New technology for improving generation efficiency of in-vitro induced hematopoietic precursor cells

Abstract

The invention relates to a novel technology for improving the generation efficiency of in-vitro induced hematopoietic precursor cells. Specifically, the invention relates to a method for preparing hematopoietic precursor cells, which comprises the following steps: taking pluripotent stem cells as starting cells, and improving the crotonylation modification level of the pluripotent stem cells. On the other hand, the invention further relates to a kit for obtaining the hematopoietic precursor cells through induction, and the kit comprises a reagent for improving the crotonylation modification level of the pluripotent stem cells.

Description

Technical Field

[0001] This invention belongs to the field of stem cell differentiation technology, and more specifically, this invention relates to a method for preparing hematopoietic progenitor cells by inducing pluripotent stem cells. Background Technology

[0002] Various types of blood cells used to treat human diseases can be effectively obtained from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs), including erythrocytes, B cells, T cells, and hematopoietic progenitor cells. Currently, methods for obtaining hematopoietic progenitor cells (HPCs) from human pluripotent stem cells include the EB method, organoid method, and adherent method. However, the EB method and organoid method have the problems of long induction cycles and high costs, with induction cycles generally around 17 days. While the adherent method has the advantage of a shorter cycle (6-8 days), the proportion of hematopoietic progenitor cells obtained is relatively low. Overall, obtaining hematopoietic progenitor cells and their downstream immune cells from human pluripotent stem cells remains a significant challenge. Summary of the Invention

[0003] The technical problem this invention aims to solve is to shorten the preparation cycle of hematopoietic progenitor cells, improve cell induction efficiency, and lay a technical foundation for downstream clinical applications such as the treatment of blood diseases.

[0004] Specifically, in the study of obtaining hematopoietic progenitor cells by induction from pluripotent stem cells, this invention has for the first time discovered that increasing the crotonylation modification level of the initiating pluripotent stem cells can improve the induction efficiency of hematopoietic progenitor cells and increase the proportion of hematopoietic progenitor cells in the product.

[0005] In this invention, improving the crotonylation modification level of pluripotent stem cells includes the following methods: adding a crotonylation enhancer such as crotonic acid to the basal culture medium of pluripotent stem cells, or downregulating the level of crotonylation-modified eraser genes (such as HDAC and SIRT family), or overexpressing crotonylation modification-related genes (such as metabolic enzyme gene ACSS2, reader gene CBP, P300, etc.) in the pluripotent stem cells.

[0006] In a specific embodiment of the present invention, the crotonylation enhancer includes any substance known in the art for promoting crotonylation modification, such as crotonic acid, crotonate, etc.

[0007] In a specific embodiment of the present invention, the induction of pluripotent stem cells into hematopoietic progenitor cells is divided into two stages: (1) obtaining mesodermal cells from pluripotent stem cells; and (2) obtaining hematopoietic progenitor cells from mesodermal cells. In this specific embodiment, a crotonylation enhancer is added throughout step (1), or a crotonylation enhancer may be added throughout both steps (1) and (2).

[0008] In specific embodiments of the present invention, the crotonylated eraser gene (also known as the crotonylated deacylate gene) includes, but is not limited to, genes from the HDAC family and the SIRT family.

[0009] In this paper, histone deacetylase (HDAC) is an evolutionarily conserved enzyme that can be used to remove acetylation modifications on histones, thereby reducing the level of crotonylation modification. In this paper, the HDAC family genes refer to the genes encoding this enzyme, including genes such as HDAC1 and HDAC4.

[0010] In this paper, sirtuins are a class of highly conserved deacetylases from bacteria to humans, which can be used to reduce the level of crotonylation modification. In this paper, the SIRT family genes refer to the coding genes corresponding to sirtuins enzymes, including genes such as SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7.

[0011] In a specific embodiment of the present invention, the gene level of the crotonylation-modified eraser in pluripotent stem cells can be downregulated by RNAi methods well known in the art to inhibit target gene expression and knock down target genes, such as siRNA and shRNA, or by the CRISPR / Cas9 system.

[0012] In a specific embodiment of the present invention, crotonylation modification-related genes refer to genes involved in crotonylation modification, including genes encoding metabolic enzymes such as ACSS2 and ACADS (wherein, ACSS2 gene ID 55902, ACADS gene ID 35), reader genes such as AF9 and MOZ (wherein, AF9 gene ID 4300, MOZ gene ID 7994), and writer genes (i.e., crotonylation-modified transferase genes) such as CBP and P300 (wherein, CBP gene ID 1387, P300 gene ID 2033).

[0013] In a specific embodiment of the present invention, the "overexpression of crotonylation-related genes" can be achieved by any conventional method in the prior art for promoting the overexpression of target genes, such as transfecting a plasmid carrying the target gene into the target cell, infecting the target cell through a lentiviral system carrying the target gene, inserting an enhancer of the target gene into the target cell, or replacing the promoter of the target gene in the target cell with a strong promoter, etc.

[0014] In summary, through long-term research, this invention has for the first time discovered that the level of crotonylation modification in pluripotent stem cells is related to the efficiency of inducing hematopoietic progenitor cells. Increasing the level of crotonylation modification in the initiating pluripotent stem cells can improve the induction efficiency of hematopoietic progenitor cells and increase the proportion of hematopoietic progenitor cells in the product. The possible mechanism is that crotonylation modification can transcribe and regulate the expression of downstream genes and can also directly affect the activity of substrate genes.

[0015] In another aspect of the invention, a kit for inducing the acquisition of hematopoietic progenitor cells is also provided, comprising a reagent for increasing the crotonylation modification level of pluripotent stem cells and a basal culture medium.

[0016] In a specific embodiment of the present invention, the basal culture medium is a conventional basal culture medium used in the prior art to induce pluripotent stem cells to differentiate into hematopoietic progenitor cells, including but not limited to DMEM / F12 or Essential 6. TM Culture medium.

[0017] In a specific embodiment of the present invention, the culture medium and culture strategy used for the differentiation of pluripotent stem cells into hematopoietic progenitor cells are both existing conventional culture techniques, such as the adherence method. The only difference is that a crotonylation enhancer is added to the culture medium, or the level of crotonylation-modified eraser genes in pluripotent stem cells is reduced, or the level of crotonylation-modified related genes in pluripotent stem cells is increased.

[0018] The culture strategy typically includes the following stages: (1) the transformation stage of pluripotent stem cells into mesodermal cells, which usually takes 2 days. This stage ends when the TBXT marker is detected in the cells. In this stage, the basic culture medium used can be Essential6. TM (1) Culture medium containing cytokines such as BMP4 and FGF2; (2) The differentiation stage of mesodermal cells into hematopoietic progenitor cells, until the production of hematopoietic progenitor cells (i.e., CD34+, CD31+ and / or CD43+ cells) is detected; In this stage, the basal culture medium used can be Essential 6 TMThe culture medium contains cytokines commonly used in the prior art to induce hematopoietic progenitor cells, including BMP4, FGF2, Activin A, CHIR99021, LY294002, VEGF, SB431542, SCF, TPO, IL3, IL6, and FLT3L. BMP4, short for bone morphogenetic protein 4 (BMP-4), is a pleiotropic ligand protein belonging to the TGF-β family. It participates in the circulatory system and can activate receptors on vascular cells. FGF2, also known as basic fibroblast growth factor (bFGF), is an important member of the fibroblast growth factor (FGF) family. Activin A mainly regulates multiple functions, including inflammation, fibrosis, and tumorigenesis, through signaling via SMAD2 / 3 proteins. It is a member of the transforming growth factor-β (TGF-β) superfamily. CHIR99021 is an aminopyrimidine derivative and a potent inhibitor of GSK3, inhibiting GSK3β (IC50 = 6.7 nM) and GSK3α (IC50 = 10 nM), acting as a WNT activator. LY294002 is a non-specific inhibitor that, in addition to class IPI3Ks, can also inhibit other types of PI3Ks, mTOR, DNA-PK, and other protein kinases, such as CK2 and Pim-1. VEGF is a highly specific vascular endothelial growth factor that plays an important physiological role in angiogenesis, maintenance, and formation. It plays a crucial role in inducing endothelial cell survival, proliferation, migration, angiogenesis, and increasing vascular permeability. SB-431542 is a TGF-β receptor kinase inhibitor (TRKI). SB-431542 inhibits the activity of ALK4, ALK5, and ALK7. Cytokines SCF, TPO, Flt3L, IL-3, and IL-6 are all commonly used hematopoietic stimulating factors.

[0019] In a specific embodiment of the present invention, a crotonylation enhancer is added to the basal culture medium in the (1) stage of pluripotent stem cell to mesodermal cell transformation, preferably in both the (1) stage of pluripotent stem cell to mesodermal cell transformation and the (2) stage of mesodermal cell to hematopoietic progenitor cell differentiation.

[0020] Specifically, the present invention relates to the following technical solutions:

[0021] 1. A method for preparing hematopoietic progenitor cells, comprising using pluripotent stem cells as starting cells and increasing the crotonylation modification level of said pluripotent stem cells;

[0022] Preferably, increasing the crotonylation modification level of pluripotent stem cells includes adding a crotonylation enhancer to the basal culture medium of pluripotent stem cells, or reducing the level of crotonylation-deleting gene in the pluripotent stem cells, or increasing the level of crotonylation-related gene in the pluripotent stem cells.

[0023] 2. The method according to Project 1, wherein the pluripotent stem cells are mammalian pluripotent stem cells, preferably human pluripotent stem cells;

[0024] Optionally, the pluripotent stem cells include embryonic stem cells or induced pluripotent stem cells, preferably mammalian cells, more preferably mouse or human cells, and most preferably human cells; wherein the embryonic stem cells are commercially available human embryonic stem cells, preferably cells of any of the following NIH-numbered cell lines: H1, H9, or NL4.

[0025] 3. According to the method described in Project 1, wherein,

[0026] The crotonylation enhancer includes crotonic acid or a salt thereof;

[0027] Optionally, the crotonylation-modified eraser gene includes HDAC family or SIRT family genes;

[0028] Optionally, the crotonylation modification-related genes include the ACSS2 gene, ACADS gene, AF9 gene, MOZ gene, CBP gene, or P300 gene.

[0029] 4. The method according to any one of items 1-3, comprising the following steps:

[0030] (1) Differentiate pluripotent stem cells to obtain mesodermal cells, wherein the mesodermal cells are TBXT+ cells;

[0031] (2) Differentiate the mesodermal cells to obtain hematopoietic precursor cells, wherein the hematopoietic precursor cells are CD34+, CD31+ and / or CD43+ cells.

[0032] 5. The method according to Project 4, wherein the crotonylation enhancer is added to the culture medium in step (1); preferably, the crotonylation enhancer is added to the culture medium in both steps (1) and (2).

[0033] 6. The method according to Project 5, wherein the concentration of the crotonylation enhancer is 5-10 mM.

[0034] 7. The method according to any one of items 1-3, wherein,

[0035] The reduction of the level of the crotonylation eraser gene in the pluripotent stem cells includes knocking down or knocking out the crotonylation eraser gene by siRNA, shRNA or CRISPR / Cas9 system;

[0036] Increasing the level of crotonylation-related genes in the pluripotent stem cells includes introducing crotonylation-related genes into the pluripotent stem cells, for example, through lentiviral transfection.

[0037] 8. Hematopoietic precursor cells obtained by any one of items 1-7.

[0038] 9. A kit, preferably used for inducing the acquisition of hematopoietic progenitor cells, wherein the kit comprises a reagent for increasing the level of crotonylation modification of pluripotent stem cells.

[0039] 10. The kit according to Project 9, wherein the reagent for enhancing the crotonylation modification level of pluripotent stem cells is a crotonylation enhancer, and optionally, the kit further comprises a basal culture medium.

[0040] 11. The kit according to Project 9, wherein the reagent for increasing the level of crotonylation modification in pluripotent stem cells is a reagent for reducing the level of crotonylation-modifying eraser genes in the pluripotent stem cells (e.g., siRNA and shRNA for knocking out or knocking down / reducing crotonylation-modifying eraser genes), or a reagent for increasing the level of crotonylation-related genes in the pluripotent stem cells (e.g., a vector or lentivirus containing crotonylation-related genes).

[0041] the term

[0042] In this invention, the crotonic acid, also known as butenoic acid, has the chemical formula C4H6O2.

[0043]

[0044] Lysine crotonylation (Kcr) is a modification produced by histone crotonyltransferase (HCT) transferring a crotonyl group to an amino acid residue of a protein using crotonyl-CoA as a substrate. It is present in core histones and some non-histone proteins in various organisms, and differs from other types of post-translational modifications (PT). M Similar to crotonylation (Kcr), which is a reversible modification with important functions such as promoting gene expression and regulation.

[0045] In this invention, the ACSS2 gene (gene ID 55902) encodes acetyl-CoA synthase 2, which is a member of the short-chain acyl-CoA synthase family. It can mediate the conversion of crotonic acid to crotonyl-CoA, thereby promoting the level of crotonylation.

[0046] In this invention, the hematopoietic progenitor cells refer to cells with the ability to differentiate into blood cells. These hematopoietic progenitor cells are CD34+, CD31+, and / or CD43+ cells, where CD34, CD31, and / or CD43 are all markers of hematopoietic progenitor cells. That is, the hematopoietic progenitor cells are cells expressing any one or more of CD34, CD31, and / or CD43. For example, the hematopoietic progenitor cells are CD34+CD31+ double-positive cells, CD31+CD43+ double-positive cells, CD34+CD43+ double-positive cells, or CD34+CD31+CD43+ triple-positive cells.

[0047] In this invention, the pluripotent stem cell (PSC), also known as a pluripotent stem cell, refers to a stem cell with unlimited self-renewal and differentiation potential in vitro. It includes embryonic stem cells or induced pluripotent stem cells, preferably mammalian cells, more preferably mouse or human cells, and most preferably human cells. The embryonic stem cells are commercially available human embryonic stem cells, preferably cells of any of the following NIH-numbered cell lines: H1, H9, etc.

[0048] Beneficial effects

[0049] This invention is the first to discover that increasing the crotonylation modification level of pluripotent stem cells can promote the induction efficiency of hematopoietic progenitor cells. Specifically, this invention improves the efficiency of inducing hematopoietic progenitor cells from human pluripotent stem cells by adding crotonic acid during the culture process to regulate the crotonylation modification level of intracellular proteins.

[0050] ① Through flow cytometry experiments on hematopoietic progenitor cells, we observed that adding crotonylation enhancers such as crotonic acid or overexpressing crotonylation-related genes such as the ACSS2 gene to the culture medium can effectively increase the proportion of hematopoietic progenitor cells.

[0051] ② By comparing the expression profiles of cells at each stage of induction, we found that after adding crotonylation enhancers such as crotonic acid to the culture medium or overexpressing crotonylation-related genes such as ACSS2, the expression of cells related to hematopoietic endothelial formation (CD34 and CD31, etc.), hematopoietic differentiation and enrichment (CD43 and CD45, etc.), and myeloid differentiation (HBZ and DMTN, etc.) was increased.

[0052] Genes related to lymphoid differentiation (such as CD4 and CD74) were upregulated, indicating that increasing the crotonylation level of pluripotent stem cells promotes the expression of hematopoietic-related genes. Attached Figure Description

[0053] Figure 1 Immunofluorescence showed that crotonic acid treatment promoted the crotonylation level of human pluripotent stem cells. Here, "mock" refers to the control group and "CA" refers to the experimental group with added crotonic acid.

[0054] Figure 2 Immunofluorescence showed that crotonic acid treatment promoted the differentiation of human pluripotent stem cells into mesodermal cells. Here, "mock" refers to the control group and "CA" refers to the experimental group with added crotonic acid.

[0055] Figure 3 The flowchart of the hematopoietic precursor cell induction method is shown;

[0056] Figure 4 The flow cytometry results show that adding 5-10 mM of crotonic acid can obtain CD31+CD43+ cells with higher efficiency.

[0057] Figure 5 The heatmap shows that hematopoietic progenitor cells differentiated from pluripotent stem cells after the addition of crotonic acid can express higher levels of hematopoietic-related marker genes.

[0058] Figure 6 The flow cytometry results show that overexpression of ACSS2 yields more efficient CD34 and CD31 cells;

[0059] Figure 7 The results of a comprehensive scoring of multiple hematopoietic-related marker genes in RNA-seq data showed that hematopoietic progenitor cells differentiated after overexpression of ACSS2 expressed higher levels of hematopoietic-related marker genes. Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0061] Unless otherwise specified, the methods used in the following examples are conventional methods, and the reagents used are commercially available reagents.

[0062] Experimental materials and procedures:

[0063] Methods for culturing hPSCs and inducing hematopoietic progenitor cells:

[0064] The procedure for inducing hematopoietic progenitor cells can be found in [reference needed]. Figure 3 As shown, the details are as follows:

[0065] hPSCs (obtained from Professor Chen Jiekai's research group at the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, or commercially available, such as from Beijing Nuowei Biotechnology Co., Ltd., catalog number 904605) were cultured in commercial mTeSR1 (STEMCELL Technologies) medium to 80-90% confluence.

[0066] After digestion with Accutase (Sigma, SCR005) trypsin solution, the cells were resuspended in fresh mTeSR1 medium and transferred to Matrigel (Corning, 356231) coated wells at a density of 1.5-3.0 × 10^6 cells / well (12-well plate), and the apoptosis inhibitor Y-27632 was added to a final concentration of 10 μM.

[0067] Induction of pluripotent stem cells into mesodermal cells: After culturing hPSCs in plates for 12-24 hours, they were cultured in D0 medium for 24 hours, then replaced with D1 medium and cultured for 24 hours until the TBXT marker appeared in the cells, which marked the end of this stage.

[0068] Induction of mesodermal cells into hematopoietic progenitor cells: Mesodermal cells are cultured in D2 medium for 48 hours, and then cultured in D4 medium for 48-96 hours to obtain hematopoietic progenitor cells. This stage is completed when CD34+, CD31+ and / or CD43+ markers appear in the cells.

[0069] The specific proportions of the culture medium for each stage are as follows:

[0070] D0 medium contains Essential 6 TM Culture medium (Thermo Fisher Scientific, A1516401), 40 ng / ml BMP4, 30 ng / ml ACTIVIN A, 20 ng / ml bFGF, 6 μM CHIR99021 and 10 μM LY294002;

[0071] D1 medium contains Essential 6 TM Culture medium: 30 ng / ml BMP4, 1 μM A83-01 and 2 μM IWR-1-endo;

[0072] D2 medium contains Essential 6 TM Culture medium: 40 ng / ml VEGF, 50 ng / ml bFGF;

[0073] D4 medium contains Essential 6 TMMedium, 40ng / ml VEGF, 50ng / ml bFGF, 10μM SB431542, 10ng / ml SCF, 50ng / ml TPO, 10ng / ml IL3, 50ng / ml IL6 and 50ng / ml FLT3L.

[0074] For the crotonic acid supplementation regimen, 5-10 mM crotonic acid was added to the culture medium from day 0 to day 8, or crotonic acid may be added only during the differentiation of pluripotent stem cells into mesodermal cells.

[0075] Flow cytometry:

[0076] Cells induced and cultured were digested with Accutase trypsin solution, neutralized with DMEM-F12 (2% BSA), centrifuged at 800g at room temperature for 5 min, resuspended and washed twice with PBS, and then resuspended in 50 μl of 0.2% BSA in DPBS solution. The cell suspension was divided into two equal volumes. The first portion was stained with 0.25 μl of APC anti-human CD31 (4A Biotech FHA031-100) and 0.25 μl of FITC anti-human CD34 (BioLegend, 343504). The second portion was stained with FITC anti-human CD43 (BD ​​Pharmingen 555475). The staining conditions were incubation at 4℃ for 15 min. After washing with 1 ml of 0.2% BSA in DPBS solution, the cells were resuspended in 300 μl and prepared for flow cytometry analysis.

[0077] RNA-seq:

[0078] At four time points—before differentiation induction (D0), 24 h (D1), 72 h (D3), and 192 h (D8)—cells were digested with Accutase trypsin solution, neutralized with DMEM-F12 (2% BSA), centrifuged at 800 g at room temperature for 5 min, resuspended and washed twice with PBS, and then used for RNA extraction using the TRIZOL method to construct RNA-seq libraries.

[0079] Example 1: Adding crotonic acid to human pluripotent stem cells to prepare mesodermal cells

[0080] Following the procedures outlined in the "Cultivation of hPSCs and Induction of Hematopoietic Progenitor Cells" section, pluripotent stem cells (obtained from the research group of Professor Chen Jiekai at the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, or commercially available, such as from Beijing Nuowei Biotechnology Co., Ltd., catalog number 904605) were passaged and seeded at 200,000 cells per well into 12-well culture plates. Both the experimental and control groups used Essential 6... TM The cells were cultured in a medium with BMP4 for 48 hours. The difference was that the experimental group was treated with an additional 10 mM crotonic acid for 48 hours. The cells were then fixed, permeabilized, and subjected to TBXT immunofluorescence staining, followed by photographic analysis.

[0081] See results Figure 2 Where "mock" refers to the control group and "CA" refers to the experimental group with added crotonic acid, which is composed of... Figure 2 It can be seen that the TBXT marker was significantly increased in the experimental group with added crotonic acid, indicating that the addition of crotonic acid can significantly promote the induced differentiation of mesodermal cells.

[0082] In addition, to further confirm that the addition of crotonic acid can indeed promote the crotonylation level of pluripotent stem cells, the inventors performed the following operation: human pluripotent stem cells were seeded into 12-well plates at a rate of 150,000-300,000 cells per well. The next day, 1 ml of fresh culture medium containing 10 mM crotonic acid was added. After 24 hours, the cells were washed once with PBS, then fixed with 500 μl of 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton-X100 for 20 min, blocked with 0.25% BSA-PBS, and after 30 min, primary antibodies (TBXT, EMD Millipore, FCMAB302F) were added and incubated overnight at 4°C. The next day, the cells were washed twice with PBS and the results were photographed and analyzed under a fluorescence microscope.

[0083] See results Figure 1 ,Depend on Figure 1 It is known that the expression of Pan-Kcr was significantly increased after the addition of crotonic acid (i.e., the CA group). The crotonylated pan-antibody Pan-Kcr is a conventional detection reagent used in the prior art to indicate crotonylation levels. Therefore, Figure 1 The results showed that, after the addition of crotonic acid, the level of crotonylation modification in the cells of the experimental group was significantly increased compared with that of the control group.

[0084] Example 2: Adding 5-10 mM crotonic acid to human pluripotent stem cells to prepare hematopoietic progenitor cells.

[0085] In this embodiment, the induced differentiation of human pluripotent stem cells into hematopoietic progenitor cells was performed.

[0086] according to Figure 3The experimental procedure and the method described above for "culture and induction of hematopoietic progenitor cells by digestion" were used to culture human pluripotent stem cells and induce the generation of hematopoietic progenitor cells. Experimental and control groups were set up. In the experimental group, crotonic acid (e.g., 5 mM or 10 mM) was added to the culture medium (e.g., throughout the culture period, from D0 to D8, or from D0 to D2), while the control group received no crotonic acid. On the afternoon of the sixth day, the cells were digested with acutase for 15 minutes, washed with DPBS containing BSA, and then incubated with antibodies. After 15 minutes, the cells were washed twice with DPBS containing BSA before flow cytometry analysis.

[0087] The results of hematopoietic precursor cell generation were detected by flow cytometry, such as Figure 4 As shown, the addition of crotonic acid resulted in the acquisition of CD31+CD43+ double-positive cells with higher efficiency. The results indicated that after the addition of crotonic acid, the proportion of CD31+ and CD43+ double-positive cells reached more than 11%, which was about 7% higher than that of the control group, indicating that the addition of crotonic acid can effectively promote the acquisition of hematopoietic precursor cells.

[0088] Following the "RNA-seq" procedure described above, RNA extraction and high-throughput sequencing were performed on cells harvested on the day of plating (D0), the second day after plating (D2), the fourth day (D4), and the eighth day (D8). The results are as follows: Figure 5 As shown, the results indicate that hematopoietic progenitor cells differentiated from pluripotent stem cells after the addition of crotonic acid can express higher levels of hematopoietic-related marker genes. Among them, genes related to hematopoietic endothelial formation (CD34 and CD31, etc.), hematopoietic differentiation and enrichment (CD43 and CD45, etc.), myeloid differentiation (HBZ and DMTN, etc.) and lymphoid differentiation (CD4 and CD74, etc.) are all upregulated, indicating that increasing the level of crotonylation has a promoting effect on the expression of hematopoietic-related genes.

[0089] Example 3

[0090] Construction of ACSS2 overexpression cell lines:

[0091] Based on the plenti-ACSS2-puro lentiviral system, overexpression of ACSS2 in human pluripotent stem cells (hPSCs) was achieved, thereby increasing the endogenous crotonylation modification in the cells. The specific procedure is as follows: CAS9 in the lentiCas9-Blast(Plasmid#52962) plasmid was excised and replaced with the ACSS2 coding sequence (gene ID 55902) to construct the lenti-ACSS2 overexpression plasmid. Lentiviral packaging uses psPAX2 and pMD2.G series plasmids. Specifically, lenti-ACSS2 expression plasmid is co-transfected with psPAX2 (#12260) and pMD2.G (#12259) into HEK-293T cells (ATCC, CRL-3216). After collecting cell culture supernatant for 48-72 hours, human embryonic stem cells H1 (obtained from Chen Jiekai's research group at Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, or commercially available, such as from Beijing Nuowei Biotechnology Co., Ltd., catalog number 904605) are infected. After 24 hours, hPSC cells successfully transfected with the ACSS2 gene are selected by screening with 1 μg / ml puromycin. These cells are then expanded and cultured for the induction and differentiation of hematopoietic progenitor cells and subsequent experiments.

[0092] Results analysis:

[0093] As described above, human embryonic stem cells H1 (obtained from the research group of Professor Chen Jiekai at the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, or commercially available, such as from Beijing Nuowei Biotechnology Co., Ltd., catalog number 904605) were used as the experimental group by lentiviral overexpression of the ACSS2 gene, while H1 cells transfected with a blank vector were used as the control group. Then, the empty vector H1 cells (as the control group) and the ACSS2-overexpressing H1 cells (as the experimental group) were induced to differentiate in parallel until day 8 (wherein, the induction differentiation method was a conventional method of existing technology, for example, see Cell Discovery, 2021, PanGuangjin et al.). Cells were collected for flow cytometry analysis, and the results are as follows: Figure 6 As shown, overexpression of ACSS2 resulted in more efficient CD34+ and CD31+ cells. Overexpression of ACSS2 increased crotonylation levels in vivo, promoting a 15.74% increase in the proportion of hematopoietic progenitor cells compared to the control group.

[0094] H1 cells without vector and H1 cells overexpressing ACSS2 were induced to differentiate to day 6. Cells were then harvested for RNA extraction and high-throughput sequencing. Results are as follows: Figure 7 As shown, it demonstrates that hematopoietic progenitor cells differentiated after overexpression of ACSS2 can express higher levels of hematopoietic-related marker genes. Figure 7The results showed that when multiple hematopoietic progenitor cell marker genes were analyzed together and scored, the upregulated scores of these marker genes (i.e., the ssGSEA score on the ordinate) were higher after ACSS2 overexpression, indicating that ACSS2 overexpression can promote the occurrence of hematopoiesis.

[0095] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing hematopoietic progenitor cells, comprising using pluripotent stem cells as starting cells and increasing the crotonylation modification level of said pluripotent stem cells; Preferably, increasing the crotonylation modification level of pluripotent stem cells includes adding a crotonylation enhancer to the basal culture medium of pluripotent stem cells, or reducing the level of crotonylation-deleting gene in the pluripotent stem cells, or increasing the level of crotonylation-related gene in the pluripotent stem cells.

2. The method according to claim 1, wherein, The pluripotent stem cells are mammalian pluripotent stem cells, preferably human pluripotent stem cells; Optionally, the pluripotent stem cells include embryonic stem cells or induced pluripotent stem cells, preferably mammalian cells, more preferably mouse or human cells, and most preferably human cells; wherein the embryonic stem cells are commercially available human embryonic stem cells, preferably cells of any of the following NIH-numbered cell lines: H1, H9, or NL4.

3. The method according to claim 1, wherein, The crotonylation enhancer includes crotonic acid or a salt thereof; Optionally, the crotonylation-modified eraser gene includes HDAC family or SIRT family genes; Optionally, the crotonylation modification-related genes include the ACSS2 gene, ACADS gene, AF9 gene, MOZ gene, CBP gene, or P300 gene.

4. The method according to any one of claims 1-3, comprising the following steps: (1) Differentiate pluripotent stem cells to obtain mesodermal cells, wherein the mesodermal cells are TBXT+ cells; (2) Differentiate the mesodermal cells to obtain hematopoietic precursor cells, wherein the hematopoietic precursor cells are CD34+, CD31+ and / or CD43+ cells.

5. The method according to claim 4, wherein, The crotonylation enhancer is added to the culture medium in step (1); preferably, the crotonylation enhancer is added to the culture medium in both steps (1) and (2).

6. The method according to claim 5, wherein, The concentration of the crotonylation enhancer is 5-10 mM.

7. The method according to any one of claims 1-3, wherein, The reduction of the level of the crotonylation eraser gene in the pluripotent stem cells includes knocking down or knocking out the crotonylation eraser gene by siRNA, shRNA or CRISPR / Cas9 system; Increasing the level of crotonylation-related genes in the pluripotent stem cells includes introducing crotonylation-related genes into the pluripotent stem cells, for example, through lentiviral transfection.

8. Hematopoietic precursor cells obtained by the method according to any one of claims 1-7.

9. A kit, preferably used for inducing the acquisition of hematopoietic progenitor cells, wherein, The kit contains reagents for increasing the level of crotonylation modification in pluripotent stem cells.

10. The kit according to claim 9, wherein, The reagent used to enhance the crotonylation modification level of pluripotent stem cells is a crotonylation enhancer, and optionally, the kit also includes a basal culture medium.

11. The kit according to claim 9, wherein, The reagents for increasing the level of crotonylation modification in pluripotent stem cells are reagents for reducing the level of crotonylation-modifying eraser genes in the pluripotent stem cells (e.g., siRNA and shRNA for knocking out or knocking down crotonylation-modifying eraser genes), or reagents for increasing the level of crotonylation-related genes in the pluripotent stem cells (e.g., vectors or lentiviruses containing crotonylation-related genes).

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