. NELFA A summary of cell contributions to the trophoblast (TE) and inner cell mass (ICM) in OE embryonic stem cell-derived chimeras is as follows: Figure 16 As shown.

[0129] Its chimeric contribution was assessed by co-staining with markers of trophectoderm (TE) and inner cell mass (ICM). The results showed that... NELFA The induced cells in chimeric blastocysts expressed CDX2-positive trophoblast markers (5 / 33 recovered embryos, 15.2%) and OCT4-positive inner cell mass markers (20 / 31 recovered embryos, 64.5%), a significantly higher proportion than WT embryonic stem cells cultured in e4CL medium. NELFA The induced human 8-cell embryo-like cells have expanded pluripotency.

[0130] 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. NELFA Application in regulating the pluripotency of human embryonic stem cells.

2. The application according to claim 1, characterized in that, The regulation includes promoting the conversion of human embryonic stem cells from the primordial state to the primitive state.

3. The application according to claim 1 or 2, characterized in that, The regulation includes: overexpression NELFA To promote the acquisition of primitive pluripotency in human embryonic stem cells; or to inhibit NELFA It is expressed in order to inhibit the conversion of human embryonic stem cells to the primitive state.

4. The application according to claim 3, characterized in that, Acquiring primitive pluripotency includes increasing the expression levels of primitive pluripotency-related factors in cells and / or causing cells to exhibit primitive-specific morphology.

5. Overexpression NELFA Application in the preparation of reagents or kits for inducing the transformation of human embryonic stem cells into 8-cell embryo-like cells.

6. The application according to claim 5, characterized in that, The human embryonic stem cells include primordial human embryonic stem cells and / or primitive human embryonic stem cells.

7. The application according to claim 5 or 6, characterized in that, The 8-cell embryo-like cells exhibit cellular characteristics of the early embryonic development stage.

8. Overexpression NELFA Application in enhancing cell expansion pluripotency; the enhancement of cell expansion pluripotency includes enhancing the expansion pluripotency of 8-cell embryo-like cells.

9. A method for inducing the transformation of human embryonic stem cells into early embryonic stage cells in vitro, characterized in that, include: Increase the concentration of human embryonic stem cells NELFA The expression or activity of.

10. An 8-cell embryonic-like cell, characterized in that, Human embryonic stem cell overexpression was induced in vitro. NELFA It was prepared.

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