Application of lysosome inhibitor in induction and maintenance of totipotency of stem cells and induction method

Abstract

In order to improve the defects of the existing in-vitro totipotent stem cell culture technology, the invention provides an application of a lysosome inhibitor in the aspects of in-vitro induction and culture of totipotent stem cells and promotion of entry of the totipotent stem cells and maintenance of a totipotent state. According to the invention, three known lysosome inhibitors are used to promote the in-vitro induced totipotent stem cells to maintain more, faster and longer totipotent states, and more operable space is provided for clinical totipotent stem cell application.

Description

Technical Field

[0001] The present invention relates to a technology for inducing pluripotent stem cells, and in particular to a technology for inducing and maintaining the pluripotency of stem cells by using a lysosome inhibitor. Background Art

[0002] Totipotency is the stemness with the most differentiation plasticity and is a key research direction in regenerative medicine. The difference between totipotency and pluripotency is that totipotent stem cells / embryos have the potential to develop into a complete individual, while pluripotent stem cells / embryos can only differentiate into most organs and lack the ability to develop into a complete individual. In mice, embryos acquire totipotency after fertilization, gradually exit totipotency after entering the four-cell stage, begin cell lineage differentiation, and finally form a pluripotent inner cell mass and blastocyst stage.

[0003] In the field of totipotency research, a large number of basic and clinical studies are difficult to carry out systematically due to the scarcity of totipotent embryos. Recently, a study has discovered an in vitro cell model of totipotency, 2-cell-like cells (2CLCs). This is a rare cell population present in mouse embryonic stem cells, which is highly similar to totipotent two-cell embryos. 2CLCs highly express totipotency-related transcripts and have differentiation capabilities similar to totipotency. Mouse embryonic stem cells will spontaneously enter the two-cell-like state and will spontaneously exit the two-cell-like state. My previous studies have pointed out that the entry into the two-cell-like state undergoes large-scale activation of totipotency-specific transcripts; the exit from the two-cell-like state undergoes the inhibition of totipotency-specific transcripts and the activation of pluripotency transcripts. Therefore, at the transcriptome level, the entry and exit process of the two-cell-like state is similar to the acquisition and exit process of embryonic totipotency, and is a reliable in vitro model of totipotency.

[0004] Naturally formed 2CLCs in mouse embryonic stem cells are very rare, accounting for less than 1% of the total number of cells, making it difficult to conduct systematic studies. To date, many research reports have suggested that compounds can induce the production of totipotent embryonic stem cells in vitro, such as the TAW cocktail drug combination of multiple compound treatments and the splicing inhibitor Pladienolide B (PlaB) treated with a single compound. In addition to compound treatments, the OBOX family, a functional homologous family in mice, is also very abundantly expressed in oocytes, one-cell and two-cell embryos. The binding sequence (motif) of OBOX is highly enriched in the open chromatin of mouse totipotent embryos. Overexpression of OBOX in mouse embryonic stem cells can also activate some mouse ZGA genes, so overexpression of OBOX can also induce the production of totipotent stem cells. In addition, the applicant and the inventor have also established an inducible two-cell-like cell line (such as) in some previous studies.Figure 1 The cell line was treated with doxycycline to induce the expression of the transcription factor Dux, which further drove the expression of the 5'LTR (2C::tdTomato) of MERVL, thereby promoting the transformation of cells into 2CLCs. At the same time, these 2CLCs can spontaneously return to a pluripotent state.

[0005] So far, many studies have reported that different compounds can induce the production of human or mouse totipotent embryonic stem cells in vitro, but there are still many aspects that need to be improved. First, the use of compound treatment can stably induce totipotent cells, but its specific biological molecular mechanism is still unclear, and the induction effect is slow and weak, leaving room for optimization. Secondly, the use of transcription factors, such as Dux, induces totipotent stem cells that are unstable and cannot be cultured for a long time. Therefore, the existing totipotent stem cell induction and culture methods have a great need for optimization.

[0006] In addition, the development of totipotent embryos undergoes multi-dimensional phenotypic changes, including remodeling of transcriptomes and epigenetic modifications, which play an important role in the regulation of totipotency. Recent studies have shown that totipotent embryonic development is also regulated by metabolic signals. Embryonic development is in a low-energy mode during the totipotency stage, with low metabolic demands, and the regulatory role of metabolic signals in totipotency has long been neglected. Therefore, we hope to optimize the existing methods of inducing and culturing totipotent stem cells from a metabolic perspective. Summary of the invention

[0007] The technical purpose of the present invention is to improve the deficiencies of existing in vitro pluripotent stem cell culture technology by using three known lysosomal inhibitors to promote in vitro induced pluripotent stem cells to maintain a pluripotent state more, faster and longer, thereby providing more operational space for clinical pluripotent stem cell applications.

[0008] In order to achieve the above technical objectives,

[0009] In a first aspect, the present invention provides a use of a lysosome inhibitor in inducing pluripotent stem cells in vitro.

[0010] Preferably, the lysosomal inhibitor may be Vacuolin-1, whose chemical structure is shown in Formula I:

[0011]

[0012] Vacuolin-1 is a potent, cell-permeable inhibitor of lysosomal exocytosis. Vacuolin-1 blocks lysosomal Ca 2+Vacuolin-1 inhibits late autophagy by impairing lysosomal maturation. Vacuolin-1 induces vacuole formation and increases the percentage of enucleated cells.

[0013] The lysosomal inhibitor may be Bafilomycin A1 (Baf A), the chemical structure of which is shown in Formula II:

[0014]

[0015] Preferably, Baf A is a specific, reversible V-ATPase inhibitor, and as a macrolide antibiotic, is an inhibitor of the late stage of autophagy. Baf A can block the fusion of autophagosomes and lysosomes, inhibit acidification and protein degradation in lysosomes of cultured cells, and also induce the occurrence of apoptosis.

[0016] Preferably, the lysosomal inhibitor may be Hydroxychloroquine (HCQ), the chemical structure of which is shown in Formula III:

[0017]

[0018] HCQ is a synthetic oral antimalarial drug that can be used in the study of malaria and autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. In addition, HCQ is also a potent autophagic flux inhibitor.

[0019] In a second aspect, the present invention provides use of the above-mentioned lysosome inhibitor in culturing totipotent stem cells.

[0020] In a third aspect, the present invention provides use of the above-mentioned lysosome inhibitor in promoting pluripotent stem cells to enter a totipotent state.

[0021] In a fourth aspect, the present invention provides use of the above-mentioned lysosome inhibitor in maintaining the pluripotent state of pluripotent stem cells.

[0022] In a fifth aspect, the present invention provides a method for inducing pluripotent embryonic stem cells using the above-mentioned lysosomal inhibitor, the induction method comprising the following specific steps:

[0023] (1) Embryonic stem cell recovery, culture, passage and cryopreservation:

[0024] (1.1) Recovery: Add gelatin solution to the cell culture grade well plate for coating.

[0025] Take the frozen mouse embryonic stem cells (mESCs) out of liquid nitrogen, thaw in a water bath, discard the supernatant after centrifugation, resuspend the cells in Serum / LIF medium, and inoculate the cells into the coated well plate. Replace with fresh Serum / LIF medium the next day for culture.

[0026] (1.2) Culture: After completing step (1.1), the mouse embryonic stem cells (mESCs) are revived and cultured with fresh PGEX medium every day. The cell status is observed until the embryonic stem cells grow well and show obvious clonal status.

[0027] (1.3) Passaging: Before passaging, coat the cell culture plate with gelatin. Then, wash the mouse embryonic stem cells (mESCs) cultured in step (1.2) with PBS once, add trypsin, place in an incubator for digestion, and add an equal volume of Serum / LIF medium to terminate the digestion. After centrifugation, discard the supernatant, resuspend the cells with Serum / LIF medium, inoculate the cells into the coated well plate, and replace with fresh Serum / LIF medium for culture the next day.

[0028] (1.4) Cryopreservation: After discarding the culture medium, wash the passaged mouse embryonic stem cells (mESCs) with PBS, then add trypsin. After the cells become round and fall off, add complete culture medium to terminate digestion. Centrifuge and remove the supernatant. Add the freezing solution containing DMSO, mix gently, and mark it. Place the cryopreservation tube in a program cooling box, put it in a -80℃ refrigerator, and transfer it to liquid nitrogen for storage after at least 2 hours.

[0029] (2) Obtaining 2CLCs:

[0030] Use 2ug ml -1 Mouse embryonic stem cells (mESCs) containing the 2C::tdTomato reporter gene and doxycycline-induced Dux expression that have completed step (1) were treated with doxycycline for 16 hours, and mouse embryonic stem cells (mESCs) without doxycycline were used as negative controls; then the proportion of tdTomato-positive 2CLCs cells was counted using the 568nm channel of a flow cytometer, or 2CLCs were sorted out for a pluripotency maintenance experiment.

[0031] (3) Maintenance of omnipotence:

[0032] The 2CLCs sorted by flow cytometry were resuspended in Serum / LIF medium and inoculated into the coated well plate. After culturing for 24 hours, the proportion of 2CLCs cells that maintained tdTomato positivity was counted using the 568 nm channel of the flow cytometer.

[0033] (4) Flow cytometer analysis of cells:

[0034] The culture medium of the cells cultured in the 24-well plate was discarded, and DPBS was added to slowly wash twice to wash away dead cells; each well was digested with trypsin, and the stop solution was added after the digestion was completed; the supernatant was discarded after centrifugation, and the cells were resuspended in DPBS, filtered and analyzed by BD LSRFortessa flow cytometer.

[0035] (5) RNA-seq and data analysis:

[0036] The samples of the control and experimental groups were collected and sent to a sequencing company to construct cDNA libraries using Smart-seq2 technology. After the library was sequenced by the illumina platform, the raw sequencing data was aligned to the mm10 genome using the STAR (v2.5.2b) software, and the repeat fragments were quantified using RepEnrich (v1.2) and seqtk (v1.3). The differentially expressed genes between different groups were calculated using the DESeq2 software package (v1.32.0). The differentially expressed genes that met the criteria of |log2FoldChange|>1 and P value <0.05 were considered statistically significant. The volcano plot was generated by ggplot2 (v3.3.5), with the x-axis representing the log2-fold expression change and the y-axis representing the logarithmic transformed p value. The pheatmap (v1.0.12) software package was used to draw the heat map. The GSEA data were analyzed using the fgsea (v1.18.0) software package.

[0037] (6) RNA extraction and reverse transcription reaction:

[0038] (6.1) Place the tissue into an RNase-free centrifuge tube, chop it into small pieces, add Trizol, break it up with a homogenizer, add Trizol again and oscillate to mix. After the cells are fully lysed, proceed to the next step.

[0039] (6.2) Add chloroform, shake vigorously to mix, let stand at room temperature, and centrifuge for 10 minutes.

[0040] (6.3) Transfer the upper aqueous phase to a new RNase-free centrifuge tube containing chloroform, shake to mix, and let stand at room temperature.

[0041] (6.4) Centrifuge once, transfer the aqueous phase to a new RNase-free centrifuge tube, add an equal volume of isopropanol, vortex to mix, and let stand at room temperature.

[0042] (6.5) Centrifuge again, aspirate the supernatant, and add 75% ethanol (RNase-free) to resuspend the white precipitate.

[0043] (6.6) Centrifuge three times, aspirate the supernatant, air-dry at room temperature, and add an appropriate volume of RNase-free water to fully dissolve.

[0044] (6.7) Prepare tissue reverse transcription system.

[0045] (6.8) After the system is prepared, mix and centrifuge, and place it in the PCR instrument.

[0046] (6.9) Add 5×qRT SuperMix II to the system in step (6.7), gently pipette to mix, and place in a PCR instrument to react to obtain cDNA stock solution.

[0047] (7) Real-time fluorescence quantitative PCR detection (RT-qPCR)

[0048] Each experiment was repeated three times, with DMSO as vehicle and GAPDH as internal reference, and the relative quantification method (2 -ΔΔCt ) was used to detect the relative expression levels of mouse embryonic stem cell pluripotency marker genes (Zscan4 and MERVL), and their gene sequences are shown in Table 4.

[0049] (8) Obtaining knockout cell lines

[0050] The CRISPR / Cas9 system was used to knock out the lysosomal inhibitor target proteins Flcn and Lamtor1 in mouse embryonic stem cells. Two specific sgRNAs were designed in the gene CDS using the LentiCrisper-v2-puromycin two-in-one plasmid, and the plasmid was integrated into the mouse embryonic stem cell genome using a lentivirus. The monoclonal cell line obtained was identified by genome extraction and Sanger sequencing of the genomic phenotype, and the positive knockout cell lines obtained by sequencing were frozen.

[0051] (9) Immunoblotting

[0052] (9.1) Lyse the sample with SDS loading buffer.

[0053] (9.2) Protein denatured samples can be stored in a -20℃ refrigerator for a short period of time.

[0054] (9.3) Prepare SDS-PAGE gel of appropriate concentration according to the size of the protein, flatten the gel surface with n-butanol, and let it stand at room temperature for more than half an hour. Wash off the n-butanol, prepare the upper concentrated gel, insert the sample comb, and let it stand at room temperature for more than half an hour.

[0055] (9.4) After loading the sample, run the gel at a constant voltage of 140 V. The running time depends on the position of the target band.

[0056] (9.5) Use a semi-dry transfer apparatus to transfer the protein samples on the gel to the PVDF membrane. The transfer time depends on the size of the target protein.

[0057] (9.6) Block the transferred PVDF membrane with milk for half an hour.

[0058] (9.7) Dilute the primary antibody with TBST buffer and place the PVDF membrane in the primary antibody buffer and incubate overnight at 4°C.

[0059] (9.8) Wash the PVDF membrane three times with TBST buffer.

[0060] (9.9) Dilute the secondary antibody of the corresponding source in TBST buffer at 1:5000 and incubate with the PVDF membrane at room temperature for half an hour.

[0061] (9.10) Wash the PVDF membrane three times with TBST buffer.

[0062] (9.11) Press an appropriate amount of X-ray film on the PVDF membrane after the ECL chemical enhanced luminescence reaction, and develop it after a short exposure. Mark the development information, scan the photo and save it.

[0063] (10) Immunofluorescence staining

[0064] Mouse embryonic stem cells were cultured overnight in gelatin-coated slides before staining. After washing with PBS, they were fixed with paraformaldehyde PFA, permeabilized with Triton X-100, and incubated overnight in primary antibody buffer (the primary antibody concentration used in this study was 1:200). The slides were then washed 3 times with PBS and incubated in secondary antibodies conjugated with the relevant resistance fluorescence coupled with the nuclear dye DAPI for 1 hour. The slides were then washed 3 times with PBS. All stained samples after sealing were observed and collected using a confocal laser scanning microscope (Olympus FV3000).

[0065] (11) Cleavage Under Targets and Tagmentation (CUT&Tag)

[0066] CUT&Tag follows the previously reported protocol to construct the library. The specific steps mainly include:

[0067] (11.1) Activate Concanavalin A beads before starting the experiment.

[0068] (11.2) Cell and tissue preparation

[0069] (11.3) Binding of Concanavalin A beads and primary antibodies

[0070] (11.4) Secondary Antibody Binding

[0071] (11.5) pAG-Tn5 enzyme binding and enzyme fragmentation

[0072] (11.6) DNA purification

[0073] (11.7) NG-Sequencing Library Construction

[0074] (11.8) After the library was built, it was sent to the company for sequencing, and the raw data was first processed using trim-galore (v0.6.7).

[0075] (11.9) The read segments were mapped to the mm10 genome using bowtie2 (v2.3.5.1) software.

[0076] (11.10) Use Samtools (v1.9) software to convert and classify SAM files into BAM format.

[0077] (11.11) Select the top 1% region of AUC and use SEACR (v1.3) to identify peak signals

[0078] (11.12) Use the bamCoverage function of Deeptools (v3.5.1) to convert the bam file to bigwig file.

[0079] (11.13)ComputeMatrix is ​​used to calculate the overall CUT&Tag signal.

[0080] (11.14)0plotProfile is used to visualize signal distribution.

[0081] (12) Data Analysis

[0082] Statistical analysis of data was performed using GraphPad (v8) or R (v4.1). Statistical significance was determined by Student's unpaired t-test or nonparametric Mann-Whitney U test. All sequencing experiments were performed in 2 biological replicates. All other experiments were performed in at least 3 biological replicates and repeated at least 2 times independently with similar results.

[0083] The beneficial effects of the present invention are:

[0084] (1) The present invention breaks through the scope of use of known lysosome inhibitors and innovatively uses lysosome inhibition to optimize the in vitro pluripotent stem cell induction culture scheme, making the pluripotent cell induction faster and more efficient, and the pluripotent cell culture more stable.

[0085] (2) The present invention optimizes the in vitro pluripotent stem cell culture by using three commonly used lysosomal inhibitors, Vacuolin-1, Baf A (Bafilomycin A1) and HCQ (Hydroxychloroquine), which can effectively promote more and faster pluripotent stem cells to enter the pluripotent state and better maintain the pluripotent state.

[0086] (3) The present invention uses molecular biological methods to analyze and identify the biological target proteins of lysosomal inhibitors in the process of omnipotence regulation, and further analyzes the regulatory molecular mechanism of intracellular lysosomal inhibitors, thereby reducing the side effects of lysosomal inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 It is an inducible two-cell-like cell line in the background art.

[0088] Figure 2 This is a schematic diagram of how lysosomal signals regulate cellular nutrient metabolism and stem cell differentiation.

[0089] Figure 3 This is the totipotent cell transcript gene set in Example 1.

[0090] Figure 4 This is the pluripotent cell transcript gene set in Example 1.

[0091] Figure 5 This is a graph showing the results of lysosomal activity detection of mouse embryonic stem cells (mESCs) and two-cell-like cells (2CLCs) in Example 1.

[0092] Figure 6 This is a flow cytometry result diagram of the conversion of in vitro induced pluripotent stem cells into 2CLCs after treatment with lysosomal inhibitors in Example 2.

[0093] Figure 7 This is a flow cytometry result showing that 2CLCs maintained their pluripotency after treatment with lysosomal inhibitors in Example 3.

[0094] Figure 8 It is a volcano plot of the transcriptome sequencing analysis in Example 4.

[0095] Fig. 9 This is a graph showing the GSEA results of the transcriptome sequencing analysis in Example 4.

[0096] Fig.10 This is a schematic diagram of the relative expression levels of two representative intracellular transcripts after lysosomal inhibitor treatment as shown in Example 4 by RT-qRCR detection.

[0097] Fig.11RT-qPCR detection in Example 4 shows the relative expression levels of two representative cell transcripts in stem cells overexpressing Obox3 and Obox5 after lysosomal inhibitor treatment.

[0098] Fig.12 This is a schematic diagram of the relative expression levels of two representative cell transcripts in PlaB-induced stem cells after treatment with the lysosomal inhibitor Vacuolin-1 as shown in Example 4 by RT-qPCR.

[0099] Fig.13 This is a schematic diagram of the relative expression levels of two representative cell transcripts in PlaB-induced stem cells after treatment with the lysosomal inhibitor Baf A as shown in Example 4 by RT-qPCR.

[0100] Fig.14 This is a ranking analysis graph of the knockout screening data of potential regulatory factors affecting pluripotency transformation in Example 5.

[0101] Fig.15 This is a diagram of the results of the immunoblotting experiment in Example 5 (Gapdh is the internal reference protein).

[0102] Fig.16 This is a flow cytometry result diagram of the transformation of in vitro induced pluripotent stem cells into 2CLCs after knockout of lysosome-related proteins Flcn and Lamtor1 in Example 5.

[0103] Fig.17 This is a flow cytometry statistical graph of the proportion of 2CLCs maintaining a pluripotent state after the lysosome-related proteins Flcn and Lamtor1 were knocked out in Example 5.

[0104] Fig.18 This is a bar graph showing the expression levels of substrate-specific selectin and substrate protein during the mouse embryonic totipotency conversion process in Example 5.

[0105] Fig.19 This is a flow cytometry statistical result diagram of the 2CLCs ratio in the Vacuolin-1 treated group after overexpression of active RagC in Example 5.

[0106] Fig. 20 This is the immunofluorescence result of the localization of transcription factors Tfe3, Tfeb and Mitf after Vacuolin-1 treatment in Example 5.

[0107] Fig.21 This is a flow cytometry statistical result diagram of the 2CLCs ratio in the Vacuolin-1 treatment group after simultaneous knockdown of Tfe3 and Tfeb in Example 5.

[0108] Fig. 22This is a schematic diagram of the relative expression levels of two representative transcripts in pluripotent stem cells induced by doxycycline after overexpression of nuclear-localized forms of Tfe3 and Tfeb as shown by RT-qPCR in Example 5.

[0109] Fig.23 This is a CUT&Tag result diagram of the binding strength of Tfeb and Tfe3 at MERVL and MT2_Mm when the stem cells enter (+DOX) and exit (-DOX) the pluripotent state after Flcn knockout in Example 5. DETAILED DESCRIPTION

[0110] In order to enable those skilled in the art to better understand the technical solution of the present invention, some technical terms of the present invention are first explained. In the text of the present invention, these explanations are mainly to help those skilled in the art understand and implement the technical solution of the present invention, rather than to limit the present invention. In addition, there may be other technical understandings of these technical terms in other disclosures outside the present invention. In order to avoid technical ambiguity, when understanding the present invention, these explanations of the present invention should be based on them.

[0111] Lysosomes and Lysosomal Inhibitors

[0112] Lysosomes are the main organelles for catabolism in cells, containing a variety of hydrolases for breaking down biomacromolecules such as proteins, nucleic acids, and polysaccharides. Ragulator-Rag complexes are clustered on the surface of lysosomes, which can sense the concentration of amino acids in lysosomes and regulate a variety of nutrient energy metabolism pathways including mammalian target of rapamycin complex 1 (mTORC1) and autophagy. Lysosomal signals not only play an important role in maintaining cellular nutrient energy balance, but also affect stem cell development and differentiation. In this process, Lamtor1, as a membrane protein specifically localized on the surface of late endosomes / lysosomes, participates in lysosomal transport and maturation, and is the anchor of the Ragulator complex to bind to the lysosome. Lysosomes can anchor MITF / TFE transcription factors through the Ragulator-Rag complex, and regulate the phosphorylation of MITF / TFE transcription factors through the RagC GTPase activating protein Flcn, causing them to remain in the cytoplasm and inhibit the transcription factor from entering the nucleus ( Figure 2 ).

[0113] The function of lysosomes in pluripotency has not yet been identified. Three common lysosome inhibitors are Vacuolin-1, Bafilomycin A1 (abbreviated as Baf A) and Hydroxychloroquine (abbreviated as HCQ). The present invention has found that inhibiting lysosomes can significantly improve the induction efficiency of pluripotent cells and the culture stability of pluripotent cells, and provides the use of the above three known commercial lysosome inhibitors in improving the in vitro pluripotent cell culture method.

[0114] Vacuolin-1 is a potent cell-permeable lysosomal exocytosis inhibitor. Vacuolin-1 blocks lysosomal Ca 2+ Vacuolin-1 inhibits late autophagy by impairing lysosomal maturation. Vacuolin-1 can induce vacuole formation and increase the percentage of enucleated cells. Its chemical structure is shown in Formula I:

[0115]

[0116] Baf A is a specific, reversible V-ATPase inhibitor. As a macrolide antibiotic, it is an inhibitor of the late stage of autophagy. Baf A can block the fusion of autophagosomes and lysosomes, inhibit acidification and protein degradation in lysosomes of cultured cells, and induce apoptosis. Its chemical structure is shown in Formula II:

[0117]

[0118] HCQ is a synthetic oral antimalarial drug that can be used in the study of malaria and autoimmune diseases (such as systemic lupus erythematosus and rheumatoid arthritis). In addition, HCQ is also an effective autophagic flux inhibitor, and its chemical structure is shown in Formula III:

[0119]

[0120] In the embodiments of the present invention, it is necessary to perform multiple groups or multiple experiments. In order to enable those skilled in the art to better implement the technical solution of the present invention, some methodologies of the present invention are first introduced. It should be noted that the methodology is not a limitation of the present invention. The specific numerical values ​​in the methodology only represent the experimental numerical values ​​(or numerical ratios) of one time, and cannot be regarded as completely absolute numerical values. Moreover, the methodology itself does not obtain the results, and the methodology needs to be combined with the embodiments to obtain the experimental results. In addition, the methodology not recorded in the present invention should generally be considered as a method in the prior art that can be directly obtained by those skilled in the art.

[0121] Reverse transcription reaction

[0122] (1) Place the tissue in a 1.5 mL RNase-free centrifuge tube, chop it into small pieces, add 500 mL Trizol, break it up with a homogenizer, and then add another 500 mL Trizol and oscillate to mix. After the cells are fully lysed, proceed to the next step.

[0123] (2) Add 200 μL of chloroform, shake vigorously to mix, let stand at room temperature for 5 minutes, and centrifuge at 12,000 rpm for 10 minutes at 4°C.

[0124] (3) Transfer the upper aqueous phase to a new 1.5 mL RNase-free centrifuge tube containing 200 μL chloroform, vortex to mix, and let stand at room temperature for 3 minutes.

[0125] (4) Centrifuge at 12,000 rpm for 15 min at 4°C. Transfer the aqueous phase to a new 1.5 mL RNase-free centrifuge tube. Add an equal volume of isopropanol, vortex to mix, and let stand at room temperature for 10 min.

[0126] (5) Centrifuge at 12,000 rpm for 20 min at 4°C, carefully aspirate the supernatant, and add 1 mL of 75% ethanol (RNase-free) to resuspend the white precipitate.

[0127] (6) Centrifuge at 12,000 rpm for 5 min at 4°C, carefully aspirate the supernatant, air-dry at room temperature for 1 min, and add an appropriate volume of RNase-free water to fully dissolve.

[0128] (7) Prepare the tissue reverse transcription system as shown in Table 1 below.

[0129] Table 1

[0130] Components content Tissue RNA 1 μg 4×gDNA wiper Mix 4μL RNase-free water Make up to 16 μL

[0131] (8) After the system is prepared, mix and centrifuge, then place in a PCR instrument at 42°C for 2 min.

[0132] (9) Add 4 μl of 5×qRT SuperMix II to the system in step (7), gently pipette and mix well, place in a PCR instrument and react at 50°C for 15 minutes and at 85°C for 2 minutes to obtain the cDNA stock solution.

[0133] Real-time fluorescence quantitative PCR (RT-qPCR)

[0134] (1) Each sample was set up in triplicate according to the following reaction system. The primer sequences are shown in Table 2 below.

[0135] Table 2

[0136]

[0137] (2) Set the PCR program as shown in Table 3 below.

[0138] Table 3

[0139]

[0140] (3) After the program is completed, check the melting curve of each primer pair and derive the CT value.

[0141] (4) Each experiment was repeated three times, with DMSO as the vehicle and GAPDH as the internal reference. The relative quantification method (2 -ΔΔCt ) was used to detect the relative expression levels of mouse embryonic stem cell pluripotency marker genes (Zscan4 and MERVL), and their gene sequences are shown in Table 4.

[0142] Table 4

[0143]

[0144] Derivation of knockout cell lines

[0145] The CRISPR / Cas9 system was used to knock out the lysosomal inhibitor target proteins Flcn and Lamtor1 in mouse embryonic stem cells. Two specific sgRNAs were designed in the gene CDS using the LentiCrisper-v2-puromycin two-in-one plasmid, and the plasmid was integrated into the mouse embryonic stem cell genome using a lentivirus. The monoclonal cell line obtained was identified by genome extraction and Sanger sequencing of the genomic phenotype, and the positive knockout cell lines obtained by sequencing were frozen.

[0146] Immunoblotting

[0147] (1) Lyse the sample with SDS loading buffer.

[0148] (2) Treat at 95°C for 10 minutes. The protein-denatured samples can be stored in a -20°C refrigerator for a short period of time.

[0149] (3) Prepare SDS-PAGE gel of appropriate concentration (10% to 15%) according to the size of the protein, flatten the gel surface with n-butanol, and let it stand at room temperature for more than half an hour. Wash off the n-butanol, prepare a 5% upper concentrated gel, insert the sample comb, and let it stand at room temperature for more than half an hour.

[0150] (4) After loading the sample, run the gel at a constant voltage of 140 V. The running time depends on the position of the target band.

[0151] (5) Use a semi-dry transfer apparatus to transfer the protein samples on the gel to the PVDF membrane. The transfer time depends on the size of the target protein.

[0152] (6) Block the transferred PVDF membrane with 5% milk (skim milk powder dissolved in TBST buffer) for half an hour.

[0153] (7) Dilute the primary antibody with TBST buffer and place the PVDF membrane in the primary antibody buffer and incubate overnight at 4°C.

[0154] (8) Wash the PVDF membrane three times with TBST buffer, 5 min each time.

[0155] (9) Dilute the secondary antibody of the corresponding source in TBST buffer at 1:5000 and incubate with the PVDF membrane at room temperature for half an hour.

[0156] (10) Wash the PVDF membrane three times with TBST buffer, 5 min each time.

[0157] (11) Press an appropriate amount of X-ray film on the PVDF membrane after the ECL chemical enhanced luminescence reaction, and develop it after a short exposure. Mark the development information, scan the photo and save it.

[0158] Immunofluorescence staining

[0159] Mouse embryonic stem cells need to be cultured overnight in gelatin-coated slides before staining. After washing with PBS, fix with 4% paraformaldehyde PFA for 15 minutes, permeabilize with 0.5% Triton X-100 for 15 minutes, and then incubate in primary antibody buffer overnight at 4°C (the primary antibody concentration used in this study was 1:200), then wash the slides 3 times with PBS, and incubate in secondary antibodies coupled with relevant resistance fluorescence coupled with nuclear dye DAPI for 1 hour, then wash the slides 3 times with PBS, and all stained samples after sealing were observed and collected using a confocal laser scanning microscope (Olympus FV3000).

[0160] Cleavage Under Targets and Tagmentation (CUT&Tag)

[0161] CUT&Tag follows the previously reported protocol to construct the library. The specific steps mainly include:

[0162] (1) Activate Concanavalin A beads before the experiment.

[0163] (2) Cell and tissue preparation,

[0164] (3) Binding of Concanavalin A beads and primary antibody,

[0165] (4) Secondary antibody binding,

[0166] (5) pAG-Tn5 enzyme binding and enzymatic fragmentation,

[0167] (6) DNA purification,

[0168] (7) NG-Sequencing library construction,

[0169] (8) After the library was constructed, it was sent to the company for sequencing. The raw data was first processed using trim-galore (v0.6.7).

[0170] (9) The reads were mapped to the mm10 genome using bowtie2 (v2.3.5.1) software.

[0171] (10) Use Samtools (v1.9) software to convert and classify SAM files into BAM format.

[0172] (11) Select the top 1% region of AUC and use SEACR (v1.3) to identify the peak signal.

[0173] (12) Use the bamCoverage function of Deeptools (v3.5.1) to convert the bam file to a bigwig file.

[0174] (13) ComputeMatrix is ​​used to calculate the overall CUT&Tag signal.

[0175] (14)0plotProfile is used to visualize signal distribution.

[0176] The present invention is described in detail, clearly and completely in the following embodiments in conjunction with the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Moreover, based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work, any modifications, equivalent substitutions, improvements, etc., should be included in the protection scope of the present invention.

[0177] Example 1, recovery, culture, passage and cryopreservation of mouse embryonic stem cells.

[0178] (1.1) Recovery: Add 1 mL of 0.1% gelatin solution to a cell culture grade plate and coat at 37°C for 30 minutes.

[0179] Take the frozen mouse embryonic stem cells (mESCs) out of liquid nitrogen, thaw in a 37°C water bath, centrifuge at 500 rcf / 5 min, discard the supernatant, resuspend the cells in Serum / LIF medium, and inoculate the cells into the coated well plate. Replace with fresh Serum / LIF medium the next day for culture.

[0180] (1.2) Culture: After completing step (1.1), the mouse embryonic stem cells (mESCs) are revived and cultured with fresh PGEX medium every day. The cell status is observed until the embryonic stem cells grow well and show obvious clonal status.

[0181] (1.3) Passaging: Before passaging, coat the cell culture plate with gelatin at 37°C for 30 minutes. Then, wash the mouse embryonic stem cells (mESCs) cultured in step (1.2) with PBS once, add trypsin, place in a 37°C incubator for digestion for 3 minutes, and add an equal volume of Serum / LIF medium to terminate the digestion. After centrifugation at 500rcf / 5min, discard the supernatant, resuspend the cells with Serum / LIF medium, inoculate the cells into the coated well plate, and replace with fresh Serum / LIF medium for culture the next day.

[0182] (1.4) Cryopreservation: After discarding the culture medium, wash the subcultured mouse embryonic stem cells (mESCs) with PBS, then add 1 ml of trypsin. After the cells become round and fall off, add 1 ml of complete culture medium to terminate digestion. Centrifuge at 1000 rpm for 4 minutes and remove the supernatant. Add 1 ml of freezing solution with a final concentration of 10% DMSO, mix gently, and mark. Place the cryopreservation tube in a program cooling box, put it in a -80°C refrigerator, and transfer it to liquid nitrogen for storage after at least 2 hours.

[0183] By performing expression analysis on published public RNA-seq database data, a two-cell (totipotent cell) transcript gene set containing 739 genes was generated ( Figure 3 ) and a four-cell (pluripotency) transcriptome gene set containing 688 genes ( Figure 4 ).

[0184] The dye was incorporated and washed according to the instructions of the intracellular lysosomal activity detection kit (Abcam, cat. no. ab234622), and the average fluorescence intensity of the cells was analyzed by flow cytometry. Finally, the data and images were analyzed and generated by FlowJo (V10) software. mESCs without any staining were used as negative controls; the lysosomal activity intensity was detected by the 488nm channel in the flow cytometer. The experimental results are shown in Figure 2. Figure 5 As shown, from Figure 5 It can be seen that the lysosomal activity of mouse stem cells is weakened when they are in the pluripotent state.

[0185] Example 2: In vitro induced pluripotent cell culture using lysosomal inhibitors.

[0186] Use 2ug ml -1 The mouse embryonic stem cells in the control group (vehicle: DMSO) and the lysosomal inhibitor treatment group were treated with doxycycline, where the concentrations of lysosomal inhibitors Vacuolin-1, Baf A and HCQ were 1 μM, 10 nM and 20 μM, respectively. After 16 hours of doxycycline treatment, the mESCs in the control group without lysosomal inhibitors were used as negative controls, and the 568 nm channel of the flow cytometer was used to sort tdTomato-positive 2CLCs and the proportion was calculated. The results are shown in Figure 6 As shown in the figure, it can be seen that the three lysosomal inhibitors can significantly promote mouse pluripotent stem cells to enter the omnipotent state.

[0187] Example 3, maintenance of the omnipotent state.

[0188] The 2CLCs cells sorted in Example 2 were resuspended in 1 mL of Serum / LIF medium and inoculated into the coated well plate. After culturing for 24 hours, the proportion of 2CLCs that maintained tdTomato positive was counted using the 568 nm channel of the flow cytometer and recorded as the proportion of 2CLCs that maintained pluripotency. Figure 7 As shown in the figure, it can be seen that the three lysosomal inhibitors can significantly promote stem cells to maintain a pluripotent state for a longer period of time.

[0189] Example 4, transcriptome sequencing analysis.

[0190] Taking Vacuolin-1 as an example, the effect of lysosomal inhibitors on the expression of two cell transcripts was analyzed. The mESCs in the vehicle and Vacuolin-1 treatment groups were collected and sent to a sequencing company for RNA-seq (performed according to the RNA-seq described in step (5) of the Summary of the Invention section), and a volcano plot was obtained ( Figure 7 ) and GSEA results ( Figure 8) showed that the expression of two-cell transcripts represented by MT2_Mm, Zscan4c, MERVL, Zscan4d and Zfp352 was significantly upregulated after Vacuolin-1 treatment.

[0191] In order to further clarify that the three lysosomal inhibitors have the effect of regulating the expression of two cell transcripts, RNA was extracted from the control group and the three lysosomal inhibitors Vacuolin-1, Baf A and HCQ treatment groups for reverse transcription and real-time quantitative fluorescence PCR experiments. The effects of the three lysosomal inhibitors on the expression of two cell transcripts represented by Zscan4 and MERVL were detected. The results showed that the three inhibitors could promote the expression of two cell transcripts ( Fig.10 ).

[0192] In order to further clarify that the situation that lysosomal inhibitors optimize the culture of totipotent stem cells in vitro is generally applicable, in this example, mouse embryonic stem cells overexpressing Obox3 and Obox5 were treated with the lysosomal inhibitor Vacuolin-1. The results showed that the lysosomal inhibitor can also further promote the expression of the two cell transcripts, that is, it can optimize the culture of totipotent stem cells obtained by different in vitro inductions ( Fig.11 ).

[0193] Lysosomal inhibitors also promote the culture of pluripotent stem cells induced by a single compound, PlaB. Vacuolin-1 and Baf A were treated on the basis of PlaB. Both inhibitors can further promote the expression of two representative cell transcripts, further indicating that lysosomal inhibitors can optimize the culture of pluripotent stem cells obtained by different in vitro inductions ( Fig.12 and Fig.13 ).

[0194] Example 5, validation of the target of lysosomal inhibitors under optimized conditions.

[0195] (1) To further clarify the target of lysosomal inhibitors in regulating the transition to the pluripotent state, the genes that affect the transition to the pluripotent state were scored using the known CRISPR / Cas9 screening data. The results showed that Lamtor1, the anchor of the Ragulator complex, and Flcn, the activating protein of the RagC GTPase, were ranked relatively high. The top 5% of lysosomal-related proteins Flcn and Lamtor1 were knocked out by lentiviral integration, and two sgRNAs were designed for each knockout protein (sequences are shown in Table 5). The results of immunoblotting showed that the knockout efficiency of each sgRNA was very high ( Fig.14 and Fig.15 ).

[0196] Table 5

[0197]

[0198] Note: For each gene, two sequences should be designed and the results should be verified repeatedly. For example, Flcn-1 and Flcn-2 are two sequences of Flcn.

[0199] (2) Use 2ug ml -1 The mouse embryonic stem cells of the control group (sgSV40) and the experimental group (sgFlcn-1, sgFlcn-2, sgLamtor1-1, sgLamtor1-2) were treated with doxycycline to induce the cells to enter the pluripotent state. After 16 hours of doxycycline treatment, mESCs without doxycycline were used as negative controls, and tdTomato-positive 2CLCs were sorted using the 568nm channel of the flow cytometer and the proportion was counted. The flow sorting results showed that knocking out the lysosome-related proteins Flcn and Lamtor1 can promote more stem cells to enter the pluripotent state ( Fig.16 ).

[0200] (3) The 2CLCs obtained by the above sorting were resuspended in 1 mL of Serum / LIF medium and inoculated into the coated well plate. After further culture for 24 hours, the proportion of 2CLCs maintaining tdTomato positive was counted using the 568 nm channel of the flow cytometer as the proportion maintaining the pluripotent state. The results showed that knocking out the lysosomal-related proteins Flcn and Lamtor1 could also significantly promote stem cells to maintain the pluripotent state more and longer ( Fig.17 ).

[0201] (4) The regulator protein Lamtor is the scaffold that fixes Rag GTP on the lysosomal membrane, and RagGTPase is a heterodimeric protein, usually composed of a RagA or RagB and a RagC or Rag D subunit. The RagC / D subunit of RagGTPase can be activated by Flcn, which activates RagC / D to recruit and regulate the phosphorylation of its downstream targets (including transcription factors such as Tfe3, Tfeb and Mitf), causing them to remain in the cytoplasm and regulate cell metabolism, translation and transcription.

[0202] To further clarify the downstream targets of lysosome regulation of the transition to the pluripotent state, we used a known public RNA-seq database (the same as in Example 1) to analyze the expression levels of RagC, RagD, Tfe3, Tfeb, Tfec, and Mitf in the process of mouse embryos entering and exiting the pluripotent state, and found that RagC and transcription factors Tfe3 and Tfeb were expressed at higher levels during the pluripotent transition process ( Fig.18 ).

[0203] (5) Overexpression of the constitutively active form of RagC (RagCS75N) significantly reversed the higher proportion of 2CLCs and the increased expression of representative 2-cell transcripts induced by Vacuolin-1 treatment, indicating that changes in RagC activity mediate the transition of lysosomal regulation totipotency ( Fig.19 ). Moreover, RT-qPCR assay showed the relative expression levels of two representative intracellular transcripts in the Vacuolin-1 treated group after overexpression of the constitutively active form of RagC.

[0204] (6) To further clarify the downstream targets of active RagC recruitment and regulation, we used immunofluorescence experiments to detect the localization of Tfe3, Tfeb, and Mitf after treatment with the lysosomal inhibitor Vacuolin-1. The experiment found that the lysosomal inhibitor Vacuolin-1 can significantly increase the nuclear translocation ratio of Tfe3 and Tfeb ( Fig. 20 ).

[0205] (7) Knockdown of Tfe3 and Tfeb (marked as DKD, where Tfe3 was knocked down with shTfe31 / 2, and Tfeb was knocked down with sgTfeb1 / 2), and the shRNA sequences shown in Table 6 were obtained. The knockdown experiment showed that the simultaneous knockdown of Tfe3 and Tfeb not only reduced the proportion of 2CLCs in the Vacuolin-1 treatment group, but also weakened the expression of representative two-cell transcripts. This indicates that the simultaneous knockdown of Tfe3 and Tfeb will reduce the promoting effect of Vacuolin-1 on the culture of pluripotent stem cells, and Tfe3 and Tfeb are downstream targets of lysosomal inhibitors in regulating the transition of pluripotent state ( Fig.21 ). Moreover, RT-qPCR detection showed the relative expression levels of two representative intracellular transcripts in the Vacuolin-1 treatment group after simultaneous knockdown of Tfe3 and Tfeb.

[0206] Table 6

[0207]

[0208] (8) Studies in the prior art have found that MERVL and MT2_Mm are retrotransposons specifically expressed in totipotent embryos, and are also alternative promoters that activate the expression of adjacent two-cell transcripts in 2CLCs and embryos, and MERVL activation has been shown to promote the transformation of stem cells to totipotency. In this example, nuclear localization forms of Tfeb and Tfe3 were overexpressed in an in vitro totipotent stem cell culture model cell line, and cell samples were collected after doxycycline treatment for 16 hours for RT-qRCR detection, and it was found that the transcription factors Tfeb and Tfe3 that entered the nucleus would activate the expression of transposons MERVL and MT2_Mm ( Fig. 22 ),

[0209] (9) To further determine whether Tfeb and Tfe3 have a direct or indirect effect on the activation of transposons MERVL and MT2_Mm, we used CUT&Tag, a technology for studying protein-DNA interactions, to demonstrate that Tfe3 and Tfeb directly bind to and activate the transcription of MERVL / MT2_Mm during the pluripotency transition, thereby providing a new mechanism by which Tfeb / 3 affects pluripotency. Here, sgFlcn is equivalent to lysosomal inhibitor treatment, and doxycycline treatment or not represents the entry and exit of stem cells into the pluripotent state. During the process of optimizing the in vitro pluripotent stem cell culture by knocking out Flcn, the DNA binding signal intensity of Tfe3 and Tfeb on MERVL and MT2_Mm was significantly increased ( Fig.23 ).

[0210] Although the preferred embodiments of the present invention have been described in the specification, once the basic creative concept is known to those skilled in the art, they may make additional changes and modifications to these embodiments or combinations of embodiments without creative work. Therefore, the protection scope of the present invention should be understood as the scope that the claims are intended to explain and cover, and should not be limited to the parts described in detail in the specification and embodiments of the present invention, and the contents described in the present invention, including the preferred embodiments, include all changes and modifications that fall within the scope of the present invention.

Claims

1. Application of lysosomal inhibitors in inducing pluripotent stem cells in vitro.

2. Application of lysosomal inhibitors in the culture of totipotent stem cells.

3. Application of lysosomal inhibitors in promoting pluripotent stem cells to enter the totipotent state.

4. Application of lysosomal inhibitors in maintaining the pluripotent state of pluripotent stem cells.

5. The use according to any one of claims 1 to 4, characterized in that: The lysosomal inhibitor is Vacuolin-1, and its chemical structure is shown in Formula I:

6. The use according to any one of claims 1 to 4, characterized in that: The lysosomal inhibitor is Bafilomycin A1, and its chemical structure is shown in Formula II:

7. The use according to any one of claims 1 to 4, characterized in that: The lysosomal inhibitor is Hydroxychloroquine, and its chemical structure is shown in Formula III:

8. A method for inducing pluripotent embryonic stem cells using a lysosomal inhibitor, characterized in that: The specific steps include: (1) Embryonic stem cell recovery, culture, passaging and cryopreservation; (2) Inducing DUX embryonic stem cells into a two-cell-like state using doxycycline; (3) Lysosomal inhibitors induce cells to enter and maintain a pluripotent state.

9. The method of inducing totipotent embryonic stem cells with a lysosomal inhibitor according to claim 8, characterized in that: Also included are analysis of totipotent embryonic stem cells, RNA-seq, and data analysis.

10. The method of inducing totipotent embryonic stem cells with lysosomal inhibitors according to claim 8, characterized in that: It also includes RNA extraction and reverse transcription reaction of totipotent embryonic stem cells, quantitative PCR detection, obtaining knockout cell lines and immunoblotting experiments.

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