Inducing hematopoietic stem group cells to differentiate into megakaryocyte progenitor cells and platelets via JAK2 / STAT3 signaling pathway

Abstract

The invention provides a combination capable of efficiently inducing differentiation of hematopoietic stem / progenitor cells to megakaryocyte lineage cells. The combination comprises three components: a first component, a second component and a third component, wherein the first component is an HSE7 gene or protein, mRNA of HSE7, an HSE7 expression cassette or an HSE7 accelerant; the second component is a histone deacetylase inhibitor; and the third component is a gamma-aminobutyric acid agonist. Also provided is a method for efficiently inducing differentiation of hematopoietic stem / progenitor cells into meganuclear lineage cells, comprising overexpressing HSE7, inhibiting HDAC expression and / or activity, and administering a GABA agonist.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically involving inducing hematopoietic stem cells to differentiate into megakaryocyte progenitor cells and platelets through the JAK2 / STAT3 signaling pathway. Background Technology

[0002] The supply of platelets in clinical blood transfusions is often insufficient to meet the ever-increasing demand. Platelet generation from stem cells offers a potential solution to reduce reliance on donor transfusions. However, current differentiation efficiency remains unsatisfactory.

[0003] Megakaryocytes (MKs) are precursors to platelets. A large number of MKs predominantly pro-thrombopoietin are essential to meet clinical requirements. However, in vitro-generated megakaryocytes often exhibit cell damage and asynchronous differentiation.

[0004] To achieve continuous and efficient platelet production, there is an urgent need to efficiently differentiate megakaryocyte progenitors (MkPs) capable of self-renewal, providing a solution for continuous platelet production. Summary of the Invention

[0005] This invention provides a method for inducing hematopoietic stem / progenitor cells to proliferate and / or differentiate into megakaryocyte progenitor cells (MKP), and also provides a combination capable of inducing hematopoietic stem / progenitor cells to proliferate and / or differentiate into megakaryocyte progenitor cells (MKP) and its use.

[0006] In a first aspect of the invention, a combination for inducing differentiation of hematopoietic stem cells into megakaryocyte lineage cells is provided, the combination comprising the following three components:

[0007] (a) HSE7 gene or protein, HSE7 mRNA, HSE7 expression cassette or HSE7 promoter;

[0008] (b) Histone deacetylase inhibitors; and

[0009] (c) Gamma-aminobutyric acid agonists.

[0010] In another preferred embodiment, the hematopoietic stem cells are selected from: hematopoietic stem cells, pluripotent progenitor cells, common myeloid progenitor cells, megakaryocyte-erythroid progenitor cells, or combinations thereof.

[0011] In another preferred embodiment, the megakaryocyte lineage cells are selected from: megakaryocyte progenitor cells, mature megakaryocytes, platelets, or combinations thereof.

[0012] In another preferred embodiment, the histone deacetylase inhibitor is a small molecule histone deacetylase inhibitor, an antibody-based histone deacetylase inhibitor, a receptor-type histone deacetylase inhibitor, or a non-receptor-type histone deacetylase inhibitor.

[0013] In another preferred embodiment, the histone deacetylase inhibitor includes (but is not limited to): MC1568, belinstat, parbistat, sodium butyrate, sodium phenylbutyrate, vorinostat, trichostatin A, sodium valproate, gembisstat, or combinations thereof.

[0014] In another preferred embodiment, the histone deacetylase inhibitor is MC1568.

[0015] In another preferred embodiment, the concentration of MC1568 is 2-10 μM, preferably 3-8 μM, and most preferably 4-6 μM.

[0016] In another preferred embodiment, the concentration of MC1568 is 5 μM.

[0017] In another preferred embodiment, the γ-aminobutyric acid agonist is γ-aminobutyric acid or a γ-aminobutyric acid pathway activator.

[0018] In another preferred embodiment, the γ-aminobutyric acid agonist is γ-aminobutyric acid.

[0019] In another preferred embodiment, the concentration of γ-aminobutyric acid is 40-120 mM, the preferred concentration of γ-aminobutyric acid is 60-100 mM, and the most preferred concentration of γ-aminobutyric acid is 70-90 mM.

[0020] In another preferred embodiment, the concentration of γ-aminobutyric acid is 80 mM.

[0021] In a second aspect of the invention, the use of the combination described in the first aspect of the invention is provided for preparing a kit for inducing the proliferation and / or differentiation of hematopoietic stem / progenitor cells into megakaryocyte progenitor cells (MKP).

[0022] In a third aspect of the invention, a kit is provided for inducing the proliferation and / or differentiation of hematopoietic stem / progenitor cells into megakaryocytic progenitor cells (MKP), the kit comprising the following three components:

[0023] (C1) HSE7 gene or protein, HSE7 mRNA, HSE7 expression cassette or HSE7 promoter;

[0024] (C2) Histone deacetylase inhibitors; and

[0025] (C3) γ-aminobutyric acid agonist.

[0026] In another preferred embodiment, the kit further includes the following component: (C4) a detection reagent for phosphorylated STAT3.

[0027] In another preferred embodiment, the phosphorylated STAT3 detection reagent is used to evaluate the effect of the combined use of the three components C1, C2, and C3 on inducing the proliferation and / or differentiation of hematopoietic stem / progenitor cells into megakaryocyte progenitor cells (MKP).

[0028] In another preferred embodiment, the kit comprises the combination described in the first aspect of the invention.

[0029] In another preferred embodiment, the phosphorylated STAT3 detection reagent is used to evaluate the effect of the combination on inducing the proliferation and / or differentiation of hematopoietic stem / progenitor cells into megakaryocyte progenitor (MKP) cells.

[0030] In a fourth aspect of the invention, a kit is provided for inducing the proliferation and / or differentiation of hematopoietic stem / progenitor cells into megakaryocyte progenitor cells (MKP), the kit comprising: hematopoietic stem cells overexpressing the HSE7 gene, a histone deacetylase inhibitor, and a γ-aminobutyric acid agonist.

[0031] In a fifth aspect of the invention, a method for inducing hematopoietic stem / progenitor cells to proliferate and / or differentiate into megakaryocyte progenitor cells (MKP) is provided, the method comprising the following steps:

[0032] (S1) HSE7 is overexpressed in hematopoietic stem cells to obtain HES7 overexpressing hematopoietic stem cells;

[0033] (S2) In the presence of HDAC inhibitors and GABA agonists, the HES7-overexpressing hematopoietic stem cells are cultured to promote the differentiation of the HES7-overexpressing hematopoietic stem cells into megakaryocyte lineage cells.

[0034] In another preferred embodiment, the overexpression of HSE7 in hematopoietic stem cells includes: overexpressing endogenous HSE7 in hematopoietic stem cells and overexpressing exogenous HSE7 in hematopoietic stem cells.

[0035] In another preferred embodiment, the overexpression of HSE7 in hematopoietic stem cells is the overexpression of HSE7 in the first and second stages of hematopoietic stem cell differentiation.

[0036] In another preferred embodiment, the method is a non-diagnostic, non-treatment method.

[0037] In a sixth aspect of the invention, there is provided the use of a phosphorylated STAT3 detection reagent for preparing a kit for evaluating the ability of the combination described in the first aspect of the invention or the kit described in the fourth aspect of the invention to induce the proliferation and / or differentiation of hematopoietic stem / progenitor cells into megakaryocyte progenitor cells (MKP).

[0038] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0039] Figure 1 The study demonstrates the effect of overexpression of certain components or combinations on MkP differentiation on day 10 of differentiation. V1: FLI1, V2: MXD3, V3: FOXB1, V4: HOXA9, V5: C-MYC.

[0040] Figure 1 (a) shows the flow cytometry results of CD34 and CD41 expression under various overexpression conditions; Figure 1 (b), 1(c), and 1(d) show Figure 1 (a) Quantitative analysis results.

[0041] Figure 2 The results showed that the VGM mixture significantly improved the differentiation of megakaryocyte progenitor cells (MkP) in vitro.

[0042] Figure 2 (a) shows a schematic diagram of the in vitro differentiation protocol. Figure 2 (b) shows the flow cytometry results of CD34 and CD41 expression in GFP and VGM group cells on day 10 of differentiation. Figure 2 (c) shows the quantitative results of CD34+CD41+ cells in (b). Figure 2 (d) shows the statistical analysis of CD34+CD41+ cell populations from 12 different cell batches.

[0043] Figure 3 The VGM mixture demonstrated its ability to promote long-term self-renewal of megakaryocyte progenitor cells (MkPs).

[0044] Figure 3 (a) shows representative images of EdU (yellow) and DAPI-labeled nuclei (blue) in GFP and VGM group cells on day 10 of differentiation. Figure 3 (b) shows the quantitative results of EdU-positive cells in (a). Figure 3 (c) shows the statistical analysis of the apoptotic cell populations in the GFP and VGM groups on day 10 of differentiation based on Annexin V and DAPI staining. Figure 3 (d) shows the cell count data for long-term culture of the GFP and VGM groups. Figure 3(e) shows the proportion of CD34+CD41+ cells in the GFP and VGM groups during long-term culture. The red curve with solid squares represents the VGM group, and the black curve with circular squares represents the GFP group.

[0045] Figure 4 The study showed that the VGM mixture promoted the production of mature megakaryocytes (MKs).

[0046] Figure 4 (a) shows the flow cytometry analysis results of CD41, CD42a and CD42b expression in GFP and VGM group cells on day 17 of differentiation. Figure 4 (b) shows the quantitative results of CD41+CD42a+ and CD41+CD42b+ cells in (a). Figure 4 (c) shows the flow cytometry analysis results of DNA multinucleation on day 17 of differentiation. PI staining is on the left; DAPI staining is on the right. Multinucleation was classified as 2N, 4N, 8N, 16N, and 32N. Figure 4 (d) shows the statistical analysis of multinucleation (2N-16N) in the GFP and VGM groups on day 17 of differentiation. Figure 4 (e) shows a representative image of CD41 (red) and DAPI (blue) stained cells on day 17 of differentiation. Figure 4 (f) shows a representative image of Wright-Giemsa staining in the VGM group on day 20 of differentiation.

[0047] Figure 5 This study demonstrated that VGM-induced megakaryocyte progenitors (MkPs) efficiently produce platelets both in vitro and in vivo.

[0048] Figure 5 (a) shows a phase-contrast microscopy image of filamentous platelets on day 20 of differentiation. Figure 5 (b) shows the flow cytometry analysis results of platelet FSC / SSC distribution and CD41 and CD42b expression in the GFP and VGM groups on day 20 of differentiation. Figure 5 (c) shows the quantitative results of CD41+CD42b+ platelets in (b). Figure 5 (d) shows the flow cytometry analysis results of CD62P expression after ADP and TRAP-6 activation. The left side shows activated platelets from human peripheral blood; the right side shows platelets from the VGM group on day 20 of differentiation. Figure 5 (e) shows a schematic diagram of the preparation of a mouse model of thrombocytopenia and the differentiation of MkPs into platelets in vivo. Figure 5 (f) shows the platelet count results on day 9 of the thrombocytopenic mouse model (n=5). Figure 5(g) shows the flow cytometry analysis results of platelet differentiation of MkPs in mice with CD41+ chimeras (mCD41-hCD41+) measured at 30 minutes, 4 hours, and 24 hours after injection. The NC (control) group was injected with PBS; S1 and S2 were injected with MkPs differentiated on days 10 and 20, respectively. Figure 5 (h) shows the quantitative results of CD41+ chimera (mCD41-hCD41+) 30 minutes after injection (n=5).

[0049] Figure 6 The study demonstrated a significant transcriptional response induced by the VGM mixture.

[0050] Figure 6 (a) shows the heatmap analysis of MkPs from the GFP group (n=3), VGM group (n=3), and PBMC group (n=3). Figure 6 (b) Shows genes that were significantly upregulated (red) and downregulated (blue) by VGM-derived MkPs compared to GFP-derived MkPs. The x-axis represents the log2 fold change, and the y-axis represents the -log10 p value. Red or blue dots indicate differentially expressed genes (DEGs) identified by p-value < 0.05 and |log2(fold change)| > 0.3. Genes of interest are marked with yellow dots. Figure 6 (c) shows the KEGG pathway analysis results of MkPs in the VGM group and MkPs in the GFP group. Figure 6 (d) shows the GO enrichment analysis results of MkPs from the VGM group and MkPs from the GFP group. Figure 6 (e) shows the TPM analysis of CD53 expression in MkPs of the GFP and VGM groups on day 10. Figure 6 (f) shows a heatmap analysis of MkPs in the GFP group (n=3), VGM group (n=3), and PBMC group (n=3) based on the MK1-MK6 gene sets.

[0051] Figure 7 The study showed that VGM mixtures promote MkP production via the JAK2-STAT3 signaling pathway.

[0052] Figure 7 (a) shows the GSEA analysis results of the JAK / STAT signaling pathway in MkPs of the VGM and GFP groups. Figure 7 (b) shows the TPM analysis of BCL2L1 in the GFP and VGM groups. Figure 7 (c) shows the TPM analysis of CCND1 in the GFP and VGM groups. Figure 7(d) shows the immunofluorescence images of cells on day 17 of differentiation, including GFP fluorescence (green) and staining results for DAPI (blue), c-MPL (grey) and p-STAT3 (red). Figure 7 (e) shows the MFI quantitative analysis of p-STAT3 in (d). Figure 7 (f) shows the Western blot analysis of JAK2, p-JAK2, STAT3 and p-STAT3 in MkP cells derived from the GFP and VGM groups on day 10 of differentiation. Figure 7 (g) shows the flow cytometry analysis results of GFP cells (untreated with VGM) and MkP cells (CD34+CD41+) in the VGM group treated with the JAK2 signaling inhibitors Baricitinib (Bar) and Tofacitinib (Tof) on day 10 of differentiation.

[0053] Figure 8 The pathways and mechanisms involved in MkP-enhanced differentiation induced by VGM mixtures are shown. Detailed Implementation

[0054] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that overexpression of HES7, combined with treatment of hematopoietic stem / progenitor cells with histone deacetylase (HDAC) inhibitors and γ-aminobutyric acid (GABA) agonists, can increase the generation of megakaryocyte progenitor cells and mature megakaryocytes, raising the differentiation efficiency of megakaryocyte progenitor cells (MkPs) to 90%, while also enhancing the self-renewal capacity of MkPs and significantly increasing the yield of platelets obtained from hematopoietic stem / progenitor cells. This invention was completed based on this discovery.

[0055] Megakaryotic progenitor cells

[0056] Hematopoietic stem cells (HSCs) gradually differentiate into megakaryocyte progenitor cells (MPCs) under the regulation of various cytokines and transcription factors. MPCs are morphologically more specialized than HSCs, gradually increasing in size and beginning to express megakaryocyte-specific molecular markers such as CD41, CD42, and CD61. During the differentiation of HSCs into MPCs, CD41 begins to be expressed on the surface of MPCs. As cells further differentiate and mature, the expression level of CD41 gradually increases. In the early stages of HSC differentiation into MPCs, CD42 expression begins to appear and gradually increases with megakaryocyte maturation.

[0057] Megakaryotic progenitors (MkPs) further differentiate into megakaryocytes (MKs). During this process, the cells continuously enlarge, undergoing intranuclear replication to become polyploid cells, with DNA content reaching 8N, 16N, or even 32N. The cytoplasm of megakaryocytes gradually matures, forming the microenvironment for platelet production, including platelet granule formation and microtubule assembly. MKs exhibit heterogeneity, comprising at least three subsets: platelet-producing MKs, immune-related MKs, and microenvironment-supporting MKs. Platelet-producing MKs extend their cytoplasm to form long pseudopodia that penetrate into the intercellular spaces of bone marrow sinusoidal endothelial cells. The megakaryocyte cytoplasm is cleaved within the sinusoids, forming individual platelets released into the peripheral blood. As megakaryotic progenitors differentiate into mature megakaryocytes, CD34 expression gradually decreases.

[0058] HES7 gene

[0059] The HES7 gene, or hair and enhancer of split 7 gene, belongs to the basic helix-loop-helix (bHLH) transcription factor family. HES7 plays a crucial role in embryonic development, primarily regulating neural crest cell differentiation and vertebrate somites. Abnormal expression of this gene can lead to developmental defects, affecting the normal development of the nervous and musculoskeletal systems. Furthermore, research on this gene provides potential research directions for fields such as regenerative medicine.

[0060] Histone deacetylase

[0061] Histone deacetylases (HDACs) are a class of proteases that play an important role in chromosome structural modification and gene expression regulation. Normally, histone acetylation facilitates the dissociation of DNA from histone octamers, relaxes nucleosome structure, and allows various transcription factors and co-transcription factors to specifically bind to DNA binding sites, activating gene transcription.

[0062] Histone deacetylases (HDACs) influence MK differentiation at different levels. For example, HDAC1 and HDAC2 activate the GATA switch by interacting with FOG-1, promoting terminal differentiation of MKs, while HDAC inhibitors delay MK maturation by inhibiting GATA1 expression. Furthermore, HDAC inhibition has been found to enhance MkP differentiation, indicating a complex regulatory mechanism of HDACs at different stages of MK lineage differentiation.

[0063] γ-Aminobutyric acid (GABA)

[0064] Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system, widely distributed in vertebrates, plants, and microorganisms. GABA is primarily known for its role in neurogenesis. Furthermore, the GABA receptor GABRR1 on hematopoietic stem cells is involved in hematopoiesis and hematopoietic stem cell transplantation.

[0065] The composition of the present invention

[0066] The composition of the present invention refers to a mixture of an HES7 overexpression inducer, a histone deacetylase inhibitor, and a γ-aminobutyric acid agonist.

[0067] In another preferred embodiment, the GABA agonist is GABA or a GABA pathway activator.

[0068] In another preferred embodiment, the GABA agonist is GABA.

[0069] In another preferred embodiment, the GABA concentration is 40-120 mM, the preferred GABA concentration is 60-100 mM, and the most preferred GABA concentration is 70-90 mM.

[0070] In another preferred embodiment, the GABA concentration is 80 mM.

[0071] In another preferred embodiment, the histone deacetylase inhibitor is MC1568.

[0072] In another preferred embodiment, the concentration of MC1568 is 2-10 μM, preferably 3-8 μM, and most preferably 4-6 μM.

[0073] In another preferred embodiment, the concentration of MC1568 is 5 μM.

[0074] In another preferred embodiment, the mixture is a VGM mixture comprising a viral vector overexpressing HES7, MC1568, and GABA.

[0075] In some specific implementation schemes, the Ctrl group consists of CD34 cells differentiated for 10 days under normal culture conditions; the GFP group consists of CD34 cells differentiated for 10 days after being infected with HES7 as a blank control group; and the VGM group consists of CD34 cells infected with HES7 lentivirus, with the optimal concentrations of GABA and MC1568 added after drug screening.

[0076] The main advantages of this invention include:

[0077] (1) The present invention provides a composition for inducing hematopoietic stem / progenitor cells to produce megakaryocytes efficiently and its use, and provides a method for inducing hematopoietic stem / progenitor cells to produce megakaryocytes efficiently, which significantly enhances the production of MkP and promotes the production of mature MKs and platelets by human HSPCs.

[0078] (2) The VGM mixture provided by the present invention significantly improves the differentiation efficiency of megakaryocyte progenitor cells (MkPs) to 90%, and has good reproducibility.

[0079] (3) The VGM mixture provided by the present invention significantly enhances the self-renewal ability of MkPs and maintains their progenitor cell state during the extended culture period.

[0080] (4) This invention is the first to discover that HES7 has a synergistic effect when combined with HDAC inhibitor MC1568 and GABA.

[0081] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0082] Example 1: Experimental Method

[0083] (1) Plasmid

[0084] The pCDH-MSCV-hCD38-EF1α+Puro cloning and expression vector (plasmid #134936) was purchased from Addgene. The cDNA sequence of the candidate gene (SEQ ID NO:1) was synthesized by GeneCreate Biotechnology Co., Ltd. and inserted under the MSCV promoter. A vector containing the inserted non-functional sequence was used as a control.

[0085] (2) Differentiation of hematopoietic stem cells

[0086] Differentiation protocols for hematopoietic stem cells, such as Figure 2As shown in a. Peripheral blood CD34+ cells were purchased from Shanghai Miaoshun Biotechnology Co., Ltd., and cultured in SFEMII (09655, StemCell Technologies) supplemented with 20 ng / mL each of hIL-3 (number 200-03, PEPROTECH), hIL-6 (number 200-06-100, PEPROTECH), TPO (number 300-18-100, PEPROTECH), hSCF (number 300-07-100, PEPROTECH) and Flt3 ligand (number 300-19-100, PEPROTECH).

[0087] In the first stage, the cells were incubated at 37°C and 5% CO2 for 10 days or until the cells were collected for analysis.

[0088] In the first stage, Mocetinostat (No. 18287, Cayman), MC1568 (No. 16265, Cayman), Quisinostat (No. 14088, Cayman), GABA (No. 0344, Tocris), Baricitinib phosphate (5.7 nM; No. HY-15315A, MCE), and Tofacitinib (20 nM; No. HY-40354, MCE) were added to the culture medium. Half of the medium was replaced daily.

[0089] In the second stage, cells were cultured in IMDM containing N2, B27 (number 17504044, Gibco), and 20 ng / mL each of TPO, hSCF, and hIL-6 at 39°C and 5% CO2 for 7-10 days. Half of the culture medium was replaced daily.

[0090] (3) Virus preparation

[0091] 293T cells were cultured in high-glucose DMEM containing 10% FBS, supplemented with 100 μM non-essential amino acids, 100 μM glutamine, and 100 U / mL penicillin-streptomycin (Gibco, catalog number 15140-122). Virus production was performed using calcium-phosphorus coprecipitation. The cDNA vector, packaging vector psPAX2 (plasmid number 12260), and envelope vector pMD2.G (plasmid number 12259) were mixed in a 3:2:1 ratio. Viral supernatants were collected at 48 and 72 hours post-transfection, filtered through a 0.45 μm filter, and concentrated to different final volumes using a 100 kDa Millipore column.

[0092] (4) Viral transduction

[0093] At 4-20 μg / cm 2 of Recombinant human fibronectin fragment (T100A, Takara) was pre-coated onto plates and incubated overnight at 4°C. After coating, the plates were blocked with PBS solution containing 2% BSA for 30 minutes, followed by washing with PBS. Viral suspension was added, and secondary infection was performed 24 hours later. Screening was performed using 1 μg / ml puromycin (HY-B1743A, MCE).

[0094] (5) Immunofluorescence staining analysis

[0095] Cells were fixed with 4% formaldehyde at room temperature for 15 minutes, followed by infiltration with 0.1% Triton X-100 for 5 minutes. After blocking with 1% BSA in PBS for 30 minutes, cells were incubated overnight at 4°C with primary antibody (dilution 1:200), followed by incubation with secondary antibody at room temperature for 1 hour. After washing three times with PBS, cells were then incubated with ProLong containing DAPI. TM Gold anti-fading mounting medium (Invitrogen) was used for mounting. Images were acquired using LEICA TCS SP5.

[0096] The antibodies used for immunofluorescence staining included: anti-CD41 (Abcam, ab181582), anti-c-MPL (Invitrogen, PA5-144648), and anti-p-STAT3 (CST, 4113S). 594 Goat Anti-Mouse IgG (Abcam, ab150116), Alexa 647 goat anti-rabbit IgG (Invitrogen, A78957).

[0097] (6) Polyploid analysis

[0098] Collect cells and first use Alexa Pre-staining with 647-labeled anti-CD41 antibody (BioLegend, 133914) followed by fixation with 70% cold ethanol overnight at 4°C. Subsequent staining with DAPI (Sigma-Aldrich, D9542), 0.1% Triton X-100, and propidium iodide (PI, Thermo Fisher, P3566) was performed, and analysis was conducted using Beckman Coulter CytoFLEX S. Data were analyzed using FlowJo.

[0099] (7) Platelet activation analysis

[0100] Platelets were purified from human peripheral blood and in vitro differentiated cells. For activation, platelets were incubated with ADP (0.2 mM) and TRAP-6 (0.05 mM) for 10 min. After activation, platelets were stained with CD41 and CD62P antibodies (BioLegend) for 30 min. Flow cytometry was then performed using BD FACSAria II to detect CD62P expression in CD41+-gated platelets. Data were analyzed using FlowJo.

[0101] (8) Flow cytometry MkP cell sorting

[0102] MkPs were obtained 10 days after the first-stage differentiation of human peripheral blood mononuclear cells (PBMCs) or CD34+ cells. Multiparameter staining was performed using the following antibodies to sort MkP cells by flow cytometry: AmCyan lineage mixture (Lin), PE-Cy7CD34, PE-Cy5CD38, APCCD123, AF700CD45RA, and PerCP-Cy5.5CD41. The resulting MkPs were identified as Lin-CD34+CD38+CD123-CD45RA-CD41+.

[0103] The MkP cell sorting strategy includes the following steps: First, Lin-cells are sorted to exclude mature lineage-positive cells, and then CD34+CD38+ cells are selected from the Lin-cell population. CD123-CD45RA- cells are further identified within the CD34+CD38+ cell population. Finally, the MkP cell population is identified using CD41+ cell gating. The sorted MkP cells are then collected for downstream analyses, including transcriptome analysis and differentiation assays.

[0104] (9) Animal model treatment

[0105] Platelet-producing capacity of MkP cells was assessed using male NXG mice (11-13 weeks old at irradiation). All mice were purchased from the Institute of Zoology, Chinese Academy of Sciences. Day 0: 24 NXG mice were irradiated with 1.8 Gy X-rays to induce thrombocytopenia. Post-irradiation care included SPF-barrier housing, antibiotic-treated drinking water (5% enrofloxacin solution, 1‰ dilution; Zhonglong Shenli, Hefei), and daily supplementation with sunflower seeds and nesting material.

[0106] Day 9: Complete blood count (CBC) and macrophage depletion. Blood samples were collected from the posterior orbital sinus for CBC. Macrophages were depleted using LIPOSOMA liposome macrophage depletion reagent (0.5 mg per 100 μL of mouse; YS Biotechnology, 40337ES08). Mice with platelet counts between 50 and 200 × 10^6 / mL were selected for further experiments.

[0107] Day 10: Approximately 24 hours after macrophage depletion, mice were infused via tail vein with either MkPs or PBS (200 μL / mouse). Mice were then divided into the following groups:

[0108] S1-D10: MkPs cultured in vitro for 10 days, injected at a cell volume of 1×10^6 (n=5);

[0109] S2-D20: MkPs cultured in vitro for 20 days, injected at a cell volume of 1×10^6 (n=5);

[0110] Control group (NC): injected with an equal volume of PBS (n=5).

[0111] (9) Restructuring assessment

[0112] Peripheral blood samples were collected via tail vein at 0.5, 4, and 24 hours post-infusion to assess donor cell chimerism (hCD41+ platelets). Flow cytometry analysis was performed using blood containing an anticoagulant (40 μL, 1:9 sodium citrate solution; Zhonglong Shenli; Hefei). In the flow cytometry analysis, hCD41+ and mCD41+ cells were identified using APC-labeled anti-mouse CD41 (133914, BioLegend) and APC-CY7-labeled anti-human CD41 (303716, BioLegend). The number of platelets produced by MkP injection was calculated as follows: Platelets / MkP = (Post-modeling platelet concentration × Total mouse blood volume × Mean hCD41 chimerism rate) / (Number of injected MkP).

[0113] (10) Protein phosphorylation analysis

[0114] Cells were lysed using RIPA buffer (Beyotime, P0013B) supplemented with a mixture of protease and phosphatase inhibitors (Beyotime, P1045). Protein concentrations were quantified using a BCA protein assay kit (Beyotime, P0010). Proteins were separated by SDS-PAGE and transferred to PVDF membranes for electrophoresis using a Bio-Rad electrophoresis system. Primary antibody probes were then used at the following dilutions: JAK2 (CST, 3230T) 1:1500, STAT3 (CST, 30835S) 1:1500, phosphorylated JAK2 (CST, 3776S) 1:1000, phosphorylated STAT3 (CST, 30835S) 1:2000, and β-actin (CST, 8457T) 1:3000. Bands were visualized using an Odyssey XF imaging system (LI-COR, Nebraska, USA) and quantified using LI-COR Image Studio software. Statistical analysis was subsequently performed.

[0115] Example 2: Enhancing the differentiation of megakaryocyte progenitor cells (MkP) through transcription factor overexpression and small molecule combination

[0116] First, the effect of overexpression of a single transcription factor on MkP differentiation was evaluated. The transcription factors included HES7, FLI1, MXD3, FOXB1, HOXA9, and C-MYC.

[0117] The results are as follows Figure 1 a and Figure 1 As shown in b, after 10 days of differentiation, overexpression of HES7 produced the highest MkP differentiation efficiency (25.13% ± 2.60%). Meanwhile, co-overexpression of HES7 with any of the other five transcription factors did not further improve the HES7-induced differentiation efficiency. Figure 1 c indicates that co-overexpression of HES7 with two, three, or four other factors does not further improve the differentiation efficiency induced by HES7. Figure 1 d indicates that the differentiation efficiency generated by HES7 overexpression is higher than that generated by overexpression of other transcription factor combinations.

[0118] The combined effects of GABA and HDAC inhibitors (HDACi) with HES7 overexpression were further investigated. Based on concentration optimization, the optimal concentration of the GABA agonist was 80 mM, and the optimal concentration of the HDAC inhibitor MC1568 was 5 μM, which induced 13.65% ± 0.21% and 29.05% ± 2.05% of MkP generation, respectively.

[0119] HES7-overexpressing CD34+ cells were co-cultured with GABA and MC1568 (i.e., VGM mixture) from day 4 to day 10.

[0120] The results are as follows Figure 2 b and Figure 2 As shown in c, by day 10, almost 90% (87.53% ± 1.11%) of the cells in the VGM group differentiated into CD34+CD41+ MkPs, while only 10.07% ± 0.45% of the cells in the GFP group differentiated into CD34+CD41+ MkPs.

[0121] like Figure 2 As shown in Figure d, the VGM group showed consistent differentiation efficiency (84.92% ± 5.65%) across different batches of cells. There was no statistically significant difference between the GFP and Ctrl groups, indicating that the blank vector had almost no effect on cell differentiation.

[0122] Furthermore, HES7 overexpression is as follows: Figure 2Throughout the process shown in a, HES7 overexpression did not adversely affect the normal growth of hematopoietic stem cells.

[0123] The results of this embodiment show that the VGM mixture can significantly induce MkP differentiation, improve differentiation efficiency, and the effect is stable.

[0124] Example 3: VGM mixture-induced MkPs exhibit enhanced self-renewal capacity

[0125] To assess the self-renewal potential of VGM-induced MkPs, the proliferation capacity of MkPs treated with GFP and VGM groups was investigated.

[0126] Proliferation status as follows Figure 3 a and Figure 3 As shown in b, on day 10, the proliferation rate of VGM-induced MkPs was 18.07% ± 0.96%, which was about twice that of the GFP group (9.67% ± 1.35%).

[0127] Apoptosis status such as Figure 3 As shown in c, at day 10, the VGM group showed a significantly lower apoptosis rate of approximately 2.06% ± 0.40% compared to the GFP group (7.31% ± 0.40%).

[0128] like Figure 3 Cell counting results showed that by day 25, the total number of cells in the VGM group was four times that of the GFP group. VGM-treated cells continued to expand and remained viable for at least 51 days.

[0129] like Figure 3 As shown in e, during the amplification process, the VGM group maintained a high proportion of CD34+CD41+ expression. Although this proportion gradually decreased during the later passages in the long-term culture, the CD34+CD41+ expression level of the VGM group was significantly higher than that of the control group within 28 days.

[0130] The results of this embodiment show that VGM treatment significantly enhances the self-renewal capacity of MkPs, specifically by promoting MkPs proliferation, inhibiting MkPs apoptosis, and maintaining their progenitor cell state during the extended culture period.

[0131] Example 4: Increased production of mature megakaryocytes (MKs) generated from VGM-induced MkPs

[0132] To assess the maturation potential of VGM-induced MkPs, cells were cultured for an additional 7 days in the second-stage differentiation medium.

[0133] The results are as follows Figure 4 a and Figure 4As shown in b, the proportion of mature megakaryocytes (CD41+CD42b+) generated in the VGM group (72.90% ± 0.42%) was significantly higher than that in the GFP group (12.15% ± 0.78%), about six times higher, confirming that VGM-induced MkPs generated an increased yield of mature megakaryocytes (MKs).

[0134] Figure 4 b further showed that approximately 63.4% of megakaryocytes in the VGM group simultaneously expressed CD41 and CD42a, compared to only 10.79% ± 1.44% in the GFP group. This result confirms that the megakaryocyte progenitors treated in the VGM group differentiated into more mature megakaryocytes.

[0135] Figure 4 c and Figure 4 The results showed that VGM-induced megakaryocytes exhibited polyploidy, with the maximum ploidy reaching 32N.

[0136] Among them, the proportion of 4N cells in the VGM group (35.77% ± 0.49%) was significantly increased compared with that in the GFP group (24.30% ± 1.04%), indicating that it was more inclined to mature multinucleated state.

[0137] Figure 4 immunofluorescence of e and Figure 4 Wright-Giemsa staining of f revealed increased cell volume and polyploidy, confirming the presence of megakaryocytes and indicating that they had entered terminal maturation and were about to form proplatelets.

[0138] The results of this embodiment indicate that the VGM mixture can significantly enhance the ability of mature megakaryocytes to be generated in vitro.

[0139] Example 5: VGM-induced MkPs efficiently generate platelets in vitro and in vivo.

[0140] Mature megakaryocytes in the VGM group were cultured for another 3 days, as follows: Figure 5 As shown in a, the preplatelet-like morphology can be clearly observed, which is characterized by multiple protrusions with long branches.

[0141] Figure 5 b and Figure 5 c indicates that the VGM group produced significantly more CD41+CD42b+ platelets (24.97% ± 1.05%) than the GFP group (21.17% ± 0.96%).

[0142] Figure 5 The activation results of platelets obtained in d and human peripheral blood-derived platelets showed that the activation pattern of these platelets was similar to that of human peripheral blood-derived platelets.

[0143] like Figure 5 As shown in e, the ability of VGM-induced MkPs to mature and release platelets in vivo was evaluated using an NXG mouse thrombocytopenia model established by irradiation.

[0144] The results are as follows Figure 5 As shown in f, on day 9, the platelet count in mice ranged from (50-200)×10^6 / mL.

[0145] After macrophage removal, VGM-induced MkPs from different culture stages (stage 1, day 10 [M1] and stage 2, day 20 [M2]) were intravenously delivered to mice. The release of human platelets in the peripheral blood of mice was assessed at 0.5 hours, 4 hours and 24 hours after injection.

[0146] The results are as follows Figure 5 g and Figure 5 As shown in h, human CD41+ platelet chimerism (mCD41-hCD41+) was successfully detected in both VGM groups [M1] and [M2], and the two groups reached similar levels after injection: 4.64% at 30 minutes, 19.4% at 4 hours, and 15.7% at 24 hours.

[0147] The results of this embodiment confirm the potential of VGM-induced MkPs to differentiate into platelets in vivo, and show that long-term in vitro culture does not affect their differentiation ability.

[0148] Example 6: VGM-induced MkPs exhibit unique gene expression characteristics

[0149] Figure 6 a demonstrates the unique gene expression patterns of the VGM and GFP groups compared to MkPs derived from peripheral blood mononuclear cells (PBMCs), highlighting significant regulatory differences.

[0150] Figure 6 b indicates that multiple MkP-related genes were upregulated in the VGM group, among which FLI1, GATA2, RUNX1 and MPL were significantly upregulated.

[0151] Figure 6 KEGG pathway analysis of c showed that NF-kappa B, MAPK, cGMP-PKG and PI3K-AKT signaling pathways were significantly enriched in VGM-induced MkPs, and these pathways are all closely related to MkP differentiation and platelet development.

[0152] Figure 6Gene ontology (GO) analysis of d further revealed a significant enrichment of gene sets related to myeloid cell development, platelet production, and cell cycle regulation in the VGM group, which is consistent with the high production of CD34+CD41+MkPs and their enhanced self-renewal capacity.

[0153] Figure 6 The study compared the CD53 expression levels of the control group, VGM group, and PBMC group. The results showed that, compared with MkPs derived from PBMC, the MkPs in the VGM group and GFP group had significantly reduced CD53 expression at the transcriptional level, indicating that our differentiation system significantly reduced the differentiation pathway of immune megakaryocytes and optimized the differentiation pathway biased towards platelet production.

[0154] Figure 6 f further demonstrated that when the gene expression profiles of VGM-treated and control MKs were mapped to six specific subtypes, the two MkP groups showed a reduction in immune-related programs (enriched in MK6) compared to PBMC-derived MkPs.

[0155] Furthermore, compared to GFP-treated MkPs, VGM-induced MkPs exhibited significant characteristics similar to the MK4 subtype supporting microenvironment MK.

[0156] Overall, the transcriptome analysis results of this embodiment indicate that VGM treatment of MkPs makes it easier to generate platelets, but still retains the characteristics of other MK subsets, showing a certain degree of heterogeneity.

[0157] Example 7: JAK2 / STAT3 signaling pathway mediates VGM-induced MK lineage differentiation

[0158] because Figure 6 c and Figure 6 The data in section d showed a significant enrichment of JAK / STAT-related gene sets (KEGG:hsa04630 and GO:0042532) in the VGM group. Therefore, further validation using GSEA confirmed the upregulation of the JAK-STAT signaling pathway gene set, as shown in the results below. Figure 7 As shown in a.

[0159] Figure 7 b and Figure 7 The results showed that in VGM-treated MkPs, the expression of CCND1 and BCL2L1 genes regulated by phosphorylated STAT3 (p-STAT3) was significantly upregulated, indicating that VGM treatment at least partially promotes MK lineage differentiation through the JAK2 / STAT3 signaling pathway.

[0160] The activation status of the JAK2 / STAT3 pathway in MkPs on day 10 of differentiation was assessed by immunofluorescence staining and Western blot analysis. Results are as follows: Figure 7 As shown in d-7f.

[0161] Figure 7 d and Figure 7 The results showed that, compared with the GFP group (20.27±4.50), the p-STAT3 level in VGM-induced MkPs was 33.38±2.53, which was a significant increase.

[0162] Figure 7 f indicates that, compared with the GFP group, the total JAK2 level, phosphorylated JAK2 level and total STAT3 level in the VGM group remained basically unchanged, but the phosphorylated STAT3 level increased significantly.

[0163] Figure 7 g indicates that the effect of the VGM mixture on MK lineage differentiation was significantly weakened when JAK2 inhibitors (Baricitinib and Tofacitinib) were introduced. The CD34+CD41+MkP populations decreased by approximately 60% and 40%, respectively.

[0164] The results of this embodiment show that the VGM mixture, through methods such as Figure 8 The JAK2 / STAT3 signaling pathway shown promotes the generation of MkP.

[0165] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A combination for inducing differentiation of hematopoietic stem cells into megakaryocyte lineage cells, characterized in that, The combination comprises the following three components: (a) HSE7 gene or protein, HSE7 mRNA, HSE7 expression cassette or HSE7 promoter; (b) Histone deacetylase inhibitors; and (c) Gamma-aminobutyric acid agonists.

2. The combination as described in claim 1, characterized in that, The hematopoietic stem cells are selected from: hematopoietic stem cells, pluripotent progenitor cells, common myeloid progenitor cells, megakaryocyte-erythroid progenitor cells, or combinations thereof.

3. The combination as described in claim 1, characterized in that, The megakaryocyte lineage cells are selected from: megakaryocyte progenitor cells, mature megakaryocytes, platelets, or combinations thereof.

4. The combination as described in claim 1, characterized in that, The γ-aminobutyric acid agonist is γ-aminobutyric acid or a γ-aminobutyric acid pathway activator.

5. The use of the combination as described in claim 1, characterized in that, This kit is used to prepare a reagent for inducing the proliferation and / or differentiation of hematopoietic stem / progenitor cells into megakaryocyte progenitor cells (MKP).

6. A kit for inducing the proliferation and / or differentiation of hematopoietic stem cells into megakaryocyte progenitor cells, characterized in that, The kit comprises the following three components: (C1) HSE7 gene or protein, HSE7 mRNA, HSE7 expression cassette or HSE7 promoter; (C2) Histone deacetylase inhibitors; and (C3) γ-aminobutyric acid agonist.

7. The kit according to claim 6, characterized in that, The kit also includes the following component: (C4) a detection reagent for phosphorylated STAT3.

8. The kit according to claim 7, characterized in that, The phosphorylated STAT3 detection reagent is used to evaluate the effect of the combination of the three components C1, C2, and C3 on inducing the proliferation and / or differentiation of hematopoietic stem cells into megakaryocyte progenitor cells (MKP).

9. A kit for inducing the proliferation and / or differentiation of hematopoietic stem cells into megakaryocytic progenitor cells (MKP), characterized in that, The kit includes: HSE7 gene-overexpressing hematopoietic stem cells, histone deacetylase inhibitor, and γ-aminobutyric acid agonist.

10. A method for inducing hematopoietic stem cells to differentiate into megakaryocyte lineage cells, characterized in that, The method includes the following steps: (S1) HSE7 is overexpressed in hematopoietic stem cells to obtain HES7 overexpressing hematopoietic stem cells; (S2) In the presence of HDAC inhibitors and GABA agonists, the HES7-overexpressing hematopoietic stem cells are cultured to promote the differentiation of the HES7-overexpressing hematopoietic stem cells into megakaryocyte lineage cells.

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