Chemical reprogramming multifunctional mesenchymal stromal cell based on glycyrrhizic acid solubilizing technology and application of chemical reprogramming multifunctional mesenchymal stromal cell

Abstract

The invention discloses a chemical reprogramming multifunctional mesenchymal stromal cell based on a glycyrrhizic acid solubilizing technology and application thereof. According to the invention, a specific chemical small molecule combination is adopted, and human fibroblasts are reprogrammed into multifunctional mesenchymal stromal cells (CiMFSCs) with high expression of NSMF / NAV2 / BMP4 / LAMA2 and other genes. The key advantage of the CiMFSCs is that the CiMFSCs can target a plurality of key links of cerebral arterial thrombosis treatment at the same time, including promotion of neurogenesis, angiogenesis, repair of blood brain barrier (BBB) and exertion of paracrine function, so that effective neural function recovery is finally realized. Compared with umbilical cord mesenchymal stem cells, neural stem cells differentiated by iPSC, vascular endothelial cells differentiated by iPSC and pericytes differentiated by iPSC, the CiMFSCs show a remarkable and superior treatment effect in a stroke model mouse: the CiMFSCs can remarkably reduce the cerebral infarction volume, improve the survival rate of the model mouse, improve the neurobehavioral score and realize comprehensive repair of nerve and blood vessel units.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary fields of cell biology, regenerative medicine and medicinal chemistry, and specifically relates to a chemical reprogramming method based on glycyrrhizic acid solubilization technology for preparing multifunctional mesenchymal stromal cells (CiMFSCs), and its application in the preparation of cell drugs for treating neurological diseases such as ischemic stroke. Background Technology

[0002] Stroke is one of the most serious diseases threatening human health worldwide, characterized by "five highs": high incidence, high recurrence, high disability, high mortality, and high economic burden. A major report titled "Practical Solutions to Reduce the Global Burden of Stroke," published jointly by *The Lancet Neurology* and the World Stroke Organization, points out that without urgent measures, the number of people dying from stroke globally is projected to increase by 50% to 9.7 million annually by 2050, resulting in economic losses of up to $2.3 trillion. my country currently has 70 million stroke patients, with 2 million new cases each year, indicating an urgent social need and a huge market potential. However, current stroke treatment faces a dual dilemma: while ischemic stroke can achieve vascular recanalization through thrombolysis (such as recombinant tissue plasminogen activator rt-PA) and mechanical thrombectomy, only about 10% of patients benefit due to the limited 4.5-hour golden time window and the risk of reperfusion injury, and the existing neurovascular damage cannot be reversed; while hemorrhagic stroke can reduce acute-phase mortality through surgical removal of hematoma, there is a lack of effective means to promote neurological function reconstruction. Both types of stroke share common pathological changes such as neurovascular unit collapse, blood-brain barrier (BBB) ​​disruption, and secondary inflammatory cascades. Traditional drugs and surgery have limited ability to repair these changes, and there is currently no particularly effective treatment strategy.

[0003] Recent advancements in cell therapy offer promising alternatives for stroke treatment. Compared to traditional therapies, they offer a wider therapeutic window, reducing stroke damage and promoting functional recovery to varying degrees (Lee et al., 2022; Li et al., 2020). Among these, neural stem / progenitor cells have demonstrated significant therapeutic potential in neuroprotection and regeneration (Jiang et al., 2019; Kim et al., 2015), while endothelial cells and parietal cells have been shown to enhance angiogenesis and promote blood-brain barrier (BBB) ​​repair (Crouch et al., 2022). Furthermore, mesenchymal stem cells (MSCs) derived from various sources, including bone marrow, umbilical cord, adipose tissue, olfactory mucosa, and dental tissue, have been shown to have therapeutic value for a variety of central nervous system (CNS) diseases due to their anti-inflammatory properties and potential to differentiate into neurons or vascular cells under suitable conditions (Gao et al., 2023; Rozenberg et al., 2016; Zhang et al., 2021). However, current cell therapy applications for stroke repair still face many limitations: a shortage of neural stem / progenitor cells; limited role of vascular endothelial cells in nerve repair; and controversy surrounding the in vivo implantation of MSCs and their transdifferentiation efficiency into functional neural / vascular cells (Ikegame et al., 2011; Ming et al., 2024; Wang et al., 2016). More importantly, the pathological mechanisms of stroke are highly complex, involving extensive cell death, demyelination, hypoperfusion, and inflammatory activation and infiltration (Li et al., 2014; Raffaele & Fumagalli, 2022). Single-function cells are insufficient to meet the diverse repair needs. To address this complexity, researchers have proposed a combined cell strategy. For example, co-transplantation of endothelial cells (ECs) or vascular progenitor cells with neural stem / progenitor cells may more effectively promote functional recovery after ischemic injury (Li et al., 2014; Nakagomi et al., 2009). Therefore, developing novel multifunctional cells capable of coping with the complex pathological environment of stroke is crucial for overcoming current therapeutic bottlenecks. Summary of the Invention

[0004] Based on recent advances in cell reprogramming, neural stem / progenitor cells or vascular cells have been successfully generated through direct reprogramming or reprogramming via induced pluripotent stem cells (iPSCs) in an intermediate state, opening new avenues for stroke treatment (Kim et al., 2015; Kokaia et al., 2017; Sun et al., 2020; Xu et al., 2022). However, the efficacy of these methods remains limited by the traditional model—reprogramming a single cell type into another single cell type with a specific function.

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for reprogramming human fibroblasts into CiMFSCs using a specific combination of small chemical molecules.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a chemically reprogrammed multifunctional mesenchymal matrix cell based on glycyrrhizic acid solubilization technology, wherein the multifunctional mesenchymal matrix cell is prepared by inducing and culturing human fibroblasts using glycyrrhizic acid solubilization technology.

[0007] Furthermore, the glycyrrhizic acid solubilization technique uses glycyrrhizic acid as a solubilizer and functional component in the induction culture medium to reprogram human fibroblasts.

[0008] Furthermore, human fibroblasts are found in skin tissue, lungs, heart, bone marrow, skin, lungs, and heart tissue from aborted fetuses, as well as human fibroblast cell lines.

[0009] A method for preparing chemically reprogrammed multifunctional mesenchymal matrix cells based on glycyrrhizic acid solubilization technology includes the following steps:

[0010] (1) Human fibroblasts were cultured and expanded using complete fibroblast culture medium;

[0011] (2) Fibroblasts were induced to generate multifunctional mesenchymal stromal cells. The induction process was divided into three stages:

[0012] First stage: Use the first induction medium and culture for 5-15 days;

[0013] Second stage: Use the second induction medium and culture for 3-10 days;

[0014] Third stage: Use the third induction medium and culture for 3-7 days;

[0015] (3) Digest and collect the induced cultured cells.

[0016] Furthermore, the complete fibroblast culture medium in step (1) includes liquid basal medium, fetal bovine serum, and penicillin / streptomycin;

[0017] The liquid basal culture medium is one or a combination of two or more of the following: 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.

[0018] 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.

[0019] Furthermore, the first induction medium in step (2) includes a complete fibroblast culture medium, as well as a first combination of small chemical molecules, cell culture nutrient additives, and cytokines;

[0020] The second induction medium includes a complete fibroblast culture medium, a second combination of small chemical molecules, cell culture nutrient supplements, and cytokines;

[0021] The third induction medium includes a complete fibroblast culture medium, a third chemical small molecule combination, cell culture nutrient supplements, and cytokines.

[0022] Furthermore, the first chemical small molecule combination includes one or more of DNA methyltransferase inhibitors, GSK3 inhibitors, or histone deacetylase inhibitors.

[0023] The second chemical small molecule combination includes one or more of the following: TGF-β receptor inhibitors, JNK inhibitors, OCT4 activators, GSK3 inhibitors, KDM1 / LSD1 inhibitors, or histone deacetylase inhibitors.

[0024] The third chemical small molecule combination includes one or more of the following: GSK3 inhibitors, KDM1 / LSD1 inhibitors, or histone deacetylase inhibitors;

[0025] The final concentrations of each component in the first chemical small molecule combination are: DNA methyltransferase inhibitor: 0–50 μM; GSK3 inhibitor: 0–50 μM; histone deacetylase inhibitor: 0–1000 μM;

[0026] The final concentrations of each component in the second chemical small molecule combination are as follows: TGF-β receptor inhibitor: 0–30 μM; JNK inhibitor: 0–20 μM; OCT4 activator: 0–50 μM; GSK3 inhibitor: 0–50 μM; KDM1 / LSD1 inhibitor: 0–20 μM; histone deacetylase inhibitor: 0–1000 μM;

[0027] The final concentrations of each component in the third chemical small molecule combination are as follows: GSK3 inhibitor: 0–50 μM; KDM1 / LSD1 inhibitor: 0–20 μM; histone deacetylase inhibitor: 0–1000 μM;

[0028] The cell culture nutrient additives are one or a mixture of two or more of the following: non-essential amino acids, GlutaMAX™, nicotinamide, sodium bicarbonate, trisodium 2-phosphate-L-ascorbate, insulin, sodium pyruvate, glucose, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, insulin-transferrin-selenium medium additive, dexamethasone, penicillin, or streptomycin.

[0029] The cytokines are selected from one or more of the following: epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor, hepatocyte growth factor, bone morphogenetic protein 4, transforming growth factor-β1, leukemia inhibitory factor, interleukin-2, interleukin-3, interleukin-4, interleukin-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, interferon-γ, nerve growth factor, brain-derived neurotrophic factor, and neurotrophic factor-3 / 4.

[0030] Furthermore, TGF-β receptor inhibitors include SB431542, LDN193189, LY2157299, LY2109761, SB525334, SB505124, GW788388, LY364974, and E-616452. (RepSox), K02288, A83-01, R26872, BIBF-0775, LDN214117, SD-208, TEW-7197, ML347, LDN212854, DMH1, Dorsomorphin, S-7701, SIS3, Galunisertib, curcumin, quercetin, tanshinone IIA, baicalein, luteolin, caffeic acid, resveratrol, artemisinin or tripterygium indicum, or isomers, salts, hydrates, precursors or combinations thereof of the above preparations;

[0031] The OCT4 activator is OAC1, OAC2, O4I1, O4I2 or Oct3 / 4-inducer-1, or isomers, salts, hydrates, precursors or combinations thereof of the above preparations;

[0032] The GSK3 inhibitor is CHIR-99021, SB216763, AT7519, CHIR-98014, TWS119, NP031112, SB415286, BIO, LY2090314, Alsterpaullone, KY19382, 1-Azakenpaullone, AZD1080, ARA014418, IM-12, WAY-119064, BRD0705, AZD2858, LiCl, berberine, quercetin, epigallocatechin gallate, luteolin, resveratrol, curcumin, or apigenin, or isomers, salts, hydrates, precursors, or combinations thereof of the above preparations;

[0033] The histone deacetylase inhibitors are Voronostat (SAHA), Entinostat (MS-275), Panobinostat (LBH589), Trichostatin A (TSA), Mocetinostat (MGCD0103), Belinostat (PXD101), Romidepsin (FK228), Dacinostat (LAQ824), CUDC-101, Quisinostat (JNJ-26481585), Pracinostat (SB939), Droxinostat, Abexinostat (PCI-24781), AR-42, Ricolinostat (ACY-1215), Fimepinostat (CUDC-907), RG2833, UF010, Sodium butyrate, Valproic Acid, or Curcumin, or isomers, salts, hydrates, precursors, or combinations thereof of the above formulations;

[0034] The KDM1 / LSD1 inhibitor is SP2509, GSK2879552, GSK-LSD1, Tranylcypromine, GSK-J4, CPI-455, IOX1, VML324, Phenelzine sulfate, α-Hydroxyglutaric acid, SKLB325, or isomers, salts, hydrates, precursors, or combinations thereof of the above preparations;

[0035] The DNA methyltransferase inhibitors mentioned above are 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, salts, hydrates, precursors or combinations thereof of the above preparations;

[0036] The JNK inhibitor is SP600125, JNK-IN-8, GDC-0134, Astragaloside IV, Gamma-Linolenic acid, Berberine sulfate, Tomatidine, JNK Inhibitor VIII, Isovitexin, Tanzisertib, JNK-IN-7, Angelicin, Bicyclol, Sinapaldehyde, Isoscopoletin, Halicin, D-JNKI-1, Vaccarin, BI-78D3, SR-3306, J30-8, Decrusinol, or Artemisic acid, or isomers, salts, hydrates, precursors, or combinations thereof of the above preparations.

[0037] The application of the above-mentioned chemically reprogrammed multifunctional mesenchymal stromal cells based on glycyrrhizic acid solubilization technology in the preparation of drugs for treating ischemic stroke or hemorrhagic stroke.

[0038] Furthermore, the cells used in the application include live cells, extracellular vesicles, cell culture supernatants, or products derived from chemically reprogrammed multifunctional mesenchymal matrix cells.

[0039] This invention employs a specific combination of small chemical molecules to reprogram human fibroblasts into a novel, highly plastic, chemically reprogrammed, multifunctional mesenchymal stromal cell (CiMFSC) type. The key advantage of CiMFSCs lies in their ability to simultaneously target multiple critical aspects of ischemic stroke treatment, including promoting neurogenesis, angiogenesis, repairing the blood-brain barrier (BBB), and exerting paracrine functions, ultimately achieving effective neurological function recovery. Compared to umbilical cord mesenchymal stem cells, iPSC-differentiated neural stem cells, iPSC-differentiated vascular endothelial cells, and iPSC-differentiated pericytes, CiMFSCs exhibit significantly superior therapeutic effects in stroke model mice: significantly reducing infarct volume, improving survival rates, enhancing neurological behavioral scores, and achieving comprehensive repair of neurovascular units.

[0040] The first aspect of the present invention is to provide a method for preparing CiMFSCs for the treatment of stroke, the method comprising the following steps:

[0041] S1. Isolation and culture of human fibroblasts: Human fibroblasts are isolated from tissues and cultured and expanded to obtain a sufficient number of cells as starting cells for reprogramming.

[0042] S2. Seeding and plating of human fibroblasts: The fibroblasts obtained in S1 were seeded into the wells of a culture plate and cultured for 12-24 hours in complete fibroblast culture medium (liquid basal medium + fetal bovine serum + penicillin / streptomycin) at 21% O2, 5% CO2, and 37°C.

[0043] S3. Reprogramming Induction of Human Fibroblasts: Reprogramming induction of human fibroblasts: Replace the cell culture medium in S2 with a first induction medium; the first induction medium is a complete fibroblast culture medium supplemented with a first chemical small molecule combination, cell culture nutrient additives, and cytokines. The first chemical small molecule combination includes one or more of DNA methyltransferase inhibitors, GSK3 inhibitors, or histone deacetylase inhibitors. The culture time in the first induction medium is 5-15 days, more preferably 7-10 days.

[0044] Next, the first induction medium in the culture system is replaced with the second induction medium. The second induction medium is a complete fibroblast culture medium supplemented with a second small chemical molecule combination, cell culture nutrients, and cytokines. The second small chemical molecule combination includes one or more of the following: TGF-β receptor inhibitors, JNK inhibitors, OCT4 activators, GSK3 inhibitors, KDM1 / LSD1 inhibitors, or histone deacetylase inhibitors. The culture time in the second induction medium is 3-10 days, preferably 5-8 days. The third induction medium is a complete fibroblast culture medium supplemented with a third small chemical molecule combination, cell culture nutrients, and cytokines. The third small chemical molecule combination includes one or more of GSK3 inhibitors, KDM1 / LSD1 inhibitors, or histone deacetylase inhibitors. The culture time in the third induction medium is 3-7 days, preferably 4-6 days.

[0045] S4. Harvesting Culture: After culturing in the third induction medium for 3-7 days, discard the medium, wash the cells twice with phosphate buffer, digest with trypsin to harvest the cells for subsequent detection and application.

[0046] In the complete fibroblast culture medium, the volume percentage of fetal bovine serum is 1% to 20%, preferably 5% to 15%.

[0047] In the complete fibroblast 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.

[0048] Preferably, the complete fibroblast culture medium contains penicillin and streptomycin in a concentration range of 1-100,000 U / mL.

[0049] In the induction culture medium, the volume percentage of fetal bovine serum is 1% to 20%, preferably 5% to 15%.

[0050] 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.

[0051] In the induction 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.

[0052] The non-essential amino acid cell culture nutrient supplements mentioned above are products from Sigma-Aldrich.

[0053] The GlutaMAX™ cell culture nutrient supplement mentioned above is a product of Thermo Fisher Scientific.

[0054] The HEPES cell culture nutrient supplement used is a product of Thermo Fisher Scientific.

[0055] The ITS cell culture nutrient supplements used are products of Thermo Fisher Scientific.

[0056] The insulin cell culture nutrient supplement mentioned above is domestically produced recombinant human insulin of cell culture grade.

[0057] Preferably, the induction culture medium contains 0.1-20% non-essential amino acids.

[0058] Preferably, the induction medium contains 0.1-20% Glutamax.

[0059] Preferably, the induction culture medium contains 0.1-100 mM nicotinamide.

[0060] Preferably, the induction culture medium contains 1-1000 μg / mL of sodium bicarbonate.

[0061] Preferably, the induction medium contains 0.1-100 μg / mL of 2-phosphate-L-ascorbic acid trisodium salt.

[0062] Preferably, the induction culture medium contains 0.1-100 μg / mL of insulin.

[0063] Preferably, the induction culture medium contains 0.1-100 mM sodium pyruvate.

[0064] Preferably, the induction medium contains 0.1-1000 μg / mL of glucose.

[0065] Preferably, the induction medium contains 0.1-1000 μg / mL of HEPES.

[0066] Preferably, the induction medium contains 0.1-20% ITS.

[0067] Preferably, the induction medium contains 0.1-1000 nM dexamethasone.

[0068] Preferably, the induction culture medium contains 1-100,000 U / mL of penicillin and streptomycin.

[0069] The cell growth factors in the induction culture medium are selected from one or more of the following: epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), bone morphogenetic protein 4 (BMP4), transforming growth factor-β1 (TGF-β1), leukemia inhibitory factor (LIF), interleukin-2, interleukin-3, interleukin-4, interleukin-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, interferon-γ, nerve growth factor, brain-derived neurotrophic factor, and neurotrophic factor-3 / 4.

[0070] Preferably, the content of the above-mentioned cytokines in the induction culture medium is 0.1-100 ng / mL.

[0071] The small molecule composition in the first induction medium includes one or a mixture of two or more of DNA methyltransferase inhibitors, GSK3 inhibitors, or histone deacetylase inhibitors.

[0072] The DNA methyltransferase inhibitor is selected from one or more of Decitabine, 5-Azacytidine, GSK-3484862, GSK-3685032, Bobcat339, or SW155246.

[0073] Preferably, the induction medium contains 0-50 μM of Decitabine.

[0074] Preferably, the induction medium contains 0-20 μM of 5-Azacytidine.

[0075] Preferably, the induction medium contains 0-20 μM of GSK-3484862.

[0076] Preferably, the induction culture medium contains 0-30 μM of GSK-3685032.

[0077] Preferably, the induction medium contains 0-10 μM of Bobcat339.

[0078] Preferably, the induction medium contains 0-50 μM of SW155246.

[0079] The GSK3 inhibitor is selected from one or more of CHIR-99021, CHIR-98014, TWS119, etc.

[0080] Preferably, the induction medium contains 0-50 μM CHIR-99021.

[0081] Preferably, the induction medium contains 0-20 μM CHIR-98014.

[0082] Preferably, the induction culture medium contains 0-50 μM of TWS119.

[0083] The histone deacetylase inhibitor is selected from one or more of Trichostatin A, Voronostat, Valproic Acid, etc.

[0084] Preferably, the induction medium contains 0-20 μM of Trichostatin A.

[0085] Preferably, the induction medium contains 0-50 μM of Vorinostat.

[0086] Preferably, the induction medium contains 0-1000 μM of Valproic Acid.

[0087] The small molecule composition in the second induction medium includes one or a mixture of two or more of the following: GF-β receptor inhibitor, JNK inhibitor, OCT4 activator, GSK3 inhibitor, KDM1 / LSD1 inhibitor, or histone deacetylase inhibitor.

[0088] The TGF-β receptor inhibitor is selected from one or more of SB431542, E-616452, A83-01, LDN193189, etc.

[0089] Preferably, the induction medium contains 0-50 μM of SB431542.

[0090] Preferably, the induction culture medium contains 0-50 μM of E-616452.

[0091] Preferably, the induction culture medium contains 0-10 μM of A83-01.

[0092] Preferably, the induction culture medium contains 0-20 μM of LY193189.

[0093] The JNK inhibitor is selected from one or more of SP600125, JNK-IN-8, GDC-0134, and Astragaloside IV.

[0094] Preferably, the induction medium contains 0-20 μM of SP600125.

[0095] Preferably, the induction medium contains 0-30 μM of JNK-IN-8.

[0096] Preferably, the induction medium contains 0-10 μM of GDC-0134.

[0097] Preferably, the induction medium contains 0-20 μM of Astragaloside IV.

[0098] The OCT4 activator is selected from one or more of OAC1, OAC2, O4I1, O4I2, or Oct3 / 4-inducer-1.

[0099] Preferably, the induction culture medium contains 0-50 μM of OAC1.

[0100] Preferably, the induction culture medium contains 0-50 μM of OAC2.

[0101] Preferably, the induction culture medium contains 0-20 μM of O4I1.

[0102] Preferably, the induction culture medium contains 0-20 μM of O4I2.

[0103] Preferably, the induction medium contains 0-20 μM of Oct3 / 4-inducer-1.

[0104] The GSK3 inhibitor is selected from one or more of CHIR-99021, CHIR-98014, TWS119, etc.

[0105] Preferably, the induction medium contains 0-50 μM CHIR-99021.

[0106] Preferably, the induction medium contains 0-20 μM CHIR-98014.

[0107] Preferably, the induction culture medium contains 0-50 μM of TWS119.

[0108] The KDM1 / LSD1 inhibitor is selected from one or more of SP2509, GSK2879552, GSK-LSD1, Tranylcypromine, etc.

[0109] Preferably, the induction medium contains 0-50 μM of SP2509.

[0110] Preferably, the induction medium contains 0-20 μM of GSK2879552.

[0111] Preferably, the induction medium contains 0-50 μM of GSK-LSD1.

[0112] Preferably, the induction medium contains 0.20 μM of tranylcypromine.

[0113] The histone deacetylase inhibitor is selected from one or more of Trichostatin A, Voronostat, Valproic Acid, etc.

[0114] Preferably, the induction medium contains 0-20 μM of Trichostatin A.

[0115] Preferably, the induction medium contains 0-50 μM of Vorinostat.

[0116] Preferably, the induction medium contains 0-1000 μM of Valproic Acid.

[0117] The small molecule composition in the third induction medium includes one or a mixture of two or more of the following: GSK3 inhibitor, KDM1 / LSD1 inhibitor, or histone deacetylase inhibitor.

[0118] The GSK3 inhibitor is selected from one or more of CHIR-99021, CHIR-98014, TWS119, etc.

[0119] Preferably, the induction medium contains 0-50 μM CHIR-99021.

[0120] Preferably, the induction medium contains 0-20 μM CHIR-98014.

[0121] Preferably, the induction culture medium contains 0-50 μM of TWS119.

[0122] The KDM1 / LSD1 inhibitor is selected from one or more of SP2509, GSK2879552, GSK-LSD1, Tranylcypromine, etc.

[0123] Preferably, the induction medium contains 0-50 μM of SP2509.

[0124] Preferably, the induction medium contains 0-20 μM of GSK2879552.

[0125] Preferably, the induction medium contains 0-50 μM of GSK-LSD1.

[0126] Preferably, the induction medium contains 0.20 μM of tranylcypromine.

[0127] The histone deacetylase inhibitor is selected from one or more of Trichostatin A, Voronostat, Valproic Acid, etc.

[0128] Preferably, the induction medium contains 0-20 μM of Trichostatin A.

[0129] Preferably, the induction medium contains 0-50 μM of Vorinostat.

[0130] Preferably, the induction medium contains 0-1000 μM of Valproic Acid.

[0131] In a second aspect, the chemically reprogrammed CiMFSCs induced by culture are characterized by simultaneously upregulating the expression of genes related to nervous system development (such as DTX1, QRFPR, LGI2, REEP1, FEFER2, SYT1, ADAMTS17, HOXB6, etc.), genes related to angiogenesis (such as KDR, LAMC3, PALMD, SOX18, SHE, DLL4, NOTUM, FOXQ1, NKD1, COL23A1, SLC6A2, etc.), genes related to development (such as WNT10A, HOXB5, BMP7, BMP4, WNT6, WNT7B, etc.), and genes related to inflammation suppression (such as IL10, IL19, IL24), while downregulating the expression of genes related to fibroblasts (such as CTGF, THY1, VIM, THBS1, SULF1, LUM, etc.) and pro-inflammatory genes (such as IL1B, IL6).

[0132] The CiMFSCs described above have stable proliferation ability and can be stably passed down for 5-15 generations;

[0133] The CiMFSCs, as detected by single-cell 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.

[0134] The CiMFSCs described above have the potential to differentiate into bone, cartilage, and adipocytes in vitro.

[0135] The CiMFSCs described above have the potential to differentiate into nerve cells in vitro.

[0136] The CiMFSCs described above have the potential to differentiate into vascular endothelial cells in vitro.

[0137] The CiMFSCs described above have the potential to differentiate into perivascular cells in vitro.

[0138] The CiMFSCs described above have been shown to have the potential to differentiate into nerve cells in vivo, according to single-cell sequencing.

[0139] The CiMFSCs described above have been shown to have the potential to differentiate into vascular endothelial cells in vivo, as determined by single-cell sequencing.

[0140] The CiMFSCs described above have been shown to have the potential to differentiate into perivascular cells in vivo, as determined by single-cell sequencing.

[0141] In a third aspect, the present invention provides the application of CiMFSCs in the treatment of stroke.

[0142] Furthermore, the stroke mentioned includes ischemic stroke and hemorrhagic stroke.

[0143] Furthermore, the ischemic stroke mentioned includes: atherosclerotic cerebral infarction of large arteries, cardioembolic cerebral infarction, lacunar infarction, non-atherosclerotic vascular disease (such as aortic dissection, vasculitis, moyamoya disease, etc.) or hypercoagulable state of blood.

[0144] Furthermore, the hemorrhagic stroke mentioned above includes: intracranial hemorrhage and subarachnoid hemorrhage.

[0145] In a preferred embodiment of the present invention, the ischemic stroke is an acute ischemic stroke.

[0146] In a preferred embodiment of the present invention, the hemorrhagic stroke is intracerebral hemorrhage.

[0147] A third aspect (or independent aspect) of the invention also provides a solubilizing system for poorly soluble small molecules to improve cell reprogramming efficiency. The solubilizing system includes, but is not limited to, glycyrrhizic acid micelles. These glycyrrhizic acid micelles can significantly increase the water solubility and bioavailability of poorly soluble small molecules, allowing compositions containing multiple poorly soluble small molecules to function more effectively in aqueous culture systems, thereby improving cell reprogramming efficiency.

[0148] The beneficial effects of this invention are as follows:

[0149] (1) CiMFSCs obtained by reprogramming human fibroblasts can proliferate stably in vitro for a long time, solving the problem of a large source of cells required for in vivo transplantation;

[0150] (2) CiMFSCs obtained by reprogramming human fibroblasts do not express major histocompatibility complex (MHC) antigen molecules in vitro and in vivo, as detected by single-cell sequencing, which can significantly reduce the risk of immune rejection caused by allogeneic cell transplantation.

[0151] (3) CiMFSCs obtained by reprogramming human fibroblasts have the potential to differentiate into osteocytes, chondrocytes, adipocytes, nerve cells, vascular endothelial cells and pericytes in vitro, and can effectively differentiate into nerve cells, vascular endothelial cells and pericytes in vivo. They can simultaneously meet the needs of central nervous system repair and regeneration for multiple targets such as nerves and blood vessels, and provide new treatment strategies for complex nervous system diseases.

[0152] (4) The culture system and preparation method provided by the present invention are simple and efficient, easy to standardize, and conducive to promotion and application. Attached Figure Description

[0153] Figure 1 These are microscopic morphological images of CiMFSCs derived from human fibroblasts in the embodiments of the present invention: (A) human fibroblasts; (B) CiMFSCs.

[0154] Figure 2 In this embodiment of the invention, quantitative real-time PCR was used to detect the downregulation of fibroblast-related gene expression in cells.

[0155] Figure 3 In this embodiment of the invention, single-cell sequencing is used to detect the expression of genes related to fibroblast, nerve, blood vessel, development and inflammation regulation in induced cells.

[0156] Figure 4 The morphology (A) and particle size distribution (B) of the micelles in the composition are shown under an electron microscope.

[0157] Figure 5 The expression of characteristic genes DRD2, NRCAM, KDR, and IL10 in CiMFSCs induced by a combination of micelles and free small molecules was detected by PCR.

[0158] Figure 6 The difference in CiMFSC gene expression induced by the combination micelles and free drugs is due to the composition.

[0159] Figure 7 This is the proliferation curve of CiMFSCs derived from human fibroblasts in the embodiments of the present invention.

[0160] Figure 8 In this embodiment of the invention, immunofluorescence staining was used to detect the differentiation potential of CiMFSCs in multiple directions in vitro, including bone, cartilage, fat, nerve, and blood vessels: (A) osteogenic differentiation; (B) chondrogenic differentiation; (C) adipogenic differentiation; (D) neural differentiation; (E) vascular differentiation. F: In vitro tube formation experiment of vascular endothelial cells induced by CiMFSCs.

[0161] Figure 9 In this embodiment of the invention, the mNSS behavioral score was used to examine the recovery of motor function in ischemic stroke model animals after CiMFSCs transplantation (n≥3).

[0162] Figure 10 In this embodiment of the invention, CiMFSCs transplantation can significantly reduce the cerebral ischemic infarct area in ischemic stroke model animals: (A) TTC staining (n=3); (B) Infarct area statistics (n=3).

[0163] Figure 11In this embodiment of the invention, single-cell sequencing was used to detect the differentiation potential of CiMFSCs in multiple directions, including nerves, blood vessels, and pericytes, in patients with ischemic stroke.

[0164] Figure 12 In this embodiment of the invention, immunofluorescence staining was used to show that some CiMFSCs (stem121+) positively expressed the neuron-specific marker SOX11 and the neural stem / progenitor cell markers PTPRZ1 and SOX2, indicating the neural differentiation of CiMFSCs.

[0165] Figure 13 In this embodiment of the invention, immunofluorescence staining was used to show that some CiMFSCs (stem121+) co-localize with CD31+ vascular endothelial cells, suggesting that CiMFSCs promote vascular differentiation and angiogenesis.

[0166] Figure 14 This refers to the mNSS behavioral score after CiMFSCs were transplanted into mice with hemorrhagic stroke in this embodiment of the invention.

[0167] Figure 15 The multifunctional stem cell CiMFSCs in this embodiment of the invention rapidly repairs the blood-brain barrier after being transplanted into mice with hemorrhagic stroke: (A) Model control, 6 days; (B) CiMFSCs transplantation group, 6 days after transplantation. Detailed Implementation

[0168] 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.

[0169] Example 1: Reprogramming primary human fibroblasts into CiMFSCs

[0170] Human fibroblasts were digested, resuspended, and then... 4Fibroblasts were seeded at a density of cells / mL in 24-well plates. They were cultured for 24 hours at 37°C and 5% CO2 using complete fibroblast culture medium (containing 10% fetal bovine serum, basal DMEM cell culture medium, 1% non-essential amino acids, 1% GlutaMAX™, 10000 U / mL penicillin and streptomycin). 24 hours later, the culture medium was changed to the first induction medium, which consisted of: basal DMEM 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™ (Thermo Fisher Scientific), 10000 U / mL penicillin and streptomycin, 25 ng / mL epidermal growth factor (EGF), 15 ng / mL basic fibroblast growth factor (bFGF), 1 μM 5-Azacytidine, 10 μM CHIR-99021, and 200 μM VPA. Change the medium every three days. After culturing in the first induction medium for 5 days, switch to the second induction medium, which consists of: basal DMEM 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™ (Thermo Fisher Scientific), 10000 U / mL penicillin and streptomycin, 20 ng / mL epidermal growth factor (EGF), 20 ng / mL basic fibroblast growth factor (bFGF), 5 μM SB431542, 2 μM JNK-IN-8, 10 μM CHIR-99021, 5 μM OAC1, 2 μM SP2509, and 200 μM VPA. The medium was changed every three days. After culturing in the second induction medium for 6 days, the medium was replaced with the third induction medium, which consisted of: basal DMEM 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™ (Thermo Fisher Scientific), 10000 U / mL penicillin and streptomycin, 20 ng / mL epidermal growth factor (EGF), 20 ng / mL basic fibroblast growth factor (bFGF), 10 μM CHIR-99021, 2 μM ranylcypromine, and 500 μM VPA. The medium was changed every three days. After culturing in the third induction medium for 6 days, CiMFSCs cells could be harvested for subsequent detection and experiments.

[0171] During culture in induction medium, the morphology of human fibroblasts changes over time, gradually transforming from an elongated spindle shape to a cobblestone-like polygon or circle, with clear cell boundaries. Figure 1 The above morphological changes suggest that human fibroblasts underwent reprogramming under the induction medium provided by this invention. qRT-PCR was used to detect the expression of fibroblast-related genes in cells 14 days after induction, revealing a significant decrease in the expression levels of fibroblast-specific marker genes such as CTGF, THY1, VIM, THBS1, SULF1, and LUM. Figure 2 Single-cell sequencing further confirmed the downregulation of fibroblast marker genes, while revealing significant upregulation of genes related to nervous system development (such as DTX1, QRFPR, LGI2, REEP1, FERMT2, SYT1, ADAMTS17, HOXB6, etc.), genes related to angiogenesis (such as KDR, LAMC3, PALMD, SOX18, SHE, DLL4, NOTUM, FOXQ1, NKD1, COL23A1, SLC6A2, etc.), genes related to development (such as WNT10A, HOXB5, BMP7, BMP4, WNT6, WNT7B, etc.), and genes related to inflammation suppression (such as IL10, IL19, IL24). Figure 3 ).

[0172] Example 2: Preparation of glycyrrhizic acid mixed polymer micelles loaded with poorly soluble small molecules using thin film dispersion method.

[0173] To improve the solubility of poorly soluble small molecules in the composition and the efficiency of cell reprogramming, this embodiment uses glycyrrhizic acid (such as sodium glycyrrhizate) to prepare mixed polymer micelles loaded with CHIR99021 via thin-film dispersion. The specific steps are as follows: CHIR99021 (0.1-10 mg), glycyrrhizic acid (1-100 mg), and anhydrous ethanol (2 mL) are mixed in a 10 mL round-bottom flask and shaken at 37°C and 230 rpm for 10-50 min. Subsequently, the anhydrous ethanol is removed by rotary evaporation at 37°C. After a uniform film forms on the inner wall of the flask, 1 mL of deionized water is added, and the mixture is hydrated by shaking at 37°C and 200 rpm for 2 h to obtain a clear and transparent micelle solution. Microscopic observation shows that the obtained micelles have a uniform particle size distribution of approximately 50-200 nm, a drug loading of 3-10%, and an encapsulation efficiency of 60-95%. Figure 4 Applying the prepared small molecule micelles to fibroblast reprogramming significantly improves the efficiency of reprogramming. Figure 5RNA transcriptome sequencing analysis showed that the expression profiles of key genes in CiMFSCs induced by this CHIR99021 micelle composition were highly similar to those in CiMFSCs induced by the free drug combination. Figure 6 This indicates that glycyrrhizic acid itself does not affect the final reprogramming state.

[0174] Example 3: Amplification and in vitro differentiation of CiMFSCs obtained in Example 1

[0175] Before cell passage, culture plates were coated with 0.1% gelatin, ensuring the bottom surface was covered, and incubated at 37°C for one hour before use. To ensure stable expansion of CiMFSCs, a third culture system was used in this study. CiMFSCs were cultured at a rate of 5 × 10⁶ cells / year. 4 Cells were seeded at a density of [cells / mL] in 6-well plates. The third culture system included 10% fetal bovine serum, 5% Knockout serum replacement (Gibco), DMEM-basic medium, 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 (Thermo Scientific), 10000 U / mL penicillin and streptomycin, 20 ng / mL EGF, 20 ng / mL β-FGF, 10 μM CHIR-99021, 1 μM A83-01, 5 μM OAC1, and 1 μM Trichostatin A. In the above culture system, CiMFSCs could stably proliferate and expand to 6-7 generations, and their proliferation curves are shown in [Figure showing proliferation curves]. Figure 7 .

[0176] Although mesenchymal stromal cells (CiMFSCs) do not differentiate as readily as mesenchymal stem cells (MSCs), they still possess a certain degree of differentiation capacity under specific induction conditions. To investigate the in vitro differentiation potential of CiMFSCs, they were seeded into gelatin-coated 24-well plates, and then induced to differentiate into multiple pathways, including osteogenic, chondrogenic, adipose, neural, and vascular processes, using different induction systems.

[0177] The osteogenic induction culture system included DMEM-low glucose, 10% fetal bovine serum, 0.1 μM dexamethasone, 50 μg / mL trisodium 2-phosphate-L-ascorbate, and 10 mM glycerophosphate. The culture medium was changed every three days. After 3 weeks of induction, positive Alizarin Red S staining confirmed the osteogenic differentiation of CiMFSCs. Figure 8 A).

[0178] The chondrogenic induction culture system included DMEM-high glucose, 1% ITS, 0.1 μM dexamethasone, 50 μg / mL ascorbic acid, 40 μg / mL L-proline, 0.9 mM sodium pyruvate, and freshly added 10 ng / mL TGF-β1. The medium was changed every three days. After three weeks of induction, positive toluidine blue staining confirmed chondrogenic differentiation of CiMFSCs. Figure 8 B).

[0179] The adipogenic induction culture system included DMEM-high glucose, 10% fetal bovine serum, 1 μg / mL insulin, 0.5 mM 3-isobutyl-1-methylxanthine, and 1 μM dexamethasone. The medium was changed every three days. After 3 weeks of induction, the adipogenic differentiation of CiMFSCs was detected by Oil Red O staining. Figure 8 C).

[0180] The neural induction culture system included DMEM / F12, 1% N2 supplement (Gibco), 2% B27 supplement (Gibco), 10 ng / mL BDNF, 10 ng / mL NT-3, 10 ng / mL NGF, 200 μM ascorbic acid, and 0.5 mM cyclic adenosine monophosphate. The culture medium was changed every two days for two weeks, followed by immunofluorescence staining for neuronal markers. The results showed positive expression of the neural markers TUJ1 and MAP2, indicating that CiMFSCs possess the potential for neural differentiation. Figure 8 D).

[0181] The vascular endothelial cell induction culture system included DMEM-basic, 2% fetal bovine serum, 1% Glutamax, 1% non-essential amino acids, 50 ng / mL VEGF, 20 ng / mL IGF, 10 ng / mL bFGF, 5 ng / mL EGF, and 22.5 mg / L heparin sodium. The culture medium was changed every two days, and the induction process was closely monitored. After 7 days of induction, vascular endothelial cells were identified using the Matrigel tube formation assay and immunofluorescence staining. The results showed positive expression of the vascular endothelial cell-specific marker CD31, and that vascular-like structures could be formed on Matrigel, indicating that CiMFSCs have the potential for vascular endothelial cell differentiation. Figure 8 EF).

[0182] Example 4: Establishment of a mouse MCAO model

[0183] Establishment of a mouse model of ischemic stroke: The modified Zea Longa method was used. The modification of this method is that the pterygopalatine artery (PPA) is not exposed, while the blood flow of the internal carotid artery (ICA) is blocked by suspending braided sutures and clamping arterial clips. Finally, an embolized suture is inserted from the external carotid artery (ECA).

[0184] 1. Experimental steps:

[0185] 1) Weigh the C57BL / 6 mice that have been fasted overnight, and anesthetize them by intraperitoneal injection with 10% chloral hydrate solution at a dose of 0.004 mL / g of mouse body weight. Prepare braided sutures for ligating or blocking arteries in advance, and soak them in physiological saline.

[0186] 2) If the mouse cannot turn over freely after a few minutes, it is considered that the required depth of anesthesia has been reached. The mouse is then tied to the dissection table, and a cotton swab is placed under the mouse's neck to elevate it. The mouse's neck is disinfected with iodine and the skin is prepared.

[0187] 3) Cut open the skin along the middle of the mouse's neck, and bluntly separate the subcutaneous tissue and fat under the magnified field of view of a stereomicroscope using ophthalmic curved forceps and ophthalmic straight forceps. Slice open the superficial fascia and platysma muscle to expose the deep cervical fascia and anterior tracheal muscle.

[0188] 4) After separating the anterior tracheal muscle, tear open the deep neck fascia at the anterior border of the right sternocleidomastoid muscle to expose the sternocleidomastoid muscle and pull it posteriorly. Continue dissecting downward along the sternocleidomastoid muscle until the carotid sheath in a deeper position is exposed.

[0189] 5) While avoiding compression of the mouse's trachea, cut open the carotid sheath, separate the common carotid artery (CCA), and thread a suture to block blood flow with a slipknot. Be careful not to damage the accompanying internal jugular vein and vagus nerve inside the sheath, as stimulation or damage to the nerves may cause respiratory arrest in the mouse.

[0190] 6) Separate upwards along the common carotid artery. At the upper border of the thyroid cartilage, the external carotid artery branches off. The external carotid artery is located superior and medial to the internal carotid artery, close to the trachea. At the point where the common carotid artery divides into the internal and external carotid arteries, there are the carotid sinus and carotid bulb. The carotid sinus is the dilated portion at the terminal end of the common carotid artery and the origin of the internal carotid artery.

[0191] 7) The small branch arising from the anteromedial wall of the external carotid artery is the superior thyroid artery, which is separated and electrocoagulated. Continuing to separate upwards along the external carotid artery, the second branch, the lingual artery, as well as the occipital and external maxillary arteries, can be found. Double ligation is performed with braided sutures near the branches of the occipital and external maxillary arteries. The external carotid artery is cut in the middle of the two ligation points, leaving the proximal end of the ligation slightly longer to facilitate subsequent embolization.

[0192] 8) Slightly lift the ligature suture at the proximal end of the external carotid artery to expose the internal carotid artery below and laterally. Before entering the skull, the internal carotid artery divides into two branches: the pterygopalatine artery on the lateral side and the intracranial branch of the internal carotid artery on the medial side. To reduce modeling surgery time and injury to the mice, the pterygopalatine artery can be left undissected; instead, a suture can be threaded under the internal carotid artery to suspend the suture and obstruct blood flow from the internal carotid artery when inserting the suture.

[0193] 9) Prepare a slipknot at the proximal end of the external carotid artery, but do not tighten it yet. Tighten it to secure the suture after it has been inserted to the designated position. Gently lift the suture at the proximal end of the external carotid artery to align the external carotid artery with the internal carotid artery. Suspend the braided suture under the internal carotid artery and temporarily clamp it with a miniature arterial clamp to control blood flow. Make a small incision at the proximal end of the external carotid artery with ophthalmic scissors. Select a suture with a rounded end, smooth body, and uniform thickness, and gently insert it through the small incision. The suture will pass through the bifurcation of the external carotid artery and enter the internal carotid artery. Note that when the suture enters the internal carotid artery, you should press down slightly to make the suture tip curl up. This will prevent the suture from being inserted into the pterygopalatine artery. It is not recommended to separate or ligate the pterygopalatine artery because blocking the pterygopalatine artery can easily lead to thrombosis in the internal carotid artery. The thrombus surrounding the suture will make thrombectomy difficult and hinder reperfusion of the model.

[0194] 10) When the suture is inserted to a depth of approximately 9-10 mm, slight resistance will be encountered. This indicates that the suture has reached the bifurcation of the anterior cerebral artery (ACA) and the middle cerebral artery (MCA), obstructing the origin of the main trunk of the middle cerebral artery. Do not continue insertion at this point. Excessive insertion may puncture the ACA and induce subarachnoid hemorrhage (SAH), leading to modeling failure or even mouse death. If significant resistance is felt when the suture is inserted to a depth of approximately 6 mm, it indicates that the suture may be too thick to enter the skull, or it may have been mistakenly inserted into the pterygopalatine artery. In this case, do not force insertion; instead, slightly withdraw the suture, change the angle, and then attempt to insert it into the intracranial branch of the internal carotid artery.

[0195] 11) After inserting the suture into the designated position, tighten the prepared slipknot on the proximal end of the external carotid artery to secure the suture. Untie the slipknot blocking the common carotid artery, and finally suture the skin. Disinfect the suture site with povidone-iodine and return the mouse to its cage.

[0196] 12) After the operation, keep the mice warm by irradiating them with infrared lamps and monitor their body temperature. Ensure that the cage is dry and free of water or dust to prevent the mice from aspirating and suffocating.

[0197] 13) Two hours later, cut the sutures, remove the embolized sutures and ligate the severed ends of the external carotid artery to restore cerebral blood perfusion in the mouse. Then, re-suture and disinfect, and put the mouse back into the cage for continued monitoring.

[0198] 2. Evaluation of the MCAO model

[0199] 1) Longa 5-point scoring method:

[0200] After the mice were awakened, the brain damage of the model was scored according to the five-level four-point scoring method of Bederson et al. and Zea Longa et al.

[0201] 0 points: No neurological signs were observed, i.e. no neurological deficit symptoms; when the mouse was suspended by its tail, its two forelimbs were extended towards the ground; when the animal was placed on a soft plastic board, the tail was gently held, and a lateral push was applied behind the mouse's shoulder to make the mouse slide about 10cm, and the resistance to pushing from left to right was equal.

[0202] 1 point: Mild neurological deficit; When the mouse is lifted by its tail, due to the weakness of the forelimb on the opposite side of the ischemia, it will be flexed, raised, with the shoulder adducted and the elbow extended. At the same time, the body will also turn to the opposite side of the ischemia. Place the animal on a soft plastic board, gently hold the mouse tail, and apply a lateral push force behind the mouse's shoulder to make the mouse slide about 10cm. The resistance to pushing on both sides should be equal.

[0203] 2 points: Moderate focal neurological deficit; the mouse circled to the side opposite to the ischemia while crawling; the animal was placed on a soft plastic board, the tail was gently held, and a lateral push was applied behind the shoulder to make the mouse slide about 10 cm. The resistance to pushing was different on the left and right sides, and the lateral push resistance on the side opposite to the ischemia was significantly reduced.

[0204] 3 points: Severe focal neurological deficit; the contralateral limb of the ischemic side cannot bear weight, and the mouse tilts to the contralateral side of the ischemia when standing.

[0205] 4 points: Severe neurological deficits; no voluntary movement, impaired consciousness, or tubular rolling.

[0206] 2) mNSS 18-point scoring method

[0207] The modified neurological severity score (mNSS) is a quantitative analysis of the severity of brain injury by comprehensively assessing the degree of deficit in four neurological functions in mice: motor function, sensory response, balance ability, and abnormal reflexes (0-18 points, with higher scores indicating more severe deficits).

[0208] Table 1. Scoring Criteria for the mNSS 18-Point Rating Method

[0209]

[0210] Mice with a Longa score of 2-3 and an mNSS score of 9-12 were selected as qualified models and entered into subsequent experiments.

[0211] Example 5: The CiMFSCs prepared in Example 1 were used in the MCAO model established in Example 4 for evaluating the therapeutic effect of cerebral ischemic stroke.

[0212] 1. Grouping

[0213] Mice with successfully established MCAO model were housed individually and randomly divided into 7 groups (G2-G8), with 10 mice in each group. Group G1 consisted of 3 mice as the sham-operated group, receiving saline injection via the tail vein; Group G2 was the MCAO model group, also receiving saline injection via the tail vein; Groups G3-G7 used human foreskin fibroblasts (HFF), human induced pluripotent stem cell-derived neural stem cells (iPSC-NSCs), human induced pluripotent stem cell-derived vascular endothelial cells (iPSC-ECs), human induced pluripotent stem cell-derived pericytes (iPSC-PCs), and umbilical cord mesenchymal stem cells (HUMSCs), respectively, as control cells; Group G8 used CiMFSCs prepared according to this invention (referred to as CiMFSCs in this embodiment), with a cell dose of 1×10⁻⁶. 6 100 μL of cells per mouse, administered via tail vein injection. Cyclophosphamide (100 mg / kg) was injected one day prior to cell reinfusion to establish immunosuppression in mice.

[0214] 2. Behavioral scoring: Animal survival and activity were observed and recorded daily after cell transplantation. Results showed that mice treated with CiMFSCs exhibited the most significant decrease in mNSS score and the highest survival rate. Figure 9 ).

[0215] 3. Infarct Area Measurement: Brain tissue was collected from each group of animals on days 3 and 7 after cell transplantation, and the ischemic infarct area was measured using TTC staining. TTC staining is a well-known experimental procedure in this field, therefore the detailed steps will not be described further. The results showed that the CiMFSCs group significantly reduced the ischemic infarct area ( Figure 10 ).

[0216] 4. Single-cell sequencing to detect the differentiation of implanted CiMFSCs in the ischemic brain: Brain tissue was collected at different time points (3 days, 7 days, 17 days, 3 months, and 10 months) after CiMFSC treatment. Brain tissue from three mice was randomly selected at each time point, and the samples were combined to prepare single-cell suspensions. Single-cell sequencing was performed using the 10×Genomics single-cell sequencing platform from Beijing Bio-Rad Laboratories Co., Ltd. The Cell Ranger counting module was used for alignment, filtering, barcode counting, and UMI counting to generate a feature barcode matrix and determine clusters. Principal component analysis (PCA) was used for dimensionality reduction, and K-means and graph-based algorithms were used to cluster the top 10 principal components. Seurat 3.0 (R package) was also used for dimensionality reduction analysis, filtering out cells with fewer than 200 genes, genes in the top 1%, or a mitochondrial gene ratio greater than 25% as abnormal data. t-SNE and UMAP algorithms were used for visualization. Cell annotation based on literature showed that transplanted CiMFSCs could effectively differentiate into various cell types in the mouse brain, including neurons, oligodendrocytes, astrocytes, ependymal cells, vascular endothelial cells, pericytes, and vascular smooth muscle cells. In addition, a large number of microglia-like immune cells were also observed. Figure 11 ).

[0217] 5. Immunofluorescence staining of pathological tissues

[0218] After perfusion of mice, brains were removed, washed twice with PBS, and fixed overnight at 4°C with 4% paraformaldehyde. Subsequently, they were subjected to gradient ethanol dehydration (70%, 85%, 95%, and 100% ethanol, 2 hours each) and xylene clearing (Cylinder I and II, 1.5 hours each). The tissues were then transferred to molten paraffin at 60°C for three immersions (1.5 hours each, with constant temperature shaking to promote penetration), and embedded in metal molds using sagittal or coronal positioning to form paraffin blocks. After pre-cooling at -20°C, sections were serially sliced ​​using a rotary microtome (4-6 μm thickness). The paraffin strips were flattened and wrinkle-free in 45°C warm water, then transferred to APES-pretreated slides to prevent detachment. Finally, the sections were baked overnight in a 60°C oven to ensure tight adhesion, sealed, and stored in a dry environment for subsequent immunofluorescence staining, using the human cell-specific antibody stem121 to indicate CiMFSCs. Results showed that transplanted TDRSCs could differentiate into neural progenitor cells and neurons in vivo. Figure 12 Furthermore, a portion of stem121-positive cells co-localize with the vascular endothelial cell marker CD31. Figure 13 ).

[0219] Example 6: CiMFSCs obtained in Example 1 were used to treat hemorrhagic stroke in mice via tail vein injection.

[0220] 1. Establishment of a mouse model of hemorrhagic stroke: This invention uses stereotactic injection of type VII collagenase to establish an ICH model. Experimental steps:

[0221] 1) Preoperative preparation: Surgical instruments and supplies required for the operation were autoclaved in advance, and mice were anesthetized intraperitoneally (IP) with pentobarbital (50 mg / mL) in advance. The unconscious mice were fixed on a stereotaxic apparatus so that the anterior and posterior fontanelles of the mice were basically on the same plane.

[0222] 2) Exposure of the skull: Shave the hair on the top of the mouse's head, wipe it with iodine solution for disinfection, make an incision of about 1 cm in the center of the disinfected area, and slowly separate the outer membrane of the skull with a cotton swab to expose the anterior fontanelle and the coronal suture.

[0223] 3) Positioning Drill: Using the anterior fontanelle as the origin, the drilling point is 0.2 mm behind the anterior fontanelle and 2.0 mm to the right. Drill a hole with a diameter of 1 mm at the positioning point using a dental drill.

[0224] 4) Targeted injection: Draw an appropriate amount of type VII collagenase (0.045 U / μL) with a microsyringe, ensuring no air bubbles are generated; fix the syringe on the stereotaxic instrument, the injection depth is 3.5 mm, the injection rate is 0.1 μL / min, and leave the needle in place for 10 minutes after each injection to avoid backflow of fluid. Slowly withdraw the needle, seal the bone hole with medical sterile bone wax, suture the scalp skin, and disinfect with povidone-iodine to prevent infection.

[0225] 5) Grouping: Two days after the operation, the mice were scored using the mNSS (Table 1). Mice with a score of 12 or higher were selected and assigned to the successful model group for subsequent experiments.

[0226] 2. Grouping and Drug Administration: Mice with successfully established cerebral hemorrhage model were housed individually and randomly divided into 4 groups (G1-G4). Group G1 was the ICH model group, receiving saline via tail vein injection; Groups G2 and G3 were control cells (iPSCs_NSCs and HUMSCs), respectively; and Group G4 received CiMFSCs. The cell dose for each group was 1.5 × 10⁻⁶ cells. 6 100 μL of cells per mouse, administered via tail vein injection. Cyclophosphamide (100 mg / kg) was injected one day prior to cell reinfusion to establish immunosuppression in mice.

[0227] 3. mNSS behavioral score: mNSS behavioral scores were assessed weekly after cell transplantation. Results showed that mice treated with CiMFSCs exhibited the fastest and most significant decrease in mNSS scores. Figure 14 ).

[0228] 4. Blood-brain barrier repair detection: Evans blue leakage test results showed that CiMFSC transplantation can significantly repair the blood-brain barrier damaged by cerebral hemorrhage. Figure 15 ).

[0229] The key design focus of this invention is to obtain chemically reprogrammed multifunctional mesenchymal matrix cells (CiMFSCs) using the glycyrrhizic acid solubilization technology provided in this application. Human fibroblasts are then induced to produce highly plastic mesenchymal matrix cell-like cells that simultaneously upregulate the expression of genes related to nervous system development, angiogenesis, development, and inflammation suppression. These cells are then used to treat stroke. The CiMFSCs prepared by this invention can effectively alleviate brain injury caused by cerebral ischemia and hemorrhage, rapidly repair and stabilize the blood-brain barrier, accelerate the recovery of neurovascular function, and promote colonization and differentiation within brain tissue, thus accelerating the repair of brain damage.

[0230] 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 chemically reprogrammed multifunctional mesenchymal matrix cell based on glycyrrhizic acid solubilization technology, characterized in that: The multifunctional mesenchymal matrix cells were prepared by inducing and culturing human fibroblasts using glycyrrhizic acid solubilization technology.

2. The chemically reprogrammed multifunctional mesenchymal matrix cells based on glycyrrhizic acid solubilization technology according to claim 1, characterized in that: The aforementioned glycyrrhizic acid solubilization technology involves using glycyrrhizic acid as a solubilizer and functional component in the induction culture medium to reprogram human fibroblasts.

3. The chemically reprogrammed multifunctional mesenchymal matrix cells based on glycyrrhizic acid solubilization technology according to claim 1, characterized in that: The human fibroblasts are skin tissue, lung, heart, bone marrow, skin, lung, and heart tissue from aborted fetuses, as well as human fibroblast cell lines.

4. A method for preparing chemically reprogrammed multifunctional mesenchymal matrix cells based on glycyrrhizic acid solubilization technology according to any one of claims 1 to 3, characterized in that... Includes the following steps: (1) Human fibroblasts were cultured and expanded using complete fibroblast culture medium; (2) Fibroblasts were induced to generate multifunctional mesenchymal stromal cells. The induction process was divided into three stages: First stage: Use the first induction medium and culture for 5-15 days; Second stage: Use the second induction medium and culture for 3-10 days; Third stage: Use the third induction medium and culture for 3-7 days; (3) Digest and collect the induced cultured cells.

5. The preparation method according to claim 4, characterized in that: The complete fibroblast culture medium in step (1) includes liquid basal culture medium, fetal bovine serum, and penicillin / streptomycin; The liquid basal culture medium is one or a combination of two or more of the following: 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. 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.

6. The preparation method according to claim 4, characterized in that: The first induction culture medium in step (2) includes a complete fibroblast culture medium, as well as a first combination of small chemical molecules, cell culture nutrient additives, and cytokines; The second induction medium includes a complete fibroblast culture medium, a second combination of small chemical molecules, cell culture nutrient supplements, and cytokines; The third induction medium includes a complete fibroblast culture medium, a third chemical small molecule combination, cell culture nutrient supplements, and cytokines.

7. The preparation method according to claim 6, characterized in that: The first chemical small molecule combination includes one or a mixture of two or more of DNA methyltransferase inhibitors, GSK3 inhibitors, or histone deacetylase inhibitors; The second chemical small molecule combination includes one or more of the following: TGF-β receptor inhibitors, JNK inhibitors, OCT4 activators, GSK3 inhibitors, KDM1 / LSD1 inhibitors, or histone deacetylase inhibitors. The third chemical small molecule combination includes one or more of the following: GSK3 inhibitors, KDM1 / LSD1 inhibitors, or histone deacetylase inhibitors; The final concentrations of each component in the first chemical small molecule combination are: DNA methyltransferase inhibitor: 0–50 μM; GSK3 inhibitor: 0–50 μM; histone deacetylase inhibitor: 0–1000 μM; The final concentrations of each component in the second chemical small molecule combination are as follows: TGF-β receptor inhibitor: 0–30 μM; JNK inhibitor: 0–20 μM; OCT4 activator: 0–50 μM; GSK3 inhibitor: 0–50 μM; KDM1 / LSD1 inhibitor: 0–20 μM; histone deacetylase inhibitor: 0–1000 μM; The final concentrations of each component in the third chemical small molecule combination are as follows: GSK3 inhibitor: 0–50 μM; KDM1 / LSD1 inhibitor: 0–20 μM; histone deacetylase inhibitor: 0–1000 μM; The cell culture nutrient additives are one or a mixture of two or more of the following: non-essential amino acids, GlutaMAX™, nicotinamide, sodium bicarbonate, trisodium 2-phosphate-L-ascorbate, insulin, sodium pyruvate, glucose, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, insulin-transferrin-selenium medium additive, dexamethasone, penicillin, or streptomycin. The cytokines are selected from one or more of the following: epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor, hepatocyte growth factor, bone morphogenetic protein 4, transforming growth factor-β1, leukemia inhibitory factor, interleukin-2, interleukin-3, interleukin-4, interleukin-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, interferon-γ, nerve growth factor, brain-derived neurotrophic factor, and neurotrophic factor-3 / 4.

8. The preparation method according to claim 7, characterized in that: The TGF-β receptor inhibitor is SB431542, LDN193189, LY2157299, LY2109761, SB525334, SB505124, GW788388, LY364974, E-616452 (RepSox), K02288, A83-01, R26872, BIBF-0775, LDN214117, SD-208, TEW-7197, ML347, LDN212854, DMH1, Dorsomorphin, S-7701, SIS3, Galunisertib, curcumin, quercetin, tanshinone IIA, baicalein, luteolin, caffeic acid, resveratrol, artemisinin, or tripterygium indicum, or isomers, salts, hydrates, precursors, or combinations thereof of the above preparations; The OCT4 activator is OAC1, OAC2, O4I1, O4I2 or Oct3 / 4-inducer-1, or isomers, salts, hydrates, precursors or combinations thereof of the above preparations; The GSK3 inhibitor is CHIR-99021, SB216763, AT7519, CHIR-98014, TWS119, NP031112, SB415286, BIO, LY2090314, Alsterpaullone, KY19382, 1-Azakenpaullone, AZD1080, ARA014418, IM-12, WAY-119064, BRD0705, AZD2858, LiCl, berberine, quercetin, epigallocatechin gallate, luteolin, resveratrol, curcumin, or apigenin, or isomers, salts, hydrates, precursors, or combinations thereof of the above preparations; The histone deacetylase inhibitors are Voronostat (SAHA), Entinostat (MS-275), Panobinostat (LBH589), Trichostatin A (TSA), Mocetinostat (MGCD0103), Belinostat (PXD101), Romidepsin (FK228), Dacinostat (LAQ824), CUDC-101, Quisinostat (JNJ-26481585), Pracinostat (SB939), Droxinostat, Abexinostat (PCI-24781), AR-42, Ricolinostat (ACY-1215), Fimepinostat (CUDC-907), RG2833, UF010, Sodium butyrate, Valproic Acid, or Curcumin, or isomers, salts, hydrates, precursors, or combinations thereof of the above formulations; The KDM1 / LSD1 inhibitor is SP2509, GSK2879552, GSK-LSD1, Tranylcypromine, GSK-J4, CPI-455, IOX1, VML324, Phenelzine sulfate, α-Hydroxyglutaric acid, SKLB325, or isomers, salts, hydrates, precursors, or combinations thereof of the above preparations; The DNA methyltransferase inhibitors mentioned above are 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, salts, hydrates, precursors or combinations thereof of the above preparations; The JNK inhibitor is SP600125, JNK-IN-8, GDC-0134, Astragaloside IV, Gamma-Linolenic acid, Berberine sulfate, Tomatidine, JNK Inhibitor VIII, Isovitexin, Tanzisertib, JNK-IN-7, Angelicin, Bicyclol, Sinapaldehyde, Isoscopoletin, Halicin, D-JNKI-1, Vaccarin, BI-78D3, SR-3306, J30-8, Decrusinol, or Artemisic acid, or isomers, salts, hydrates, precursors, or combinations thereof of the above preparations.

9. The use of chemically reprogrammed multifunctional mesenchymal stromal cells based on glycyrrhizic acid solubilization technology according to any one of claims 1 to 3 in the preparation of a medicament for treating ischemic stroke or hemorrhagic stroke.

10. The application according to claim 9, characterized in that: The cells used in the application include live cells, extracellular vesicles, cell culture supernatants, or products derived from chemically reprogrammed multifunctional mesenchymal matrix cells.

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