Application of lysosomal inhibitors in inducing and maintaining stem cell pluripotency and induction methods

Abstract

To address the shortcomings of existing in vitro pluripotent stem cell culture technologies, this invention provides a method for using a lysosome inhibitor to induce and culture pluripotent stem cells in vitro, as well as to promote the initiation and maintenance of pluripotent stem cells in a pluripotent state. By using three known lysosome inhibitors, this invention promotes the maintenance of pluripotent stem cells in a pluripotent state in vitro, increasing their number, speed, and duration, thus providing greater potential for clinical pluripotent stem cell applications.

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 pluripotency of stem cells by using lysosome inhibitors. Background Art

[0002] Totipotency is the most plastic stem cell with differentiation potential 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 eventually form a pluripotent inner cell mass and

[0003] In the field of totipotency research, the scarcity of totipotent embryos has hindered the systematic conduct of numerous basic and clinical studies. Recently, a study has identified an in vitro cell model of totipotency: 2-cell-like cells (2CLCs). These cells, a rare population present in mouse embryonic stem cells, closely resemble totipotent, two-cell embryos. 2CLCs highly express totipotency-associated transcripts and possess totipotency-like differentiation capabilities. Mouse embryonic stem cells spontaneously enter and exit the 2-cell-like state. My previous research has shown that entry into the 2-cell-like state involves a massive activation of totipotency-specific transcripts, while exit from the 2-cell-like state involves the repression of totipotency-specific transcripts and the activation of pluripotency-specific transcripts. Therefore, at the transcriptomic level, the entry and exit processes of 2-cell-like cells mimic those of embryonic totipotency, making them 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 cell number, 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 treated with multiple compounds and the splicing inhibitor Pladienolide B (PlaB) treated with a single compound. In addition to compound treatment, the OBOX family, a functional homologous family in mice, is also highly 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 asFigure 1 2CLCs were enriched using a doxycycline-treated cell line. Doxycycline treatment induced the expression of the transcription factor Dux, which in turn drove the expression of the 5'LTR (2C::tdTomato) of MERVL, promoting the transformation of cells into 2CLCs. These 2CLCs could then spontaneously revert to a pluripotent state.

[0005] To date, 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 biomolecular mechanism is still unclear, and the induction effect is slow and weak, leaving room for optimization. Second, the use of transcription factors, such as Dux, totipotent stem cells induced are unstable and cannot be cultured for a long time. Therefore, there is a great need for optimization of existing totipotent stem cell induction and culture methods.

[0006] Furthermore, totipotent embryonic development undergoes multidimensional phenotypic changes, including transcriptome and epigenetic remodeling, which play a crucial role in regulating totipotency. Recent studies have shown that totipotent embryonic development is also regulated by metabolic signals. During the totipotent stage, embryonic development is in a low-energy mode with low metabolic demands, yet the regulatory role of metabolic signals in totipotency has long been overlooked. Therefore, we aim to optimize existing methods for 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 shortcomings of existing in vitro totipotent stem cell culture technology by using three known lysosomal inhibitors to promote in vitro induced totipotent stem cells to maintain a totipotent state more, faster and longer, thereby providing more operational space for clinical totipotent 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 and cell-permeable lysosomal exocytosis inhibitor. Vacuolin-1 blocks lysosomal Ca 2+Vacuolin-1 inhibits late autophagy by impairing lysosomal maturation and induced vacuole formation, increasing the percentage of enucleated cells.

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

[0014]

[0015] Preferably, 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.

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

[0017]

[0018] HCQ is a synthetic, oral antimalarial drug used in the research of malaria and autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. 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 totipotent state of pluripotent stem cells.

[0022] In a fifth aspect, the present invention provides a method for inducing totipotent 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] Remove cryopreserved mouse embryonic stem cells (mESCs) from liquid nitrogen, thaw in a water bath, centrifuge, discard the supernatant, resuspend the cells in Serum / LIF medium, and seed the cells onto the coated wells. Replace with fresh Serum / LIF medium the next day.

[0026] (1.2) Culture: After completing step (1.1), culture the mouse embryonic stem cells (mESCs) by replacing them with fresh PGEX medium daily and observing the cell status until the embryonic stem cells grow well and show obvious clonal status.

[0027] (1.3) Passaging: Before passaging, coat the cell culture plates with gelatin. Wash the mouse embryonic stem cells (mESCs) cultured in step (1.2) once with PBS, add trypsin, and digest in an incubator. Add an equal volume of Serum / LIF medium to terminate digestion. After centrifugation, discard the supernatant and resuspend the cells in Serum / LIF medium. Plate the cells onto the coated wells and replace with fresh Serum / LIF medium the next day.

[0028] (1.4) Cryopreservation: After discarding the culture medium, wash the passaged mouse embryonic stem cells (mESCs) once with PBS. Then, add trypsin. Once the cells have rounded and detached, add complete culture medium to terminate digestion. Centrifuge and remove the supernatant. Add freezing solution containing DMSO, mix gently, and label the tubes. Place the cryovials in a programmed cooling box and place in a -80°C freezer. After at least 2 hours, transfer to liquid nitrogen for storage.

[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 had 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 for pluripotency maintenance experiments.

[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 cells cultured in 24-well plates was discarded, and DPBS was added to slowly wash twice to remove dead cells; each well was digested with trypsin, and stop solution was added after digestion was completed; after centrifugation, the supernatant was discarded, and the cells were resuspended in DPBS, filtered, and analyzed using a BD LSRFortessa flow cytometer.

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

[0036] Samples from the control and experimental groups were collected and sent to a sequencing company for cDNA library construction using Smart-seq2 technology. After high-throughput sequencing using the Illumina platform, the raw sequencing data were aligned to the mm10 genome using STAR (v2.5.2b) software. Repeat fragment quantification was performed using RepEnrich (v1.2) and seqtk (v1.3). Differentially expressed genes between groups were calculated using the DESeq2 software package (v1.32.0). Differentially expressed genes meeting the criteria of |log2FoldChange|>1 and a P value <0.05 were considered statistically significant. Volcano plots were generated using ggplot2 (v3.3.5), with the x-axis representing the log2-fold change in expression and the y-axis representing the log-transformed p-value. Heatmaps were created using the pheatmap (v1.0.12) software package. 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 in an RNase-free centrifuge tube, mince it, add Trizol, and break it up using a homogenizer. Add Trizol again and vortex to mix thoroughly. Once 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 it 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 the tissue reverse transcription system.

[0045] (6.8) After the system is prepared, mix and centrifuge, then 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 (RT-qPCR)

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

[0049] (8) Obtaining knockout cell lines

[0050] The lysosomal inhibitor target proteins Flcn and Lamtor1 were knocked out in mouse embryonic stem cells using the CRISPR / Cas9 system. Two specific sgRNAs were designed at the gene CDS using the LentiCrisper-v2-puromycin two-in-one plasmid. The plasmids were integrated into the mouse embryonic stem cell genome using lentivirus. The resulting monoclonal cell lines were analyzed for genomic phenotype by genome extraction and Sanger sequencing, 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 an SDS-PAGE gel of appropriate concentration based on protein size. Flatten the gel surface with n-butanol and let it stand at room temperature for at least half an hour. Rinse off the n-butanol, prepare the top layer of concentrated gel, insert the sample comb, and let it stand at room temperature for at least half an hour.

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

[0056] (9.5) Use a semi-dry transfer apparatus to transfer the protein sample from the gel to a 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 incubate the PVDF membrane in the primary antibody buffer at 4°C overnight.

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

[0060] (9.9) Dilute the corresponding secondary antibody in TBST buffer at a dilution of 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 onto the PVDF membrane after the ECL chemically enhanced luminescence reaction. After a brief exposure, develop the film. Mark the development information and scan the film for storage.

[0063] (10) Immunofluorescence staining

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

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

[0066] CUT&Tag follows a previously reported protocol to construct a library. The specific steps 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 to primary antibodies

[0070] (11.4) Secondary Antibody Binding

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

[0072] (11.6) DNA purification

[0073] (11.7) NG-Sequencing Library Construction

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

[0075] (11.9) The reads 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 a 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 was performed using GraphPad (v8) or R (v4.1). Statistical significance was determined by Student's unpaired t-test or the nonparametric Mann-Whitney U test. All sequencing experiments were performed in duplicate. All other experiments were performed in at least three replicates and independently repeated at least twice 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 and culture scheme, making the induction of pluripotent cells faster and more efficient, and the pluripotent cell culture more stable.

[0085] (2) The present invention optimizes 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 pluripotent stem cells to enter the pluripotent state faster 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 signaling regulates cellular nutrient metabolism and stem cell differentiation.

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

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

[0091] Figure 5 1 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 of converting 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 maintain 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] Figure 9 This is a graph showing the GSEA results of the transcriptome sequencing analysis in Example 4.

[0096] Figure 10 Schematic diagram of the relative expression levels of two representative intracellular transcripts after lysosomal inhibitor treatment as shown in Example 4 by RT-qPCR assay.

[0097] Figure 111 is a schematic diagram showing the relative expression levels of two representative cell transcripts in stem cells overexpressing Obox3 and Obox5 after treatment with lysosomal inhibitors as shown in Example 4 by RT-qPCR.

[0098] Figure 12 1 is a schematic diagram showing 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] Figure 13 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 assay.

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

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

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

[0103] Figure 17 This is a flow cytometric statistical graph showing the proportion of 2CLCs maintaining a pluripotent state after knockout of the lysosome-related proteins Flcn and Lamtor1 in Example 5.

[0104] Figure 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] Figure 19 This is a flow cytometry statistical result of the 2CLCs ratio in the Vacuolin-1-treated group after overexpression of active RagC in Example 5.

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

[0107] Figure 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] Figure 22Schematic 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-q PCR in Example 5.

[0109] Figure 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] To help those skilled in the art better understand the technical solutions of the present invention, we first explain some of the technical terms of the present invention. In the context of the present invention, these explanations are primarily intended to help those skilled in the art understand and implement the technical solutions of the present invention, and are not intended to limit the present invention. Furthermore, other disclosures outside of the present invention may provide different technical interpretations of these technical terms. To avoid technical ambiguity, these explanations should be considered in conjunction with the present invention when understanding the present invention.

[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. The Ragulator-Rag complex accumulates on the surface of lysosomes, which can sense the concentration of amino acids within the lysosome and regulate various nutrient and energy metabolism pathways, including the mammalian target of rapamycin complex 1 (mTORC1) and autophagy. Lysosomal signaling not only plays an important role in maintaining cellular nutrient and energy balance, but also affects stem cell development and differentiation. In this process, Lamtor1, a membrane protein specifically localized on the surface of late endosomes / lysosomes, participates in lysosomal trafficking and maturation, serving as the anchor of the Ragulator complex for binding to lysosomes. 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 inhibiting the transcription factor's nuclear import ( Figure 2 ).

[0113] The function of lysosomes in totipotency has not yet been identified. Three common lysosomal inhibitors are Vacuolin-1, Bafilomycin A1 (Baf A), and Hydroxychloroquine (HCQ). The present invention discovers that inhibiting lysosomes can significantly improve the induction efficiency of totipotent cells and the culture stability of totipotent cells. It also provides the use of these three known commercial lysosomal inhibitors in improving in vitro totipotent cell culture methods.

[0114] Vacuolin-1 is a potent cell-permeable lysosomal exocytosis inhibitor. 2+ Vacuolin-1 inhibits late autophagy by impairing lysosomal maturation and inhibits lysosomal exocytosis. 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 research of malaria and autoimmune diseases (such as systemic lupus erythematosus and rheumatoid arthritis). In addition, HCQ is also a potent autophagic flux inhibitor. Its chemical structure is shown in Formula III:

[0119]

[0120] In the embodiments of the present invention, it is necessary to carry out multiple groups or multiple experiments. In order to enable those skilled in the art to better implement the technical scheme of the present invention, some methodologies of the present invention are first introduced. It should be noted that methodology is not a limitation of the present invention. The specific numerical values ​​in the methodology only represent one experimental value (or numerical ratio) and cannot be regarded as completely absolute values. Moreover, the methodology itself does not obtain results. The methodology needs to be combined with the embodiments to obtain 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 into a 1.5 mL RNase-free centrifuge tube, mince it, add 500 mL Trizol, break it up with a homogenizer, and then add another 500 mL Trizol and shake 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 at 4°C for 10 minutes.

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

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

[0126] (5) Centrifuge at 12,000 rpm for 20 minutes 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 minutes at 4°C, carefully aspirate the supernatant, air-dry at room temperature for 1 minute, and add an appropriate volume of RNase-free water to fully dissolve.

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

[0129] Table 1

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

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

[0132] (9) Add 4 μl of 5×qRT SuperMix II to the system in step (7), gently pipette to mix, and place in a PCR instrument for reaction 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, and the relative quantification method (2 -ΔΔCt ) were used to detect the relative expression levels of mouse embryonic stem cell pluripotency marker genes (Zscan4 and MERVL). The gene sequences are shown in Table 4.

[0142] Table 4

[0143]

[0144] Derivation of knockout cell lines

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

[0146] Immunoblotting experiments

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

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

[0149] (3) Prepare an SDS-PAGE gel of appropriate concentration (10% to 15%) according to the protein size, 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% top layer of 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 is determined according to the position of the target band.

[0151] (5) Use a semi-dry transfer apparatus to transfer the protein sample from the gel to a 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 minutes each time.

[0155] (9) Dilute the corresponding secondary antibody in TBST buffer at a dilution of 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 minutes each time.

[0157] (11) Press an appropriate amount of X-ray film onto the PVDF membrane after the ECL chemically enhanced luminescence reaction, briefly expose it, and then develop it. Mark the development information and scan the photo to save it.

[0158] Immunofluorescence staining

[0159] Mouse embryonic stem cells were cultured overnight on gelatin-coated slides before staining. After washing with PBS, the slides were fixed with 4% paraformaldehyde (PFA) for 15 minutes and permeabilized with 0.5% Triton X-100 for 15 minutes. The samples were then incubated overnight at 4°C in primary antibody buffer (the primary antibody concentration used in this study was 1:200). The slides were then washed three times with PBS and incubated in a secondary antibody conjugated to the relevant fluorescent dye DAPI for 1 hour. The slides were then washed three times with PBS. All stained samples, after mounting, were observed and imaged using a confocal laser scanning microscope (Olympus FV3000).

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

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

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

[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 built, 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 into 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 below by way of embodiments, with reference to the accompanying drawings. It is apparent that the embodiments described are merely a portion of the embodiments of the present invention, and not all of them. Furthermore, any modifications, equivalent substitutions, improvements, etc. made by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort shall be included within the scope of protection 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] Remove cryopreserved mouse embryonic stem cells (mESCs) from 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 seed the cells onto the coated wells. Replace with fresh Serum / LIF medium the next day.

[0180] (1.2) Culture: After completing step (1.1), culture the mouse embryonic stem cells (mESCs) by replacing them with fresh PGEX medium daily and observing the cell status 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. Wash the mouse embryonic stem cells (mESCs) cultured in step (1.2) once with PBS, add trypsin, and digest in a 37°C incubator for 3 minutes. Add an equal volume of Serum / LIF medium to terminate digestion. Centrifuge at 500 rcf / 5 min and discard the supernatant. Resuspend the cells in Serum / LIF medium and seed the coated wells. Replace with fresh Serum / LIF medium the next day.

[0182] (1.4) Cryopreservation: After discarding the culture medium, wash the passaged mouse embryonic stem cells (mESCs) once with PBS. Then, add 1 ml of trypsin. Once the cells become round and detach, 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 label. Place the cryovial in a programmed cooling box in a -80°C freezer. After at least 2 hours, transfer to liquid nitrogen for storage.

[0183] By performing expression analysis on published public RNA-seq database data, a two-cell (totipotent cell) transcriptome 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). The mean fluorescence intensity of the cells was analyzed by flow cytometry. Finally, the data and images were analyzed and generated using FlowJo (V10) software. Unstained mESCs served as a negative control. The lysosomal activity intensity was detected by the 488 nm 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 lysosomal activity is weakened when mouse stem cells are in a pluripotent state.

[0185] Example 2: Lysosomal inhibitors inducible pluripotent cell culture in vitro.

[0186] Use 2ug ml -1 Mouse embryonic stem cells were treated with doxycycline in the control group (vehicle: DMSO) and the lysosomal inhibitor treatment group, with the concentrations of lysosomal inhibitors Vacuolin-1, Baf A, and HCQ at 1 μM, 10 nM, and 20 μM, respectively. After 16 hours of doxycycline treatment, mESCs in the control group without lysosomal inhibitors were used as negative controls. tdTomato-positive 2CLCs were sorted using the 568 nm channel of a flow cytometer and their proportions were calculated. 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 totipotent state.

[0187] Example 3, maintenance of the pluripotent 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 24 hours of culture, the proportion of 2CLCs that maintained tdTomato positive was counted using the 568 nm channel of a 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, we analyzed the effect of lysosomal inhibitors on the expression of two cell transcripts. mESCs from the vehicle and Vacuolin-1 treatment groups were collected and sent to a sequencing company for RNA-seq (according to the RNA-seq described in step (5) of the invention summary), and the volcano plot ( 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] To further clarify that the three lysosomal inhibitors all have the effect of regulating the expression of dicellular transcripts, RNA was extracted from the control group and the three lysosomal inhibitors Vacuolin-1, Baf A and HCQ treated groups for reverse transcription and real-time quantitative fluorescence PCR experiments. The effects of the three lysosomal inhibitors on the expression of dicellular transcripts represented by Zscan4 and MERVL were detected. The results showed that the three inhibitors could promote the expression of dicellular transcripts ( Figure 10 ).

[0192] To further clarify the universal applicability of lysosomal inhibitors in optimizing the culture of totipotent stem cells in vitro, 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 could also further promote the expression of the two cell transcripts, that is, it could optimize the culture of totipotent stem cells obtained by different in vitro inductions ( Figure 11 ).

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

[0194] Example 5, Target Verification of Lysosomal Inhibitors under Optimized Conditions.

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

[0196] Table 5

[0197]

[0198] Note: For each gene, design two sequences and verify the results 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. The 568nm channel of the flow cytometer was used to sort tdTomato-positive 2CLCs and the proportion was calculated. The flow sorting results showed that knocking out the lysosomal-related proteins Flcn and Lamtor1 could promote more stem cells to enter the pluripotent state ( Figure 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 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 the pluripotent state. The results showed that knocking out the lysosomal-related proteins Flcn and Lamtor1 could also significantly promote the stem cells to maintain the pluripotent state for a longer period of time ( Figure 17 ).

[0201] (4) The regulatory 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. We found that RagC and the transcription factors Tfe3 and Tfeb were expressed at higher levels during the pluripotent transition ( Figure 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 ( Figure 19 Furthermore, RT-qPCR assays 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 ( Figure 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. Knockdown experiments showed that simultaneous knockdown of Tfe3 and Tfeb not only reduced the proportion of 2CLCs in the Vacuolin-1 treated group, but also weakened the expression of representative two-cell transcripts. This indicates that simultaneous knockdown of Tfe3 and Tfeb will reduce the promoting effect of Vacuolin-1 on pluripotent stem cell culture, and Tfe3 and Tfeb are downstream targets of lysosomal inhibitors that regulate the transition to pluripotent state ( Figure 21 ). In addition, RT-qPCR assay showed the relative expression levels of two representative cell 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 2CLCs and adjacent two-cell transcripts in embryos. 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. It was found that the nuclear transcription factors Tfeb and Tfe3 activated the expression of transposons MERVL and MT2_Mm ( Figure 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 technique 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, thus providing a new mechanism by which Tfeb / 3 affect 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 ( Figure 23 ).

[0210] Although the specification has described the preferred embodiments of the present invention, those skilled in the art may make additional changes and modifications to these embodiments or combinations of embodiments without inventive effort once they understand the basic inventive concepts. Therefore, the scope of protection of the present invention should be understood as the scope intended to be interpreted and covered by the claims, and should not be limited to the details described in the specification and embodiments of the present invention. Moreover, 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; The lysosomal inhibitor is selected from at least one of the following three: Vacuolin-1, whose chemical structure is shown in Formula I: Formula I; Bafilomycin A1, whose chemical structure is shown in Formula II: Formula II; Hydroxychloroquine, whose chemical structure is shown in Formula III: Formula III.

2. Application of lysosomal inhibitors in the culture of totipotent stem cells; The lysosomal inhibitor is selected from at least one of the following three: Vacuolin-1, whose chemical structure is shown in Formula I: Formula I; Bafilomycin A1, whose chemical structure is shown in Formula II: Formula II; Hydroxychloroquine, whose chemical structure is shown in Formula III: Formula III.

3. Application of lysosomal inhibitors in promoting pluripotent stem cells to enter the totipotent state; The lysosomal inhibitor is selected from at least one of the following three: Vacuolin-1, whose chemical structure is shown in Formula I: Formula I; Bafilomycin A1, whose chemical structure is shown in Formula II: Formula II; Hydroxychloroquine, whose chemical structure is shown in Formula III: Formula III.

4. Application of lysosomal inhibitors in maintaining the totipotent state of pluripotent stem cells; The lysosomal inhibitor is selected from at least one of the following three: Vacuolin-1, whose chemical structure is shown in Formula I: Formula I; Bafilomycin A1, whose chemical structure is shown in Formula II: Formula II; Hydroxychloroquine, whose chemical structure is shown in Formula III: Formula III.

5. A method for inducing totipotent embryonic stem cells using a lysosomal inhibitor, characterized in that: The specific steps include: (1) Embryonic stem cell recovery, culture, passage and cryopreservation; (2) DUX embryonic stem cells were induced to enter a two-cell state by doxycycline; (3) Lysosomal inhibitors induce cells to enter and maintain a pluripotent state; The lysosomal inhibitor is selected from at least one of the following three: Vacuolin-1, whose chemical structure is shown in Formula I: Formula I; Bafilomycin A1, whose chemical structure is shown in Formula II: Formula II; Hydroxychloroquine, whose chemical structure is shown in Formula III: Formula III.

6. The method of inducing totipotent embryonic stem cells using a lysosomal inhibitor according to claim 5, characterized in that: Also included are analysis of totipotent embryonic stem cells, RNA-seq, and data analysis.

7. The method of inducing totipotent embryonic stem cells using a lysosomal inhibitor according to claim 5, 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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