Energizing mesenchymal stem cell as well as preparation method and application thereof

Abstract

The invention provides an energizing mesenchymal stem cell and a preparation method thereof. The energizing mesenchymal stem cell is applied to myocardial infarction treatment. According to the invention, a chemical reprogramming strategy without genetic modification is adopted, and an endogenous signal channel is activated in a time sequence manner, so that the mesenchymal stem cells highly express the endowed mesenchymal stem cells (eMSCs) of genes such as WISP1 / GPNMB and the like. When the medicine is applied to myocardial infarction treatment, myocardial remodeling can be improved and cardiac function recovery can be promoted by promoting angiogenesis, resisting myocardial apoptosis and inhibiting inflammation, fibrosis and other mechanisms. The invention provides a novel safe treatment strategy based on non-genetically modified cells for myocardial infarction.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a method for preparing enhanced mesenchymal stem cells (eMSCs) that are induced by a combination of non-genetically modified chemical small molecules to produce mesenchymal stem cells with high expression of beneficial genes such as WISP1 and GPNMB, and the application of the cell products obtained thereby in the preparation of drugs for the treatment of myocardial infarction. Background Technology

[0002] Cardiovascular disease is one of the leading causes of death among Chinese residents, with acute myocardial infarction (AMI) being particularly high due to its high incidence, high disability rate, and high mortality rate.

[0003] Current clinical revascularization strategies (including percutaneous coronary intervention and thrombolytic therapy) can restore myocardial perfusion in a timely manner and save endangered myocardium, but they cannot reverse necrotic myocardial tissue, nor can they effectively inhibit the subsequent pathological ventricular remodeling process, ultimately leading to the occurrence and development of heart failure.

[0004] Cell therapy, a significant breakthrough in regenerative medicine, offers a novel treatment strategy for myocardial infarction. Mesenchymal stem cells (MSCs), due to their multipotent differentiation potential, low immunogenicity, and potent paracrine properties, have become a research hotspot in the field of myocardial repair. MSCs improve cardiac function by secreting various bioactive factors, regulating immune inflammatory responses, promoting angiogenesis, and inhibiting cardiomyocyte apoptosis and fibrosis. However, natural MSCs face limitations such as limited therapeutic efficacy, low post-transplant survival rates, and significant functional heterogeneity. While genetic engineering techniques can enhance the therapeutic efficacy of MSCs, the use of viral vectors introduces safety risks such as insertional mutations, tumorigenicity, and immunogenicity, severely restricting their clinical translation prospects.

[0005] Recent studies have revealed that WISP1 (Wnt1-induced signaling pathway protein 1) and GPNMB (transmembrane glycoprotein NMB) play crucial roles in cardioprotection. WISP1 has important functions in promoting angiogenesis and inhibiting cardiomyocyte apoptosis; GPNMB exhibits significant anti-inflammatory and anti-fibrotic effects. However, how to safely and efficiently upregulate the expression of WISP1 and GPNMB in MSCs without gene editing and apply them to the treatment of myocardial infarction has not yet been reported.

[0006] Therefore, developing a new technology that can simultaneously enhance the expression of WISP1 and GPNMB in MSCs without gene manipulation is of great clinical significance and application value for addressing unmet clinical needs in the treatment of myocardial infarction and reducing the socioeconomic burden. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application of mesenchymal stem cells (eMSCs) that highly express WISP1 / GPNMB in the preparation of drugs for the treatment of myocardial infarction.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: an energized mesenchymal stem cell, wherein the energized mesenchymal stem cell is prepared by mesenchymal stem cell induction culture, and the energized mesenchymal stem cell highly expresses genes related to immune regulation, promoting angiogenesis, inhibiting cell apoptosis and improving energy metabolism.

[0009] Furthermore, mesenchymal stem cells are obtained by isolating from bone marrow, adipose tissue, umbilical cord, placenta, amniotic fluid, dental pulp, periodontal ligament, synovium, muscle, dermis, peripheral blood, liver, and lungs.

[0010] Furthermore, the genes related to mesenchymal stem cell immune regulation, angiogenesis promotion, apoptosis inhibition, and energy metabolism improvement are WISP1, GPNMB, LRRC32, NEU1, CD109, PDGFD, IL18BP, PPARGC1B, UCN2, RPOS1, TDO2, PTGDS, LIPG, PIK3R3, TNC, CLU, NEU1, and CD276.

[0011] Furthermore, it empowers mesenchymal stem cells to express mesenchymal stromal cell markers CD29, CD44, CD73, CD90, and CD105, but does not express stem cell-specific marker genes, hematopoietic markers, or major histocompatibility complex II (MHC-II) antigen molecules, and expresses HLA-DPB1, HLA-DPA1, HLA-DMA1, HLA-DOA, and HLA-DOB at very low levels.

[0012] The stem cell-specific marker genes are NANOG, POU5F1, KLF4, C-MYC, and LIN2; the hematopoietic markers are CD11b, CD34, CD45, and CD14; and the major histocompatibility complex-II (MHC-II) antigen molecules are HLA-DRB1, HLA-DOA, HLA-DQA1, HLA-DRB5, HLA-DQB2, HLA-DQA2, HLA-DRA, HLA-DMB, and HLA-DQB1.

[0013] A method for preparing the above-mentioned empowered mesenchymal stem cells includes the following steps:

[0014] (1) Mesenchymal stem cells were cultured and expanded using complete mesenchymal stem cell culture medium;

[0015] (2) The expanded mesenchymal stem cells were induced to grow using an induction medium:

[0016] (3) Digest and collect the induced cultured cells.

[0017] Furthermore, the complete culture medium for mesenchymal stem cells in step (1) includes liquid basal medium, fetal bovine serum, cytokines, cell culture additives, and penicillin / streptomycin;

[0018] The liquid basal culture medium is one or a mixture of two or more of the following: DMEM / F12 cell culture medium, DMEM-low glucose cell culture medium, DMEM-basic cell culture medium, DMEM-high glucose cell culture medium, 1640RPMI cell culture medium, or F12 cell culture medium.

[0019] The cytokines are one or a mixture of two or more of the following: epidermal growth factor (EGF), basic fibroblast growth factor (b-FGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), bone morphogenetic protein 4 (BMP4), bone morphogenetic protein 2 (BMP2), transforming growth factor-β1 (TGF-β1), leukemia inhibitory factor (LIF), interleukin-2 (IL-2), interleukin-3, interleukin-4, interleukin-1β (IL-1β), interleukin-6, Flt-3 ligand, stem cell factor, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, thrombopoietin, granulocyte-macrophage colony-stimulating factor, Activin A, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), nerve growth factor, brain-derived neurotrophic factor, or neurotrophic factor-3 / 4.

[0020] The fetal bovine serum volume fraction in the complete mesenchymal stem cell culture medium is 1%~20%;

[0021] The culture and expansion process involves seeding cells onto cell culture plates and culturing them at 21% O2, 5% CO2, and 37°C for 12–24 hours. The cell culture plates are 96-well, 48-well, 24-well, 12-well, or 6-well plates; the cell seeding density is 5 × 10⁶ cells / well. 3 ~1×10 5 cesulls / mL.

[0022] Furthermore, the induction culture medium in step (2) includes a complete mesenchymal stem cell culture medium and a combination of chemical small molecules, wherein the combination of chemical small molecules is one or more of histone deacetylase inhibitors (HDAC inhibitors), DNA methyltransferase inhibitors (DNMT inhibitors), GSK3 inhibitors, and histone methyltransferase inhibitors.

[0023] The final concentrations of the chemical small molecule combination in solution state are: HDAC inhibitor 0–1000 μM; DNMT inhibitor 0–20 μM; GSK3 inhibitor 0–50 μM; histone methyltransferase inhibitor 0–50 μM;

[0024] The induction culture time is 5 to 14 days.

[0025] Furthermore, the HDAC inhibitor is Voronostat, Entinostat, Panobinostat, Trichostatin A, Mocetinostat, Belinostat, Romidepsin, Dacinostat, CUDC-101, Quisinostat, Pracinostat, Droxinostat, Abexinostat, AR-42, Ricolinostat, Fimepinostat, RG2833, UF010, Sodium butyrate, Valproic Acid, or Curcumin, or isomers of the above reagents and / or their salts, hydrates, or precursors, or combinations of two or more of the above reagents;

[0026] The DNMT inhibitor is Decitabine, 5-Azacytidine, GSK-3484862, GSK-3685032, Bobcat339, DNMT-IN-5, USP7-IN-18, DNMT1-IN-5, RG108, SGI-1027, 6-Thioguanine, Hinokitiol, MY-1B, Zebularine, DY-46-2, DNMT3A-IN-1, CM-272, Levetiracetam, EGR-1-IN-1, METTL16-IN-1, Lomeguatrib, Guadecitabine sodium, 5-Methyl-2'-deoxycytidine, Psammaplin A or SW155246, or isomers of the above reagents and / or their salts, hydrates or precursors, or combinations of two or more of the above reagents;

[0027] The GSK3 inhibitor is CHIR-99021, SB216763, AT7519, CHIR-98014, TWS119, NP031112, Kenpaullone, SB415286, BIO, LY2090314, Alsterpaullone, KY19382, 1-Azakenpaullone, AZD1080, ARA014418, IM-12, WAY-119064, BRD0705, AZD2858, Lithium chloride hydrate, TDZD-8 or BRD3731, or isomers of the above reagents and / or their salts, hydrates or precursors, or combinations of two or more of the above reagents;

[0028] The histone methyltransferase inhibitors are GSK126, EPZ-6438 (Tazemetostat), EI1, GSK503, UNC1999, BIX-01294, UNC0638, A-366, DCG066, BRD4770, EPZ-5676 (Pinometostat), SGC0946, EPZ004777, and GSK3326595. (Zelenirstat), MS023, EPZ015666 (GSK3235025), C7280948, LLY-507, AZ505, BCI-121, MM-401, OICR-9429, Chaetocin, Sinefungin, AMI-1, Ryuvidine, UNC1215 or AZ191, or isomers of the above reagents and / or their salts, hydrates or precursors, or combinations of two or more of the above reagents.

[0029] Furthermore, the digestion and collection of induced cultured cells in step (3) includes: washing the cells twice with phosphate buffer after discarding the culture medium, and collecting the cells by trypsin digestion.

[0030] The application of the above-mentioned empowered mesenchymal stem cells in the preparation of a drug for treating myocardial infarction.

[0031] To prepare the above-mentioned eMSCs, the present invention provides the following technical solution:

[0032] The first aspect of the present invention is to provide a method for preparing eMSCs for the treatment of myocardial infarction, the method comprising the following steps:

[0033] (1) Expansion of mesenchymal stem cells: Mesenchymal stem cells were expanded by culturing them in complete mesenchymal stem cell culture medium.

[0034] (2) Mesenchymal stem cell seeding and plating: The cells obtained in step (1) are seeded into cell culture plates and cultured for 12-24 hours under the conditions of 21% O2, 5% CO2 and 37℃.

[0035] (3) Induction of eMSCs: Replace the complete mesenchymal stem cell culture medium in step (2) with induction culture medium;

[0036] (4) Harvest eMSCs: Digest and collect the cells from step (3) for subsequent experiments and tests.

[0037] The complete mesenchymal stem cell culture medium contains: liquid basal culture medium, fetal bovine serum, cell culture nutrient supplements, cytokines, and penicillin / streptomycin.

[0038] In the complete culture medium for mesenchymal stem cells, the volume percentage of fetal bovine serum is 1%-20%, preferably 5%-15%, and more preferably 10%.

[0039] In the complete mesenchymal stem cell culture medium, the basal culture medium is selected from: DMEM / F12 cell culture medium, low glucose DMEM cell culture medium, basal DMEM cell culture medium, high glucose DMEM cell culture medium, RPMI 1640 cell culture medium or F12 cell culture medium; preferably, high glucose DMEM cell culture medium.

[0040] Preferably, the complete culture medium for mesenchymal stem cells contains penicillin and streptomycin in a concentration range of 1-100,000 U / mL.

[0041] The induction medium comprises: liquid basal medium, fetal bovine serum, a combination of small chemical molecules (selected from one or more of HDAC inhibitors, DNMT inhibitors, GSK3 inhibitors, and histone methyltransferase inhibitors), cell culture nutrient supplements, and cytokines.

[0042] In the induction culture medium, the volume percentage of fetal bovine serum is 1% to 20%, preferably 5% to 15%.

[0043] In the induction medium, the basal medium is selected from one or more combinations of DMEM / F12 cell culture medium, low-glucose DMEM cell culture medium, basal DMEM cell culture medium, high-glucose DMEM cell culture medium, RPMI 1640 cell culture medium, or F12 cell culture medium. Preferably, the basal medium in the induction medium is DMEM-basic cell culture medium.

[0044] In the complete culture medium and induction culture medium, the cell culture nutrient additives are selected from one or more of the following components: non-essential amino acids, GlutaMAX™, nicotinamide, sodium bicarbonate, trisodium 2-phosphate-L-ascorbate, insulin, sodium pyruvate, glucose, HEPES, ITS, dexamethasone, penicillin, and streptomycin.

[0045] The non-essential amino acid cell culture nutrient supplements mentioned above are products from Sigma-Aldrich.

[0046] The GlutaMAX™ cell culture nutrient supplement mentioned above is a product of Thermo Fisher Scientific.

[0047] The HEPES cell culture nutrient supplement used is a product of Thermo Fisher Scientific.

[0048] The ITS cell culture nutrient supplements mentioned above are products of Thermo Fisher Scientific.

[0049] The insulin cell culture nutrient supplement mentioned above is domestically produced recombinant human insulin of cell culture grade.

[0050] Preferably, the induction culture medium contains 0.1-20% non-essential amino acids.

[0051] Preferably, the induction medium contains 0.1-20% Glutamax.

[0052] Preferably, the induction culture medium contains 0.1-100 mM nicotinamide.

[0053] Preferably, the induction culture medium contains 1-1000 μg / mL of sodium bicarbonate.

[0054] Preferably, the induction medium contains 0.1-100 μg / mL of 2-phosphate-L-ascorbic acid trisodium salt.

[0055] Preferably, the induction culture medium contains 0.1-100 μg / mL of insulin.

[0056] Preferably, the induction culture medium contains 0.1-100 mM sodium pyruvate.

[0057] Preferably, the induction medium contains 0.1-1000 μg / mL of glucose.

[0058] Preferably, the induction medium contains 0.1-1000 μg / mL of HEPES.

[0059] Preferably, the induction medium contains 0.1-20% ITS.

[0060] Preferably, the induction medium contains 0.1-1000 nM dexamethasone.

[0061] Preferably, the induction culture medium contains 1-100,000 U / mL of penicillin and streptomycin.

[0062] The cell growth factors in the complete culture medium and the induction culture medium are selected from one or more of the following: epidermal growth factor (EGF), basic fibroblast growth factor (b-FGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), bone morphogenetic protein 4 (BMP4), bone morphogenetic protein 2 (BMP2), transforming growth factor-β1 (TGF-β1), leukemia inhibitory factor (LIF), interleukin-2 (IL-2), interleukin-3, interleukin-4, interleukin-1β (IL-1β), interleukin-6, Flt-3 ligand, stem cell factor, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, thrombopoietin, granulocyte-macrophage colony-stimulating factor, Activin A, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), nerve growth factor, brain-derived neurotrophic factor, and neurotrophic factor-3 / 4.

[0063] Preferably, the induction medium contains 0.1-100 ng / mL of EGF.

[0064] Preferably, the induction medium contains 0.1-100 ng / mL of b-FGF.

[0065] Preferably, the induction medium contains 0.1-100 ng / mL of IGF.

[0066] Preferably, the induction culture medium contains 0.1-100 ng / mL of HGF.

[0067] Preferably, the induction medium contains 0.1-100 ng / mL of BMP4.

[0068] Preferably, the induction medium contains 0.1-100 ng / mL of TGF-β1.

[0069] Preferably, the induction medium contains 0.1-100 ng / mL of LIF.

[0070] Preferably, the induction medium contains 0.1-100 ng / mL of IL-2.

[0071] Preferably, the induction culture medium contains 0.1-100 ng / mL of TNF-α.

[0072] Preferably, the induction medium contains 0.1-100 ng / mL of IL-1β.

[0073] Preferably, the induction medium contains 0.1-100 ng / mL of IFN-γ.

[0074] In the induction medium, the combination of small chemical molecules is selected from one or more of HDAC inhibitors, DNMT inhibitors, GSK3 inhibitors, and histone methyltransferase inhibitors.

[0075] The HDAC inhibitors mentioned include, but are not limited to, histone deacetylase inhibitors or compounds of the same class that have the same function or induce the same target, such as Vorinostat, Entinostat, Panobinostat, Trichostatin A, Mocetinostat, Belinostat, Romidepsin, Dacinostat, CUDC-101, Quisinostat, Pracinostat, Droxinostat, Abexinostat, AR-42, Ricolinostat, Fimepinostat, RG2833, UF010, Sodium butyrate, Valproic Acid, and Curcumin, or equivalent pharmaceutical preparations, analogs, isomers and / or their salts, hydrates or precursors, or combinations thereof.

[0076] Preferably, the induction medium contains 0-20 μM of Trichostatin A.

[0077] Preferably, the induction medium contains 0-50 μM of Vorinostat.

[0078] Preferably, the induction medium contains 0-1000 μM of Valproic Acid.

[0079] Preferably, the induction medium contains 0-20 μM of Romidepsin.

[0080] Preferably, the induction medium contains 0-50 μM of sodium butyrate.

[0081] Preferably, the induction medium contains 0-1000 μM of Curcumin.

[0082] The DNMT inhibitors mentioned include, but are not limited to: Decitabine, 5-Azacytidine, GSK-3484862, GSK-3685032, Bobcat339, DNMT-IN-5, USP7-IN-18, DNMT1-IN-5, RG108, SGI-1027, 6-Thioguanine, Hinokitiol, MY-1B, Zebularine, DY-46-2, DNMT3A-IN-1, CM-272, Levetiracetam, EGR-1-IN-1, METTL16-IN-1, Lomeguatrib, Guadecitabine sodium, 5-Methyl-2'-deoxycytidine, and Psammaplin. Inhibitors or compounds of the same class of histone deacetylases, such as A and SW155246, that have the same function or induce the same target, or equivalent pharmaceutical products, analogs, isomers and / or their salts, hydrates or precursors, or combinations thereof; preferably, the DNMT inhibitor is selected from Decitabine, 5-Azacytidine, GSK-3484862 or RG108.

[0083] Preferably, the induction medium contains 0-10 μM Decitabinine.

[0084] Preferably, the induction medium contains 0-20 μM of 5-Azacytidine.

[0085] Preferably, the induction medium contains 0-15 μM of GSK-3484862.

[0086] Preferably, the induction medium contains 0-10 μM DNMT-IN-5.

[0087] Preferably, the induction culture medium contains 0-10 μM of RG108.

[0088] Preferably, the induction medium contains 0-20 μM of SGI-1027.

[0089] Preferably, the induction culture medium contains 0-15 μM of zebularine.

[0090] Preferably, the induction medium contains 0-10 μM of Levetiracetam.

[0091] The GSK3 inhibitors mentioned include, but are not limited to: CHIR-99021, SB216763, AT7519, CHIR-98014, TWS119, NP031112, Kenpaullone, SB415286, BIO, LY2090314, Alsterpaullone, KY19382, 1-Azakenpaullone, AZD1080, ARA014418, IM-12, WAY-119064, BRD0705, AZD2858, and Lithium chloride. Represented by hydrate, TDZD-8, BRD3731, etc., these are GSK3 pathway inhibitors or compounds with the same function or inducing the same target, or equivalent pharmaceutical products, analogs, isomers and / or their salts, hydrates or precursors, or combinations thereof; preferred are GSK3 inhibitors CHIR-99021, CHIR-98014, BRD3731 or TWS119.

[0092] Preferably, the induction medium contains 0-50 μM CHIR-99021.

[0093] Preferably, the induction medium contains 0-20 μM CHIR-98014.

[0094] Preferably, the induction culture medium contains 0-50 μM of TWS119.

[0095] Preferably, the induction medium contains 0-50 μM of BIO.

[0096] Preferably, the induction medium contains 0-20 μM of Alsterpaullone.

[0097] Preferably, the induction medium contains 0-50 μM of AZD1080.

[0098] The histone methyltransferase inhibitors mentioned include, but are not limited to: GSK126, EPZ-6438 (Tazemetostat), EI1, GSK503, UNC1999, BIX-01294, UNC0638, A-366, DCG066, BRD4770, EPZ-5676 (Pinometostat), SGC0946, EPZ004777, GSK3326595 (Zelenirstat), MS023, and EPZ015666. Represented by GSK3235025, C7280948, LLY-507, AZ505, BCI-121, MM-401, OICR-9429, Chaetocin, Sinefungin, AMI-1, Ryuvidine, UNC1215, AZ191, etc., these are monoamine oxidase inhibitors or compounds of the same class with the same function or inducing the same target, or equivalent pharmaceutical products, analogs, isomers and / or their salts, hydrates or precursors, or combinations thereof; preferably, histone methyltransferase inhibitors UNC1999, BIX-01294, UNC0638 or A-366.

[0099] Preferably, the induction medium contains 0-50 μM of UNC1999.

[0100] Preferably, the induction medium contains 0-20 μM of BIX-01294.

[0101] Preferably, the induction medium contains 0-50 μM of UNC0638.

[0102] Preferably, the induction culture medium contains 0-50 μM of A-366.

[0103] Preferably, the induction medium contains 0-20 μM of BRD4770.

[0104] Preferably, the induction medium contains 0-50 μM of EPZ004777.

[0105] In a second aspect, the induced MSCs are characterized in that, compared with mesenchymal stem cells, in addition to expressing mesenchymal stromal cell markers such as CD29, CD44, CD73, CD90, and CD105, they also highly express genes related to immune regulation, angiogenesis promotion, apoptosis inhibition, and energy metabolism improvement, such as WISP1, GPNMB, LRRC32, NEU1, CD109, PDGFD, IL18BP, PPARGC1B, UCN2, RPOS1, TDO2, PTGDS, LIPG, PIK3R3, TNC, CLU, NEU1, and CD276.

[0106] The eMSCs described above have stable proliferation capacity and can be stably passaged for 5-20 generations.

[0107] The eMSCs, as detected by transcriptome sequencing, do not express major histocompatibility complex II (MHC-II) antigen molecules (such as HLA-DRB1, HLA-DOA, HLA-DQA1, HLA-DRB5, HLA-DQB2, HLA-DQA2, HLA-DRA, HLA-DMB, HLA-DQB1, etc.) in vitro, and express HLA-DPB1, HLA-DPA1, HLA-DMA1, HLA-DOA, HLA-DOB, etc. at very low levels.

[0108] The eMSCs mentioned herein are administered via one or more of the following methods for the treatment of myocardial infarction: intravenous injection (e.g., tail vein injection), intraperitoneal injection, intramuscular injection, or subcutaneous injection.

[0109] A third aspect of the invention provides the application of eMSCs in the treatment of myocardial infarction.

[0110] The beneficial effects of this invention are as follows:

[0111] (1) Significantly improved safety: The reprogramming strategy induced by completely chemical small molecules avoids the use of viral vector transduction or gene editing technology, fundamentally eliminating potential risks such as insertion mutation, tumorigenicity and immunogenicity, and has higher clinical translational safety.

[0112] (2) Synergistic enhancement of therapeutic function: The obtained eMSCs can specifically and continuously express two key therapeutic factors, WISP1 and GPNMB. WISP1 strongly promotes angiogenesis and inhibits cardiomyocyte apoptosis, while GPNMB effectively inhibits inflammatory response and tissue fibrosis. The two have a synergistic effect, and compared with traditional MSCs, the therapeutic efficacy against the pathological links of myocardial infarction is significantly enhanced.

[0113] (3) The preparation process is standardized and controllable: The induction scheme with clear chemical composition and precise controllable concentration has been adopted, which has realized the standardization and reproducibility of the preparation process. It effectively solves the problems of large cell functional heterogeneity and unstable quality caused by donor differences and culture process of traditional MSCs, and is more in line with the requirements of industrial production.

[0114] (4) Broad prospects for clinical translation: This invention provides a novel treatment strategy based on non-genetically modified cells for the prevention and treatment of heart failure in the later stage of myocardial infarction, which has important clinical application value and market prospects. Attached Figure Description

[0115] Figure 1 This is the cell morphology of eMSCs induced by human umbilical cord mesenchymal stem cells (HUMSC) in this embodiment of the invention, with scarbar=100μm.

[0116] Figure 2 This is an embodiment of the invention, showing the differential gene expression characteristics of eMSCs compared to HUMSCs.

[0117] Figure 3 This is an example of an embodiment of the present invention, showing the echocardiogram of a mouse model of myocardial infarction treated with eMSCs 30 days later.

[0118] Figure 4 In this embodiment of the invention, after 30 days of treatment with eMSCs in a mouse model of myocardial infarction, cardiac function indicators were measured by ultrasound. ****, P<0.00001; ***, P<0.0001; *, P<0.05.

[0119] Figure 5 In this embodiment of the invention, after treating a mouse model of myocardial infarction with eMSCs for 30 days, Masson staining was used to detect myocardial fibrosis. Detailed Implementation

[0120] The present invention will now be described in detail with reference to the embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0121] Example 1: Preparation of eMSCs

[0122] Human umbilical cord mesenchymal stem cells (HUMSCs) were digested, resuspended, and then... 4The cells were seeded at a density of cells / mL in 24-well plates. Umbilical cord mesenchymal stem cells were cultured for 24 hours at 37°C and 5% CO2 using complete culture medium (containing 10% fetal bovine serum, DF12 cell culture medium, 1% non-essential amino acids, 10 ng / mL bFGF, 10 ng / mL EGF, 10000 U / mL penicillin and streptomycin).

[0123] After 24 hours, the medium was replaced with induction medium consisting of: DF12 cell culture medium, 10% fetal bovine serum, 1% non-essential amino acids, 10 mM nicotinamide, 600 μg / mL sodium bicarbonate, 20 μg / mL trisodium 2-phosphate-L-ascorbate, 1% ITS, 1% GlutaMAX™, 10000 U / mL penicillin and streptomycin, 15 ng / mL EGF, 15 ng / mL bFGF, 500 mM MVPA, 0.5 μM 5-Azacytidine, 2 μM CHIR-98014, and 2 μM UNC0638. Cells were cultured in the induction medium for 10 days, after which they transformed into epithelial-like cell morphologies. Figure 1 ), thus obtaining eMSCs.

[0124] Example 2: Transcriptome sequencing to detect gene expression characteristics of eMSCs

[0125] Total RNA was collected from EMSCs induced on day 10 and from uninduced control cells using Trizol reagent (Invitrogen, USA). The specific procedure was as follows: RNA extraction and purification were performed according to the Trizol instructions, including cell lysis, chloroform phase separation, isopropanol precipitation, and washing with 75% ethanol, followed by dissolution in RNase-free water. RNA concentration and purity (A260 / A280 ≥ 1.8, A260 / A230 ≥ 2.0) were assessed using a NanoDrop ND-1000 spectrophotometer, and RNA integrity (RIN ≥ 7.0) was confirmed using an Agilent 2100 Bioanalyzer (RNA Nano 6000 kit). Residual genomic DNA was digested with DNase I, and RNA was purified using a kit (such as RNeasy MinElute) and its concentration was retested. Total RNA (≥1 μg) was used to construct libraries using a strand-specific library construction kit (e.g., NEBNext Ultra II Directional). After quality control using Agilent Bioanalyzer (fragment distribution) and Qubit (concentration), PE150 paired-end sequencing was performed on an Illumina NovaSeq 6000 platform, with ≥3 biological replicates per group. After quality control, alignment (HISAT2 / STAR), and quantification (featureCounts), differential expression analysis (DESeq2 / edgeR, |log2FC|>1 & FDR<0.05), functional enrichment analysis (GO / KEGG, clusterProfiler), and PCA were performed. Gene expression characterization analysis confirmed that, in addition to expressing mesenchymal stromal cell markers such as CD29, CD44, CD73, CD90, and CD105, it also highly expresses genes related to immune regulation, promoting angiogenesis, inhibiting apoptosis, and improving energy metabolism, such as WISP1, GPNMB, LRRC32, NEU1, CD109, PDGFD, IL18BP, PPARGC1B, UCN2, RPOS1, TDO2, PTGDS, LIPG, PIK3R3, TNC, CLU, NEU1, and CD276, which together constitute its unique molecular marker. Figure 2 ).

[0126] Example 3: eMSCs treatment of a mouse model of myocardial infarction

[0127] To evaluate the therapeutic effect of eMSCs on myocardial infarction, we established an immunodeficient mouse model of myocardial infarction induced by left anterior descending artery ligation. Twenty-four hours after modeling, mice were randomly divided into four groups: sham operation group (Sham), myocardial infarction model group (MI), myocardial infarction + HUMSC transplantation group (MI + HUMSC, as a negative control), and myocardial infarction + eMSCs transplantation group (MI + eMSCs). Mice were injected via tail vein with an equal volume of PBS and 1×10⁻⁶ eMSCs, respectively. 6 One HUMSC or 1×10 6 Four weeks post-transplantation, cardiac function was assessed by echocardiography, and heart tissue was analyzed using Masson staining and immunofluorescence staining. Results showed that compared to the MI + HUMSC group, the MI + eMSCs group exhibited significantly increased left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), and significantly decreased left ventricular end-systolic diameter (LVESD) and end-diastolic diameter (LVEDD). Histological analysis further indicated a significant reduction in myocardial infarction area and collagen volume fraction in the infarcted region in the MI + eMSCs group. These results demonstrate that, compared to unreprogrammed HUMSCs, eMSC transplantation can significantly alleviate post-myocardial infarction pathological remodeling by improving cardiac function and inhibiting fibrosis.

[0128] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the technical scope of the present invention.

Claims

1. A method for empowering mesenchymal stem cells, characterized in that: The empowered mesenchymal stem cells are prepared by inducing and culturing mesenchymal stem cells, and the empowered mesenchymal stem cells highly express genes related to immune regulation, promoting angiogenesis, inhibiting cell apoptosis and improving energy metabolism.

2. The empowered mesenchymal stem cells according to claim 1, characterized in that: The mesenchymal stem cells were obtained by isolating from bone marrow, adipose tissue, umbilical cord, placenta, amniotic fluid, dental pulp, periodontal ligament, synovium, muscle, dermis, peripheral blood, liver, and lungs.

3. The empowered mesenchymal stem cells according to claim 1, characterized in that: The genes related to the immune regulation, angiogenesis promotion, apoptosis inhibition, and energy metabolism improvement of mesenchymal stem cells are WISP1, GPNMB, LRRC32, NEU1, CD109, PDGFD, IL18BP, PPARGC1B, UCN2, RPOS1, TDO2, PTGDS, LIPG, PIK3R3, TNC, CLU, NEU1, and CD276.

4. The empowered mesenchymal stem cells according to claim 1, characterized in that: The empowered mesenchymal stem cells express mesenchymal stromal cell markers CD29, CD44, CD73, CD90, and CD105, but do not express stem cell-specific marker genes, hematopoietic markers, or major histocompatibility complex II (MHC-II) antigen molecules, and express HLA-DPB1, HLA-DPA1, HLA-DMA1, HLA-DOA, and HLA-DOB at very low levels. The stem cell-specific marker genes are NANOG, POU5F1, KLF4, C-MYC, and LIN2; the hematopoietic markers are CD11b, CD34, CD45, and CD14; and the major histocompatibility complex-II (MHC-II) antigen molecules are HLA-DRB1, HLA-DOA, HLA-DQA1, HLA-DRB5, HLA-DQB2, HLA-DQA2, HLA-DRA, HLA-DMB, and HLA-DQB1.

5. A method for preparing empowered mesenchymal stem cells according to any one of claims 1 to 4, characterized in that: Includes the following steps: (1) Mesenchymal stem cells were cultured and expanded using complete mesenchymal stem cell culture medium; (2) The expanded mesenchymal stem cells were induced and cultured using an induction medium: (3) Digest and collect the induced cultured cells.

6. The preparation method according to claim 5, characterized in that: The complete culture medium for mesenchymal stem cells in step (1) includes liquid basal culture medium, fetal bovine serum, cytokines, cell culture additives, and penicillin / streptomycin; The liquid basal culture medium is one or a mixture of two or more of the following: DMEM / F12 cell culture medium, DMEM-low glucose cell culture medium, DMEM-basic cell culture medium, DMEM-high glucose cell culture medium, 1640RPMI cell culture medium, or F12 cell culture medium. The cytokines are one or a mixture of two or more of the following: epidermal growth factor (EGF), basic fibroblast growth factor (b-FGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), bone morphogenetic protein 4 (BMP4), bone morphogenetic protein 2 (BMP2), transforming growth factor-β1 (TGF-β1), leukemia inhibitory factor (LIF), interleukin-2 (IL-2), interleukin-3, interleukin-4, interleukin-1β (IL-1β), interleukin-6, Flt-3 ligand, stem cell factor, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, thrombopoietin, granulocyte-macrophage colony-stimulating factor, Activin A, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), nerve growth factor, brain-derived neurotrophic factor, or neurotrophic factor-3 / 4. The fetal bovine serum volume fraction in the complete mesenchymal stem cell culture medium is 1%~20%; The culture and expansion process involves seeding cells onto cell culture plates and culturing them at 21% O2, 5% CO2, and 37°C for 12–24 hours. The cell culture plates are 96-well, 48-well, 24-well, 12-well, or 6-well plates; the cell seeding density is 5 × 10⁶ cells / well. 3 ~1×10 5 cesulls / mL.

7. The preparation method according to claim 5, characterized in that: The induction culture medium in step (2) includes complete mesenchymal stem cell culture medium and a combination of small chemical molecules, wherein the combination of small chemical molecules is one or more of histone deacetylase inhibitors (HDAC inhibitors), DNA methyltransferase inhibitors (DNMT inhibitors), GSK3 inhibitors, and histone methyltransferase inhibitors. The final concentrations of the chemical small molecule combination in solution state are: HDAC inhibitor 0–1000 μM; DNMT inhibitor 0–20 μM; GSK3 inhibitor 0–50 μM; histone methyltransferase inhibitor 0–50 μM; The induction culture time is 5 to 14 days.

8. The preparation method according to claim 7, characterized in that: The HDAC inhibitor is Voronostat, Entinostat, Panobinostat, Trichostatin A, Mocetinostat, Belinostat, Romidepsin, Dacinostat, CUDC-101, Quisinostat, Pracinostat, Droxinostat, Abexinostat, AR-42, Ricolinostat, Fimepinostat, RG2833, UF010, Sodium butyrate, Valproic Acid, or Curcumin, or isomers of the above reagents and / or their salts, hydrates, or precursors, or a combination of two or more of the above reagents; The DNMT inhibitor is Decitabine, 5-Azacytidine, GSK-3484862, GSK-3685032, Bobcat339, DNMT-IN-5, USP7-IN-18, DNMT1-IN-5, RG108, SGI-1027, 6-Thioguanine, Hinokitiol, MY-1B, Zebularine, DY-46-2, DNMT3A-IN-1, CM-272, Levetiracetam, EGR-1-IN-1, METTL16-IN-1, Lomeguatrib, Guadecitabine sodium, 5-Methyl-2'-deoxycytidine, Psammaplin A or SW155246, or isomers of the above reagents and / or their salts, hydrates or precursors, or combinations of two or more of the above reagents; The GSK3 inhibitor is CHIR-99021, SB216763, AT7519, CHIR-98014, TWS119, NP031112, Kenpaullone, SB415286, BIO, LY2090314, Alsterpaullone, KY19382, 1-Azakenpaullone, AZD1080, ARA014418, IM-12, WAY-119064, BRD0705, AZD2858, Lithium chloride hydrate, TDZD-8 or BRD3731, or isomers of the above reagents and / or their salts, hydrates or precursors, or combinations of two or more of the above reagents; The histone methyltransferase inhibitors are GSK126, EPZ-6438 (Tazemetostat), EI1, GSK503, UNC1999, BIX-01294, UNC0638, A-366, DCG066, BRD4770, EPZ-5676 (Pinometostat), SGC0946, EPZ004777, GSK3326595 (Zelenirstat), MS023, and EPZ015666. (GSK3235025), C7280948, LLY-507, AZ505, BCI-121, MM-401, OICR-9429, Chaetocin, Sinefungin, AMI-1, Ryuvidine, UNC1215 or AZ191, or isomers of the above reagents and / or their salts, hydrates or precursors, or combinations of two or more of the above reagents.

9. The preparation method according to claim 5, characterized in that: The digestion and collection of induced cultured cells in step (3) includes: washing the cells twice with phosphate buffer after discarding the culture medium, and collecting the cells by trypsin digestion.

10. The use of empowered mesenchymal stem cells according to any one of claims 1 to 4 in the preparation of a medicament for treating myocardial infarction, wherein the cells used in the application include live cells of empowered mesenchymal stem cells, extracellular vesicles, cell culture supernatant, or products derived from the cells.

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