Small molecule compound combination and method for preparing vascular endothelial cells by inducing differentiation of cells using the same

Abstract

The application discloses a kind of small molecule compound combination and the method for preparing vascular endothelial cell using the small molecule compound combination induced differentiation cell.The method is directed to induce differentiated cell, and finally prepare vascular endothelial cell;The directional induction includes inhibiting the activity of lysine deacetylase, inhibiting the signal path of TGF-β, inhibiting the activity of DNA methyltransferase (DNMT), activating the signal path of WNT / β-catenin and activating the signal path of cAMP.The present application can be converted into vascular endothelial cell by small molecule compound combination induction, and each step can realize accurate quality control, which is convenient for standardized operation and large-scale production.The method provided by the present application has wide donor sources, and the patient himself can be used as a donor, so that the vascular endothelial cells required for basic research, clinical treatment or tissue engineering production can be obtained in a short time.

Description

Technical Field

[0001] This invention relates to the fields of cell biology, tissue engineering, and regenerative medicine, and in particular to a combination of small molecule compounds and a method for preparing vascular endothelial cells using cells induced to differentiate by the combination of small molecule compounds. Background technology:

[0002] Vascular endothelial cells (VECs), serving as the structural lining of blood vessels, play a crucial role in maintaining normal vascular morphology and physiological function. Metabolically active VECs exhibit antithrombotic effects in vivo, making them key to ensuring long-term patency after tissue-engineered vessel implantation and forming the basis for angiogenesis in clinical treatment. With the increasing number of patients undergoing trauma, organ transplantation, and coronary artery bypass grafting for cardiovascular diseases, the market demand for VECs or their precursor cells—serving as seed cells for tissue-engineered vessels or regenerative medicine—is also increasing annually. Currently, the VECs required for regenerative medicine or tissue engineering are mainly derived from allogeneic donations or induced from embryonic stem cells, iPS cells, or adult stem cells. However, these methods suffer from limited availability, complex acquisition processes, low cell purity, and risks of immunogenicity and tumorigenesis, thus limiting their clinical application.

[0003] Various cell types, such as skin fibroblasts, offer advantages such as abundant availability, ease of acquisition, and the ability to be expanded and cultured in large quantities in vitro over extended periods. Currently, cell transdifferentiation technology allows for the direct induction of differentiated cells, such as skin fibroblasts, into various functional cell types, including myoblasts, neurons, hepatocytes, and osteoblasts; or, first, induction into pluripotent stem cells, followed by further directed induction into corresponding functional cells. The functional cells obtained directly or indirectly from specifically differentiated cells, such as skin fibroblasts, using transdifferentiation technology no longer retain the molecular characteristics and functions of the source cells, but acquire the typical molecular characteristics and cellular functions of the target cells. Currently, these induced functional cells are increasingly being applied in disease model research, clinical treatment research, and tissue engineering research.

[0004] Traditional cell transdifferentiation requires the introduction of specific exogenous genes, sometimes synergistically with small molecule compounds, cytokines, or recombinant proteins. Numerous studies have reported on using specific exogenous genes to induce one type of differentiated cell into another functionally differentiated cell. For example, there are reports of using BMP-2, BMP-7, and LMP3, alone or in combination, to transdifferentiate skin fibroblasts into osteoblasts, which exhibit bone-forming activity both in vitro and in vivo. However, the introduction of exogenous genes carries tumorigenic risks and may induce immunogenicity in target cells, hindering widespread application. In 2013, Deng Hongkui reported that small molecule compounds or their combinations could be used to reprogram mouse skin fibroblasts into neural cells, demonstrating that this transdifferentiation technology has advantages such as a short induction process, a stable induction system, easy quality control, low cost, no tumorigenic risks associated with exogenous gene insertion, and that the obtained target cells exhibit good safety and stability without immunogenicity, indicating potential clinical application value and industrialization prospects. Subsequently, Chinese patent application No. 201410075246.5 provided a method for inducing differentiated cells to transdifferentiate into neural stem cells and its application. Specifically, this application involves using a combination of histone deacetylase (HDAC) inhibitors, glycogen synthase kinase (GSK-3) inhibitors, and transforming growth factor β (TGF-β) signaling pathway inhibitors to induce the transdifferentiation of differentiated cells such as fibroblasts and epithelial cells into neural stem cells with good pluripotency and passage stability under normal physiological hypoxia. Chinese patent application No. 201610213644.8 discloses an induction medium, method, and application for inducing fibroblast transdifferentiation into cardiomyocytes. The induction medium comprises a basal medium and an inducing small molecule combination, wherein the inducing small molecule combination is 6TCFOW or SCFOV, where 6 is E61541, T is phenylcyclopropane, C is CHIR99021, F is trichomoniasisin, O is Dorsomorphin, W is IWR-1, S is SB431542, and V is valproic acid. This induction medium can induce fibroblast transdifferentiation into cardiomyocytes. Currently, results have been reported in inducing the differentiation of human cells, such as skin fibroblasts, into Schwann cells (THOMA EC, et al., 2014), nerve cells (HU W, et al., 2015), and pancreatic islet cells (Sheng Ding, et al., 2015), using simple small molecule compounds or combinations thereof.

[0005] Because humans and mice share approximately 25% genetic differences, the feasibility of applying the patented technology that was successful in mouse cell experiments to transdifferentiate human cells is not high. On the other hand, because the specific theoretical basis and technical means of inducing transdifferentiation of human cells to obtain different target cells are not the same, the applicant repeated the experiments using the reported technology, but failed to successfully apply the transdifferentiation technology used in mouse cells to transdifferentiate human cells, nor did it induce human differentiated cells to transdifferentiate into vascular endothelial cells. Summary of the Invention

[0006] This invention provides a combination of small molecule compounds and a method for preparing vascular endothelial cells using cells induced to differentiate from this combination of small molecule compounds. The method provided by this invention, based on the regulation of corresponding signaling pathways and epigenetic modifications, utilizes a combination of small molecule compounds to treat differentiated cells to rapidly and stably obtain a large number of vascular endothelial cells and / or their products.

[0007] In a first aspect, the present invention provides a method for preparing vascular endothelial cells from induced differentiated cells, wherein the method involves directed induction of differentiated cells to ultimately obtain vascular endothelial cells; the directed induction includes inhibiting the activity of lysine deacetylases inhibitors (KDACIs), inhibiting the TGF-β signaling pathway, activating the WNT / β-catenin signaling pathway, activating the cAMP signaling pathway, and inhibiting the activity of DNA methyltransferase (DNMT) and / or inhibiting the activity of histone methyltransferase (HMT).

[0008] Furthermore, the directed induction further includes inhibiting the activity of histone demethylases and / or inhibiting the JNK signaling pathway and / or inhibiting the ROCK signaling pathway and / or inhibiting the activity of PKC and / or activating the RA (Retinoic acid) signaling pathway.

[0009] Furthermore, according to the signaling pathways and / or enzymes proposed in this invention, small molecule compounds can be used to regulate the corresponding signaling pathways and / or enzymes. That is, the directed induction involves contacting differentiated cells with inducing compounds to ultimately prepare vascular endothelial cells. The inducing compounds include inhibitors of lysine deacetylases, TGF-β receptor inhibitors, WNT / β-catenin agonists, cAMP agonists, and DNA methyltransferase (DNMT) inhibitors.

[0010] Furthermore, the inducing compound also includes at least one of histone methyltransferase (HMT) inhibitors, histone demethylase inhibitors, JNK inhibitors, ROCK (Rho-associated protein kinase) inhibitors, PKC inhibitors, and RAR (Retinoic acid Receptor) agonists.

[0011] Furthermore, the inducing compound also includes at least one of ascorbate, cell growth factor b-FGF, EGF, VEGF, and IGF-1; or the ascorbate and / or cell growth factor b-FGF and / or EGF and / or VEGF and / or IGF-1 are contacted with the corresponding cells or cell products after the above methods are performed.

[0012] The differentiated cells are derived from mammals such as humans and include fibroblasts, epithelial cells, and adipocytes. Preferably, the differentiated cells are skin fibroblasts.

[0013] In a second aspect, the present invention also provides a small molecule compound combination, said small molecule compound comprising the inducing compound, or comprising compounds that regulate the above-mentioned signaling pathways and / or enzyme activity (including inhibitors or activators of signaling pathways, and inhibitors or activators of enzymes).

[0014] The lysine deacetylase inhibitors in the small molecule compound combination include sodium phenylbutyrate, butyrate, sodium butyrate, MC1568, CI994 (Tacedinaline), chidamide, CAY10683 (SantacruzaMate A), CUDC-907, M344 (Histone Deacetylase Inhibitor III), LAQ824 (NVP-LAQ824, Dacinostat), Pracinostat (SB939), VPA, Scriptaid, Apidin, LBH-589 (Panobinostat), MS-275, SAHA (Vorinostat), Trichostatin (TSA), and Psammaplin. A, PCI-24781 (Abexinostat), Rocilinostat (ACY-1215), Mocetinostat (MGCD0103), 4-Phenylbutyrate (4PB), splitomicin, SRT1720, resveratrol, Sirtinol, APHA, CI-994, Depudecin, FK-228, HC-Toxin, ITF-2357 (Givinostat), Chidamide, RGFP 966, PHOB, BG45, Nexturastat A, TMP269, CAY10603, MGCD-0103, Niltubacin, PXD-101 (Belinostat), Pyroxamide, Tubacin, EX-527, BATCP, Cambinol, MOCPAC, PTACH, MC1568, NCH51 and TC-H106 at least one of the following:

[0015] The TGF-β receptor inhibitors include at least one of 616452, LY2109761, Pirfenidone, Repsox (E-616452), SB431542, A77-01, Tranilast, Galunisertib (LY2157299), A8301, GW788388, ITD-1, SD208, SB525334, LY364947, ASP3029, D4476, and SB505124;

[0016] The WNT / β-catenin activator includes at least one of MAY-262611, CHIR98014, CHIR99021, LiCl, Li2CO3, TD114-2, AZD2858, AZD1080, BIO, Kenpaullone, TWS119, LY2090314, CBM1078, SB216763 and AR-A014418;

[0017] The cAMP activator includes at least one of EPAC / RAP1 activator, 8-Bromo-cAMP, Dibutyryl-Camp and Sp-8-Br-cAMPs;

[0018] The EPAC / RAP1 activator includes at least one of Forskolin, IBMX, Prostaglandin E2 (PGE2), NKH477, 8-pCPT-2′-O-Me-cAMP, GSK256066, Apremilast (CC-10004), Roflumilast, Cilmimilast, Rolipram, and Milrinone;

[0019] The ROCK inhibitors include at least one of Y-27632, Y-27632 2HCl, Thiazovivin, Ripasudil (K-115), Fasudil, Fasudil (HA-1077)HCl, GSK429286A, RKI-1447 and PKI-1313;

[0020] The JNK inhibitors include at least one of SP600125, JNK Inhibitor IX, AS601245, AS602801, and JNK-IN-8;

[0021] The DNMT inhibitors include at least one of RG108, Thioguanine, 5-Aza-2'-deoxycytidine (Decitabine), SGI-1027, Zebularine, and 5-Azacytidine (AZA);

[0022] The HMT inhibitors include at least one of EPZ004777, EPZ5676, GSK503, BIX 01294 and SGC0946;

[0023] The histone demethylase inhibitors include at least one of parnate (tranylcypromine), tranylcypromine (2-PCPA)HCl, SP2509, 4SC-202, ORY-1001 (RG-6016), GSKJ1, and GSK-LSD1.

[0024] The PKC inhibitors include at least one of Go6983, Ro31-8220Mesylate, Go6976, and BisindolylmaleimideI (GF109203X).

[0025] The RAR agonist includes at least one of TTNPB, Bexarotene, Ch55, Tamibarotene, Retinol, AM580, ATRA, Vitamin A, Vitamin A derivatives, and 13-cis RA;

[0026] More preferably, the combination of small molecule compounds adopts any one of the following combinations:

[0027] VPA+CHIR99021+Repsox+Rolipram+EPZ004777;

[0028] VPA+CHIR99021+Repsox+Forskolin+TSA+5-Aza-2'-deoxycytidine;

[0029] VPA+CHIR99021+Repsox+Forskolin+TSA+RG108;

[0030] VPA+CHIR99021+Repsox+Rolipram+EPZ004777+RG108;

[0031] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+Go6983+5-Aza-2'-deoxycytidine;

[0032] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine+EPZ004777;

[0033] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108;

[0034] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+-Aza-2'-deoxycytidine;

[0035] VPA+CHIR99021+Repsox+Forskolin+TSA+RG108+Go6983;

[0036] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine+Go6983;

[0037] VPA+CHIR99021+Repsox+Forskolin+RG108;

[0038] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine;

[0039] VPA+CHIR99021+Repsox+Forskolin+TSA+5-Aza-2'-deoxycytidine+SP600125;

[0040] VPA+CHIR99021+Repsox+Forskolin+TSA+5-Aza-2'-deoxycytidine+Go6983;

[0041] VPA+CHIR99021+Repsox+Forskolin+Go6983+5-Aza-2'-deoxycytidine+SP600125;

[0042] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580;

[0043] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+Go6983;

[0044] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+SP600125;

[0045] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+SP600125+Go6983;

[0046] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+SP600125+Go6983+Y-27632;

[0047] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+SP600125+Go6983+Y-27632+sodium butyrate;

[0048] VPA+CHIR99021+Repsox+Forskolin+RG108+Go6983+Y-27632+sodium butyrate;

[0049] VPA+CHIR99021+Repsox+Forskolin+RG108+SP600125+Go6983+Y-27632;

[0050] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+SP600125+Y-27632;

[0051] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+Y-27632+Go6983;

[0052] VPA+CHIR99021+Repsox+Rolipram+EPZ004777+Y-27632;

[0053] VPA+CHIR99021+Repsox+Forskolin+TSA+5-Aza-2'-deoxycytidine+sodiumbutyrate;

[0054] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine+sodiumbutyrate;

[0055] VPA+CHIR99021+Repsox+Forskolin+TSA+5-Aza-2'-deoxycytidine+sodiumbutyrate+Go6983;

[0056] VPA+CHIR99021+Repsox+Forskolin+Y-27632+5-Aza-2'-deoxycytidine+ascorbate;

[0057] VPA+CHIR99021+Repsox+Forskolin+Y-27632+5-Aza-2'-deoxycytidine+ascorbate+Go6983;

[0058] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+SP600125+Y-27632+ascorbate;

[0059] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+Y-27632+ascorbate;

[0060] VPA+CHIR99021+Repsox+Forskolin+TSA+5-Aza-2'-deoxycytidine+Y-27632+Go6983;

[0061] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108;

[0062] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Y-27632;

[0063] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+Y-27632;

[0064] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Y-27632+ascorbate;

[0065] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+ascorbate;

[0066] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+Y-27632+ascorbate;

[0067] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Go6983;

[0068] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+Go6983;

[0069] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Y-27632+Go6983;

[0070] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+Y-27632+Go6983;

[0071] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Y-27632+ascorbate+Go6983;

[0072] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+ascorbate+Go6983;

[0073] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+Y-27632+ascorbate+Go6983;

[0074] VPA+CHIR99021+Repsox+Forskolin+TSA+5-Aza-2'-deoxycytidine;

[0075] VPA+CHIR99021+Repsox+Forskolin+TSA+RG108;

[0076] VPA+CHIR99021+Repsox+Forskolin+RG108;

[0077] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine;

[0078] VPA+CHIR99021+Repsox+Forskolin+TSA+5-Aza-2'-deoxycytidine+Go6983;

[0079] VPA+CHIR99021+Repsox+Forskolin+TSA+RG108+Go6983;

[0080] VPA+CHIR99021+Repsox+Forskolin+RG108+Go6983;

[0081] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine+Go6983;

[0082] VPA+CHIR99021+Repsox+Forskolin+TSA+5-Aza-2'-deoxycytidine+TTNPB;

[0083] VPA+CHIR99021+Repsox+Forskolin+TSA+RG108+TTNPB;

[0084] VPA+CHIR99021+Repsox+Forskolin+RG108+TTNPB;

[0085] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine+TTNPB;

[0086] VPA+CHIR99021+Repsox+Forskolin+TSA+5-Aza-2'-deoxycytidine+Go6983+TTNPB;

[0087] VPA+CHIR99021+Repsox+Forskolin+TSA+RG108+Go6983+TTNPB;

[0088] VPA+CHIR99021+Repsox+Forskolin+RG108+Go6983+TTNPB;

[0089] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine+Go6983+TTNPB;

[0090] VPA+CHIR99021+Repsox+Forskolin+RG108+SP600125+Go6983+Y-27632+sodiumbut yrate;

[0091] VPA+CHIR99021+Repsox+Forskolin+RG108+Go6983+Y-27632+sodium butyrate;

[0092] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine+Go6983+Y-27632+sodium butyrate;

[0093] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine+Go6983+Y-27632+sodium butyrate + ascorbate;

[0094] VPA+CHIR99021+Repsox+Forskolin+TSA+5-Aza-2'-deoxycytidine+Y-27632;

[0095] VPA+CHIR99021+Repsox+Forskolin+TSA+RG108+Y-27632;

[0096] VPA+CHIR99021+Repsox+Forskolin+RG108+Y-27632;

[0097] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine+Y-27632;

[0098] VPA+CHIR99021+Repsox+Forskolin+TSA+5-Aza-2'-deoxycytidine+Y-27632+ascorbate;

[0099] VPA+CHIR99021+Repsox+Forskolin+TSA+RG108+Y-27632+ascorbate;

[0100] VPA+CHIR99021+Repsox+Forskolin+RG108+Y-27632+ascorbate;

[0101] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine+Y-27632+ascorbate;

[0102] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580;

[0103] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108;

[0104] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Y-27632;

[0105] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+Y-27632;

[0106] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Y-27632+ascorbate;

[0107] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+ascorbate;

[0108] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+Y-27632+ascorbate;

[0109] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Go6983;

[0110] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+Go6983;

[0111] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Y-27632+Go6983;

[0112] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+Y-27632+Go6983;

[0113] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Y-27632+ascorbate+Go6983;

[0114] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+ascorbate+Go6983;

[0115] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+Y-27632+ascorbate+Go6983;

[0116] VPA+CHIR99021+Repsox+Forskolin+RG108+SP600125+Go6983+Y-27632+sodiumbut yrate+TTNPB;

[0117] VPA+CHIR99021+Repsox+Forskolin+RG108+Go6983+Y-27632+sodium butyrate+TTNPB;

[0118] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine+Go6983+Y-27632+sodium butyrate+TTNPB;

[0119] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine+Go6983+Y-27632+sodium butyrate + ascorbate+TTNPB;

[0120] VPA+CHIR99021+Repsox+Forskolin+TSA+5-Aza-2'-deoxycytidine+Y-27632+TTNPB;

[0121] VPA+CHIR99021+Repsox+Forskolin+TSA+RG108+Y-27632+TTNPB;

[0122] VPA+CHIR99021+Repsox+Forskolin+RG108+Y-27632+TTNPB;

[0123] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine+Y-27632+TTNPB;

[0124] VPA+CHIR99021+Repsox+Forskolin+TSA+5-Aza-2'-deoxycytidine+Y-27632+ascorbate+TTNPB;

[0125] VPA+CHIR99021+Repsox+Forskolin+TSA+RG108+Y-27632+ascorbate+TTNPB;

[0126] VPA+CHIR99021+Repsox+Forskolin+RG108+Y-27632+ascorbate+TTNPB;

[0127] VPA+CHIR99021+Repsox+Forskolin+5-Aza-2'-deoxycytidine+Y-27632+ascorbate+TTNPB;

[0128] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+TTNPB;

[0129] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+TTNPB;

[0130] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Y-27632+TTNPB;

[0131] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+Y-27632+TTNPB;

[0132] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Y-27632+ascorbate+TTNPB;

[0133] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+ascorbate+TTNPB;

[0134] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+Y-27632+ascorbate+TTNPB;

[0135] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Go6983+TTNPB;

[0136] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+Go6983+TTNPB;

[0137] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Y-27632+Go6983+TTNPB;

[0138] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Y-27632+Go6983+TTNPB+SP600125;

[0139] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Y-27632+Go6983+TTNPB+SP600125+ascorbate;

[0140] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+Y-27632+Go6983+TTNPB;

[0141] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+Y-27632+ascorbate+Go6983+TTNPB;

[0142] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+ascorbate+Go6983+TTNPB;

[0143] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+AM580+RG108+Y-27632+ascorbate+Go6983+TTNPB;

[0144] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+Parnate;

[0145] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+Parnate;

[0146] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+SP600125+Parnate;

[0147] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+SP600125+Parnate;

[0148] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+SP600125+Y-27632+Parnate;

[0149] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+Go6983+Parnate;

[0150] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+Go6983+Parnate;

[0151] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+Go6983+Y-27632+Parnate;

[0152] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+SP600125+Y-27632+5-Aza-2'-deoxycytidine+Parnate;

[0153] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+SP600125+Y-27632+Go6983+ascorbate+Parnate;

[0154] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+Go6983+Y-27632+ascorbate+Parnate;

[0155] VPA+CHIR99021+Repsox+Forskolin+EPZ004777+Y-27632+ascorbate+Parnate.

[0156] In a third aspect, the present invention also provides a vascular endothelial cell, which is prepared by means of the method described or the combination of the small molecule compounds described, and the vascular endothelial cell possesses the characteristics of a standard vascular endothelial cell.

[0157] In a fourth aspect, the present invention also provides the application of the vascular endothelial cells and their products, wherein the vascular endothelial cells are used to prepare or induce compounds for vascular endothelial cells or regenerative medicine seed cells, tissue engineering seed cells and their products derived from vascular endothelial cells, and can be used for basic research, clinical treatment and tissue engineering product development and production, and can also be used to prepare small molecule compound combinations of vascular endothelial cells.

[0158] In a fifth aspect, the invention also provides a kit or culture medium / base comprising the combination of said small molecule compounds.

[0159] In a sixth aspect, this invention also provides the application of small molecule compound combinations, kits or culture media containing said small molecule compound combinations, for the in vivo mobilization and induction or in vitro induction of vascular endothelial cells or regenerative medical seed cells, tissue-engineered seed cells and their products derived therefrom; these can be used for basic research, clinical treatment and the research and development and production of tissue-engineered products.

[0160] The method of this invention is carried out under conditions suitable for generating induced vascular endothelial cells, including, for example, the composition and concentration of the culture medium, the culture temperature, the culture time, and other conditions. Based on the ample teachings of the prior art and in conjunction with the exemplary description of this invention, those skilled in the art can readily determine the above culture conditions without excessive experimentation. The key lies in selecting the cell signaling pathways to be inhibited or activated and determining the order in which these pathways act.

[0161] Some embodiments of the present invention have specific effective concentrations of small molecule compounds as follows. The concentration ranges given below are for reference only and can be adapted accordingly. If other small molecules are substituted for the following small molecules, the concentrations can also be adjusted accordingly.

[0162] Forskolin concentration: 2 μM–20 μM; Repsox concentration: 2–15 μM; CHIR99021 concentration: 1 μM–10 μM; VPA concentration: 0.5 mM–1.5 mM; TTNPB concentration: 3 μM–8 μM; AM580 concentration: 0.03–0.08 μM; EPZ004777 concentration: 3–8 μM; Go6983 concentration: 1–15 μM; Y-27632 concentration: 3–15 μM; L-Ascorbin acid 2-phosphate concentration: 0.15–0.25 mM; SP600125 concentration: 1–15 μM; 5-Aza-2'-deoxycytidine concentration: 0.5–15 μM.

[0163] The method of this invention, or an adaptive adjustment based on the method of this invention, can also be used to reprogram differentiated cells into vascular smooth muscle cells. If other cells besides vascular endothelial cells and vascular smooth muscle cells are prepared using the combination of small molecule compounds provided by this invention, the concentration of the corresponding small molecule compounds or the combination of small molecule compounds can be adjusted according to actual needs.

[0164] The mechanism of this invention is as follows: This invention activates reprogrammed endogenous transcription factors by treating differentiated cells with histone acetylation and methylation, and then achieves dedifferentiation of differentiated cells by inhibiting the TGF-beta signaling pathway, activating the WNT / β-catenin signaling pathway, activating the cAMP signaling pathway, inhibiting the activity of histone methyltransferase (HMT), inhibiting the activity of histone demethylase, inhibiting the JNK signaling pathway, and inhibiting the ROCK signaling pathway, thereby regenerating vascular endothelial cells.

[0165] Numerous studies in this field have reported small molecules applicable to various signaling pathways, and those skilled in the art continue to develop such molecules. In this invention, there are no particular limitations on the structure or classification of the small molecule compounds used, but they are required to achieve inhibitory or activating functions against lysine deacetylases, TGF-β, DNMT, HMT, JNK, ROCK, WNT / β-catenin, and cAMP. Therefore, this invention covers all molecules capable of achieving the corresponding inhibitory or activating functions against lysine deacetylases, TGF-β, DNMT, HMT, histone demethylases, JNK, ROCK, WNT / β-catenin, and cAMP, and also covers alternatives capable of achieving corresponding inhibitory or activating functions against the aforementioned targets.

[0166] Compared with existing technologies, this invention has the following technical advantages: It utilizes combinations of small molecule compounds to obtain a large number of vascular endothelial cells or their products, requiring a small sample size, facilitating collection, utilizing a wide range of donor sources, and enabling standardized operation and precise quality control. It allows for the large-scale or personalized preparation of vascular endothelial cells and tissue-engineered blood vessels and related products. Furthermore, this invention can provide cell models or intervention methods for research on cardiovascular and cerebrovascular diseases, and has broad applications in basic medical research, clinical research, clinical treatment, or vascular tissue engineering, demonstrating promising application prospects. Attached Figure Description

[0167] Figure 1 Human skin fibroblasts were transdifferentiated into vascular endothelial cells. Figure A shows human skin fibroblasts, which grow in a flattened, spindle-shaped morphology. Figure B shows vascular endothelial cells obtained after treatment with forskolin, VPA, Repsox, CHIR99021, 5-Aza-2'-deoxycytidine, and TSA, which grow in a cobblestone-like morphology. Figure C shows vascular endothelial cells obtained after treatment with forskolin, VPA, Repsox, CHIR99021, TTNPB, AM580, EPZ004777, Go6983, Y-27632, L-Ascorbin acid 2-phosphate 15, and sodium butyrate (20–150 mM).

[0168] Figure 2Molecular markers of transdifferentiated vascular endothelial cells were identified. Figure A shows the expression of FLK1, a gene highly expressed in early vascular endothelial cell development, in HuFib (human skin fibroblasts, negative control), HuEC (human umbilical vein endothelial cells isolated in vivo, positive control), and IEC (experimentally transdifferentiated vascular endothelial cells). As shown in the figure, FLK1 is significantly highly expressed in transdifferentiated vascular endothelial cells. Figure B shows genes highly expressed in early vascular endothelial cell development; CD31 was also highly expressed in HuFib (human skin fibroblasts, negative control), HuEC (human umbilical vein endothelial cells isolated in vivo, positive control), and IEC (experimentally transdifferentiated vascular endothelial cells). Figure 1 shows the expression of CD31 in transdifferentiated vascular endothelial cells (CESCs) and IECs (experimentally transdifferentiated vascular endothelial cells). Figure 2 shows the expression of CD31 in transdifferentiated vascular endothelial cells. Figure 3 shows the expression of VWF, a gene highly expressed in the early stages of vascular endothelial cell development, in HuFib (human skin fibroblasts, negative control), HuEC (human umbilical vein endothelial cells isolated in vivo, positive control), and IECs (experimentally transdifferentiated vascular endothelial cells). Figure 4 shows the expression of VWF in transdifferentiated vascular endothelial cells. Figure 5 shows the immunofluorescence staining of VWF in transdifferentiated vascular endothelial cells. The results show that the transdifferentiated cells are positive for staining.

[0169] Figure 3 This is an experimental diagram of AC-LDL uptake in transdifferentiated vascular endothelial cells. Ac-LDL labeled with fluorescent probes was added and co-cultured with HuFib and IEC cells, respectively. After 5 hours, fluorescence detection was performed. Figure A shows the detection of AC-LDL uptake by HuFib, and the result is negative. Figure B shows the detection of AC-LDL uptake by IEC cells, and the result is positive.

[0170] Figure 4 To assess the tubular formation ability of transdifferentiated vascular endothelial cells using a tubule formation assay, Figure A shows the morphology of cells immediately after seeding onto a matrix gel and photographed after 15 hours of incubation at 37°C. Figure B shows the morphology of IEC cells after 15 hours. Figure C shows the morphology of HuEC cells after 15 hours. Figure D shows the morphology of HuFib cells after 15 hours. Detailed Implementation

[0171] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following experimental scheme.

[0172] Example 1

[0173] 1. Isolation of skin fibroblasts

[0174] 1.1 Obtain a skin tissue block with a diameter of approximately 1 cm from any location on the donor, isolate skin fibroblasts using the adherence method, and culture the isolated cells in a basal culture medium, which is: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM.

[0175] 1.2 Cell passage expansion was performed, with cell passages between 3 and 12, for induction of transdifferentiation into vascular endothelial cells. On Day 1, the cell density was seeded at 4–6 x 10⁻⁶ cells. 3 / cm2 was cultured in an incubator at 37℃ and 5% CO2.

[0176] 2. Directional induction of skin fibroblasts into vascular endothelial cells

[0177] 2.1 When initiating transdifferentiation (Day 0), completely replace the basal culture medium with the induction culture medium. Culture the cells for 2–12 days. Culture medium A consists of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM ​​medium (Gibco) + forskolin (1 μM–30 μM) + Repsox (2–15 μM) + CHIR99021 (1 μM–15 μM) + VPA (0.5 mM–1.5 mM) + 1 μM–20 μM 5-Aza-2'-deoxycytidine + 50 nM–1 μM TSA. In this culture system, the 10% fetal bovine serum can also be replaced by a serum substitute (Invitrogen) at a concentration of 10%–20%. The 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) are optional. Cells were cultured at 37°C in a 5% CO2 environment.

[0178] 2.2 After the cells were treated as described above, cell growth factors were added to the induction culture medium, including b-FGF (5 ng / ml to 25 ng / ml), EGF (1 ng / ml to 10 ng / ml) and IGF-1 (5 ng / ml to 50 ng / ml). The cells were cultured for 4 to 16 days at 37°C and 5% CO2.

[0179] 3. Expansion and culture of induced vascular endothelial cells

[0180] Subsequently, the culture medium was replaced with standard vascular endothelial cell culture medium or commercially available vascular endothelial cell culture medium (Lonza), and the cells were cultured at 37°C in a 5% CO2 environment. The standard vascular endothelial cell culture medium consisted of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high-glucose DMDM ​​medium (Gibco) + FBS (10% FBS) + HYDROCORTISONE (0.2 μg / ml) + VEGF (0.5 ng / ml) + R3-IGF-1 (20 ng / ml) + ASCORBIC ACID (1 μg / ml) + hFGF-B (10 ng / ml). In this culture system, the 10% fetal bovine serum could be replaced with a serum substitute (Invitrogen) at a concentration of 10%–20%; the 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) could be omitted.

[0181] 4. Detection of induced vascular endothelial cells, see [link to relevant documentation]. Figures 1-4 ;

[0182] 4.1 Molecular markers of vascular endothelial cells were detected by immunohistochemical staining and RT-PCR. The main molecular markers were as follows: CD31; VWF; Enos; VE;

[0183] 4.2 In vitro functional verification:

[0184] 4.2.1 Detection of AC-LDL uptake by endothelial cells:

[0185] (1) Cell preparation: IEC and HuFib were plated in 24-well plates at a density of 1×10^4 cells / ml.

[0186] (2) Wash twice with PBS.

[0187] (3) The Ac-LDL-594, IEC and HuFib were diluted 1:100 and loaded with 4 l Ac-LDL + 396 l EBM Mdedium and 4 l Ac-LDL + 396 l DMEM Mdedium, respectively.

[0188] (4) Incubate the cells in a 37℃ incubator for 5 hours.

[0189] (5) Observe and photograph under a microscope.

[0190] 4.2.2 Formation of vascular tubular structures

[0191] (1) Before the experiment, place the matrigel in a 4°C refrigerator for 24-48 hours to melt it into a liquid. Place the pipette tip in the refrigerator to pre-cool it. The operation is carried out on ice.

[0192] (2) Spread Matrigel 50ul / well (96-well plate) at room temperature or 37℃ for 30-60 minutes. Seed IEC and HuFib at a cell density of 2*10^4 / well. Add EBM Medium to IEC and DMEM Medium to HuFib.

[0193] (3) Observe and record photos at 37℃ for 0h, 6h, 10h, 15h and 24h.

[0194] Example 2

[0195] 1. Isolation of skin fibroblasts

[0196] 1.1 Obtain a skin tissue block with a diameter of approximately 1 cm from any location on the donor, isolate skin fibroblasts using the adherence method, and culture the isolated cells in a basal culture medium, which is: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM.

[0197] 1.2 Cell passage expansion was performed, with cell passages between 3 and 12, for induction of transdifferentiation into vascular endothelial cells. On Day 1, the cell density was seeded at 4–6 x 10⁻⁶ cells. 3 / cm2 was cultured in an incubator at 37℃ and 5% CO2.

[0198] 2. Directional induction of skin fibroblasts into vascular endothelial cells

[0199] When initiating transdifferentiation (Day 0), completely replace the basal culture medium with the induction culture medium and culture the cells for 10–28 days. Culture medium A consists of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM ​​medium (Gibco) + forskolin (1 μM–30 μM) + Repsox (2–15 μM) + CHIR99021 (1 μM–15 μM) + VPA (0.5 mM–1.5 mM) + 1 μM–20 μM 5-Aza-2'-deoxycytidine + 50 nM–1 μM TSA. In this culture system, the 10% fetal bovine serum can also be replaced by a serum substitute (Invitrogen) at a concentration of 10%–20%. The 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) can be omitted. Cells were cultured at 37°C in a 5% CO2 environment.

[0200] 3. Expansion and culture of induced vascular endothelial cells

[0201] Subsequently, the culture medium was replaced with standard vascular endothelial cell culture medium or commercially available vascular endothelial cell culture medium (Lonza), and the cells were cultured at 37°C in a 5% CO2 environment. The standard vascular endothelial cell culture medium consisted of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high-glucose DMDM ​​medium (Gibco) + FBS (10% FBS) + HYDROCORTISONE (0.2 μg / ml) + VEGF (0.5 ng / ml) + R3-IGF-1 (20 ng / ml) + ASCORBIC ACID (1 μg / ml) + hFGF-B (10 ng / ml). In this culture system, the 10% fetal bovine serum could be replaced with a serum substitute (Invitrogen) at a concentration of 10%–20%; the 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) could be omitted.

[0202] 4. Detection of induced vascular endothelial cells

[0203] 4.1 Molecular markers of vascular endothelial cells were detected by immunohistochemical staining and RT-PCR. The main molecular markers were as follows: CD31; VWF; Enos; VE;

[0204] 4.2 In vitro functional verification:

[0205] 4.2.1 Detection of AC-LDL uptake by endothelial cells:

[0206] (1) Cell preparation: IEC and HuFib were mixed at a ratio of 1×10⁻⁶. 4 Cells / ml density was used for plating in 24-well plates.

[0207] (2) Wash twice with PBS.

[0208] (3) The Ac-LDL-594, IEC and HuFib were diluted 1:100 and loaded with 4 l Ac-LDL + 396 l EBM Mdedium and 4 l Ac-LDL + 396 l DMEM Mdedium, respectively.

[0209] (4) Incubate the cells in a 37℃ incubator for 5 hours.

[0210] (5) Observe and photograph under a microscope.

[0211] 4.2.2 Formation of vascular tubular structures

[0212] (1) Before the experiment, place the matrigel in a 4°C refrigerator for 24-48 hours to melt it into a liquid. Place the pipette tip in the refrigerator to pre-cool it. The operation is carried out on ice.

[0213] (2) Spread Matrigel 50ul / well (96-well plate) at room temperature or 37℃ for 30-60 minutes. Seed IEC and HuFib at a cell density of 2*10^4 / well. Add EBM Medium to IEC and DMEM Medium to HuFib.

[0214] (3) Observe and record photos at 37℃ for 0h, 6h, 10h, 15h and 24h.

[0215] Example 3

[0216] 1. Isolation of skin fibroblasts

[0217] 1.1 Obtain a skin tissue block with a diameter of approximately 1 cm from any location on the donor, isolate skin fibroblasts using the adherence method, and culture the isolated cells in a basal culture medium, which is: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM.

[0218] 1.2 Cell passage expansion was performed, with cell passages between 3 and 12, for induction of transdifferentiation into vascular endothelial cells. On Day 1, the cell density was seeded at 4–6 x 10⁻⁶ cells. 3 / cm2 was cultured in an incubator at 37℃ and 5% CO2.

[0219] 2. Directional induction of skin fibroblasts into vascular endothelial cells

[0220] When initiating transdifferentiation (Day 0), completely replace the basal culture medium with the induction culture medium and culture the cells for 10–28 days. Culture medium A consists of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM ​​medium (Gibco) + forskolin (1 μM–30 μM) + Repsox (2–15 μM) + CHIR99021 (1 μM–15 μM) + VPA (0.5 mM–1.5 mM) + 1 μM–20 μM 5-Aza-2'-deoxycytidine + 50 nM–1 μM TSA. In this culture system, the 10% fetal bovine serum can also be replaced by a serum substitute (Invitrogen) at a concentration of 10%–20%. The 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) can be omitted. Cells were cultured at 37°C in a 5% CO2 environment.

[0221] 3. Expansion and culture of induced vascular endothelial cells

[0222] Subsequently, the culture medium was replaced with standard vascular endothelial cell culture medium or commercially available vascular endothelial cell culture medium (Lonza), and the cells were cultured at 37°C in a 5% CO2 environment. The standard vascular endothelial cell culture medium consisted of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high-glucose DMDM ​​medium (Gibco) + FBS (10% FBS) + HYDROCORTISONE (0.2 μg / ml) + VEGF (0.5 ng / ml) + R3-IGF-1 (20 ng / ml) + ASCORBIC ACID (1 μg / ml) + hFGF-B (10 ng / ml). In this culture system, the 10% fetal bovine serum could be replaced with a serum substitute (Invitrogen) at a concentration of 10%–20%; the 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) were optional.

[0223] 4. Detection of induced vascular endothelial cells

[0224] Molecular markers of vascular endothelial cells were detected by immunohistochemical staining and RT-PCR. The main molecular markers were CD31, VWF, Enos, and VE.

[0225] Example 4

[0226] 1. Isolation of skin fibroblasts

[0227] 1.1 Obtain a skin tissue block with a diameter of approximately 1 cm from any location on the donor, isolate skin fibroblasts using the adherence method, and culture the isolated cells in a basal culture medium, which is: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM.

[0228] 1.2 Cell passage expansion was performed, with cell passages between 3 and 12 generations, for induction of transdifferentiation into vascular endothelial cells. On Day 1, the cell density was seeded at 4–6 × 10⁶ cells / year. 3 / cm 2 Incubated in an incubator at 37℃ and 5% CO2.

[0229] 2. Directional induction of skin fibroblasts into vascular endothelial cells

[0230] When initiating transdifferentiation (Day 0), completely replace the basal culture medium with the induction culture medium. Culture cells for 10–28 days. Culture medium A consists of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high-glucose DMDM ​​medium (Gibco) + forskolin (1 μM–30 μM) + Repsox (2–15 μM) + CHIR99021 (1 μM–15 μM) + VPA (0.5 mM–1.5 mM) + 1 μM–20 μM 5-Aza-2'-deoxycytidine + 50 nM–1 μM TSA+Go6983 (1–15 μM) was used in this culture system. 10% fetal bovine serum could be replaced with a serum substitute (Invitrogen) at a concentration of 10%–20%. 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) were not required. Cells were cultured at 37°C in a 5% CO2 environment.

[0231] 3. Expansion and culture of induced vascular endothelial cells

[0232] Subsequently, the culture medium was replaced with standard vascular endothelial cell culture medium or commercially available vascular endothelial cell culture medium (Lonza), and the cells were cultured at 37°C in a 5% CO2 environment. The standard vascular endothelial cell culture medium consisted of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high-glucose DMDM ​​medium (Gibco) + FBS (10% FBS) + HYDROCORTISONE (0.2 μg / ml) + VEGF (0.5 ng / ml) + R3-IGF-1 (20 ng / ml) + ASCORBIC ACID (1 μg / ml) + hFGF-B (10 ng / ml). In this culture system, the 10% fetal bovine serum could be replaced with a serum substitute (Invitrogen) at a concentration of 10%–20%; the 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) could be omitted.

[0233] 4. Detection of induced vascular endothelial cells, same as in Example 1.

[0234] Example 5

[0235] 1. Isolation of skin fibroblasts

[0236] 1.1 Obtain a skin tissue block with a diameter of approximately 1 cm from any location on the donor, isolate skin fibroblasts using the adherence method, and culture the isolated cells in a basal culture medium, which is: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM.

[0237] 1.2 Cell passage expansion was performed, with cell passages between 3 and 12, for induction of transdifferentiation into vascular endothelial cells. On Day 1, the cell density was seeded at 4–6 x 10⁻⁶ cells. 3 / cm2 was cultured in an incubator at 37℃ and 5% CO2.

[0238] 2. Directional induction of skin fibroblasts into vascular endothelial cells

[0239] When initiating transdifferentiation (Day 0), completely replace the basal culture medium with the induction culture medium. Culture cells for 10–28 days. Culture medium A consists of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high-glucose DMDM ​​medium (Gibco) + forskolin (1 μM–30 μM) + Repsox (2–15 μM) + CHIR99021 (1 μM–15 μM) + VPA (0.5 mM–1.5 mM) + 1 μM–20 μM 5-Aza-2'-deoxycytidine + 50 nM–1 μM The culture system consists of TSA + Go6983 (1–15 μM) + Y-27632 (3–15 μM). 10% fetal bovine serum can be replaced with a serum substitute (Invitrogen) at a concentration of 10%–20%. 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) are optional. Cells are cultured at 37°C in a 5% CO2 environment.

[0240] 3. Expansion and culture of induced vascular endothelial cells

[0241] Subsequently, the culture medium was replaced with standard vascular endothelial cell culture medium or commercially available vascular endothelial cell culture medium (Lonza), and the cells were cultured at 37°C in a 5% CO2 environment. The standard vascular endothelial cell culture medium consisted of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high-glucose DMDM ​​medium (Gibco) + FBS (10% FBS) + HYDROCORTISONE (0.2 μg / ml) + VEGF (0.5 ng / ml) + R3-IGF-1 (20 ng / ml) + ASCORBIC ACID (1 μg / ml) + hFGF-B (10 ng / ml). In this culture system, the 10% fetal bovine serum could be replaced with a serum substitute (Invitrogen) at a concentration of 10%–20%; the 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) could be omitted.

[0242] 4. Detection of induced vascular endothelial cells, same as in Example 1.

[0243] Example 6

[0244] 1. Isolation of skin fibroblasts

[0245] 1.1 Obtain a skin tissue block with a diameter of approximately 1 cm from any location on the donor, isolate skin fibroblasts using the adherence method, and culture the isolated cells in a basal culture medium, which is: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM.

[0246] 1.2 Cell passage expansion was performed, with cell passages between 3 and 12, for induction of transdifferentiation into vascular endothelial cells. On Day 1, the cell density was seeded at 4–6 x 10⁻⁶ cells. 3 / cm2 was cultured in an incubator at 37℃ and 5% CO2.

[0247] 2. Directional induction of skin fibroblasts into vascular endothelial cells

[0248] When initiating transdifferentiation (Day 0), completely replace the basal culture medium with the induction culture medium. Culture cells for 10–28 days. Culture medium A consists of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high-glucose DMDM ​​medium (Gibco) + forskolin (2 μM–20 μM) + Repsox (2–15 μM) + CHIR99021 (1 μM–10 μM) + VPA (0.5 mM–1.5 mM) + TTNPB (3 μM–8 μM) + AM580 (0.03–0.08 μM) + EPZ004777 (3–8 μM) + Go6983 (1–15 μM) + Y-27632 (3–15 μM) + L-Ascorbin acid 2-phosphate (0.15–0.25 mM). In this culture system, 10% fetal bovine serum can be replaced by a serum substitute (invitrogen) at a concentration of 10%–20%; 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) can be omitted. Cells are cultured at 37°C and 5% CO2.

[0249] 3. Expansion and culture of induced vascular endothelial cells

[0250] Subsequently, the culture medium was replaced with standard vascular endothelial cell culture medium or commercially available vascular endothelial cell culture medium (Lonza), and the cells were cultured at 37°C in a 5% CO2 environment. The standard vascular endothelial cell culture medium consisted of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high-glucose DMDM ​​medium (Gibco) + FBS (10% FBS) + HYDROCORTISONE (0.2 μg / ml) + VEGF (0.5 ng / ml) + R3-IGF-1 (20 ng / ml) + ASCORBIC ACID (1 μg / ml) + hFGF-B (10 ng / ml). In this culture system, the 10% fetal bovine serum could be replaced with a serum substitute (Invitrogen) at a concentration of 10%–20%; the 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) could be omitted.

[0251] 4. Detection of induced vascular endothelial cells, same as in Example 1.

[0252] Example 7

[0253] 1. Isolation of skin fibroblasts

[0254] 1.1 Obtain a skin tissue block with a diameter of approximately 1 cm from any location on the donor, isolate skin fibroblasts using the adherence method, and culture the isolated cells in a basal culture medium, which is: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM.

[0255] 1.2 Cell passage expansion was performed, with cell passages between 3 and 12, for induction of transdifferentiation into vascular endothelial cells. On Day 1, the cell density was seeded at 4–6 x 10⁻⁶ cells. 3 / cm2 was cultured in an incubator at 37℃ and 5% CO2.

[0256] 2. Directional induction of skin fibroblasts into vascular endothelial cells

[0257] When initiating transdifferentiation (Day 0), completely replace the basal culture medium with the induction culture medium. Culture cells for 10–28 days. Culture medium A consists of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high-glucose DMDM ​​medium (Gibco) + forskolin (2 μM–20 μM) + Repsox (2–15 μM) + CHIR99021 (1 μM–10 μM) + VPA (0.5 mM–1.5 mM) + TTNPB (3 μM–8 μM) + AM580 (0.03–0.08 μM) + EPZ004777 (3–8 μM) + Go6983 (1–15 μM) + Y-27632 (3–15 μM) + L-Ascorbin acid 2-phosphate (0.15–0.25 mM) + sodium Butyrate (20–150 mM) can be used in this culture system. 10% fetal bovine serum can be replaced by a serum substitute (Invitrogen) at a concentration of 10%–20%. 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) can be omitted. Culture cells at 37°C and 5% CO2.

[0258] 3. Expansion and culture of induced vascular endothelial cells

[0259] Subsequently, the culture medium was replaced with standard vascular endothelial cell culture medium or commercially available vascular endothelial cell culture medium (Lonza), and the cells were cultured at 37°C in a 5% CO2 environment. The standard vascular endothelial cell culture medium consisted of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high-glucose DMDM ​​medium (Gibco) + FBS (10% FBS) + HYDROCORTISONE (0.2 μg / ml) + VEGF (0.5 ng / ml) + R3-IGF-1 (20 ng / ml) + ASCORBIC ACID (1 μg / ml) + hFGF-B (10 ng / ml). In this culture system, the 10% fetal bovine serum could be replaced with a serum substitute (Invitrogen) at a concentration of 10%–20%; the 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) could be omitted.

[0260] 4. Detection of induced vascular endothelial cells, same as in Example 1.

[0261] Example 8

[0262] 1. Isolation of skin fibroblasts

[0263] 1.1 Obtain a skin tissue block with a diameter of approximately 1 cm from any location on the donor, isolate skin fibroblasts using the adherence method, and culture the isolated cells in a basal culture medium, which is: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM.

[0264] 1.2 Cell passage expansion was performed, with cell passages between 3 and 12, for induction of transdifferentiation into vascular endothelial cells. On Day 1, the cell density was seeded at 4–6 x 10⁻⁶ cells. 3 / cm2 was cultured in an incubator at 37℃ and 5% CO2.

[0265] 2. Directional induction of skin fibroblasts into vascular endothelial cells

[0266] When initiating transdifferentiation (Day 0), completely replace the basal culture medium with the induction culture medium and culture the cells for 10–28 days. Culture medium A consists of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high glucose DMDM ​​medium (Gibco) + forskolin (1 μM–30 μM) + Repsox (2–15 μM) + CHIR99021 (1 μM–15 μM) + VPA (0.5 mM–1.5 mM) + 1 μM–20 μM 5-Aza-2'-deoxycytidine + sodium butyrate (20–150 mM). In this culture system, the 10% fetal bovine serum can also be replaced by a serum substitute (Invitrogen) at a concentration of 10%–20%. The 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) can be omitted. Cells were cultured at 37°C in a 5% CO2 environment.

[0267] 3. Expansion and culture of induced vascular endothelial cells

[0268] Subsequently, the culture medium was replaced with standard vascular endothelial cell culture medium or commercially available vascular endothelial cell culture medium (Lonza), and the cells were cultured at 37°C in a 5% CO2 environment. The standard vascular endothelial cell culture medium consisted of: 10% fetal bovine serum (Hyclone) + 100 U / ml penicillin (Sigma) + 100 μg / ml streptomycin (Sigma) + high-glucose DMDM ​​medium (Gibco) + FBS (10% FBS) + HYDROCORTISONE (0.2 μg / ml) + VEGF (0.5 ng / ml) + R3-IGF-1 (20 ng / ml) + ASCORBIC ACID (1 μg / ml) + hFGF-B (10 ng / ml). In this culture system, the 10% fetal bovine serum could be replaced with a serum substitute (Invitrogen) at a concentration of 10%–20%; the 100 U / ml penicillin (Sigma) and 100 μg / ml streptomycin (Sigma) could be omitted.

[0269] 4. Detection of induced vascular endothelial cells, same as in Example 1.

[0270] The vascular endothelial cells obtained in the above embodiments and the detection results are shown in [the original text]. Figures 1-4 The skin fibroblasts used in the examples can be replaced with other differentiated cells, such as blood cells, fat cells, etc.

[0271] It should be understood that, within the scope of this invention, the above-described technical features of this invention, along with the various technical features specifically described below (such as in the embodiments), can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

Claims

1. A method for preparing vascular endothelial cells from induced differentiated cells, characterized in that, The method is operated as follows: the differentiated cells are directionally induced using an induction culture medium, and then expanded after induction culture to obtain vascular endothelial cells; the differentiated cells are human fibroblasts; The induction culture medium is selected from any combination of the following small molecule compounds: forskolin+Repsox+CHIR99021+VPA+5-Aza-2'-deoxycytidine+TSA; forskolin+Repsox+CHIR99021+VPA+5-Aza-2'-deoxycytidine+TSA+Go6983; forskolin+Repsox+CHIR99021+VPA+5-Aza-2'-deoxycytidine+TSA+Go6983+Y-27632; forskolin+Repsox+CHIR99021+VPA+TTNPB+AM580+EPZ004777+Go6983+Y-27632+L-Ascorbin acid 2-phosphate + sodium butyrate; forskolin+Repsox+CHIR99021+VPA+5-Aza-2'-deoxycytidine+sodium butyrate; The fibroblasts were induced to expand and cultured. The expansion culture medium consisted of the following components: 10% fetal bovine serum or 10-20% serum substitute + high glucose DMEM medium + 0.2ug / ml HYDROCORTISONE + 0.5ng / ml VEGF + 20ng / ml R3-IGF-1 + 1ug / ml ASCORBIC ACID + 10ng / ml hFGF-B.

2. The method for preparing vascular endothelial cells from induced differentiated cells according to claim 1, characterized in that, In addition to the addition of small molecule compounds, cell growth factors are added to the induction culture medium for further induction culture. The growth factors include b-FGF, EGF and IGF-1, and the culture time is 4 to 16 days.

3. The method for preparing vascular endothelial cells from induced differentiated cells according to claim 1, characterized in that, The concentrations of the small molecules in the combination of the small molecule compounds are as follows: forskolin: 1 μM to 30 μM; Repsox: 2 to 15 μM; CHIR99021: 1 μM to 15 μM; VPA: 0.5 mM to 1.5 mM; 5-Aza-2'-deoxycytidine: 1 μM to 20 μM; TSA: 50 nM to 1 μM; Go6983: 1 to 15 μM; Y-27632: 3 to 15 μM; TTNPB: 3 μM to 8 μM; AM580: 0.03 to 0.08 μM. The concentration of EPZ004777 is 3~8μM; the concentration of L-Ascorbin acid 2-phosphate is 0.15~0.25mM; and the concentration of sodium butyrate is 20~150mM.

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