A method of promoting differentiation of target cells into neural cells

Abstract

The application discloses a method for promoting differentiation of target cells into nerve cells, and the operation is as follows: after the target cells are cultured in a nerve cell inducing solution for 5-15 days, the target cells are transferred into a nerve cell culture solution, and are continuously subcultured to obtain nerve cells; the nerve cell inducing solution is Mix Neu: HG-DMEM culture solution containing 10% FBS, and includes A8301, bFGF, EPZ004777, RG108, parnate, CHIR99021, Forskolin, VPA, AM580, BIX 01294, or the nerve cell inducing solution is DMEM / F12 culture solution containing 10% KOSR, and includes bFGF, Y-27632, VPA, EPZ004777, Forskolin and Repsox; and the target cells are rapidly and successfully differentiated into nerve cells under the action of small molecule compounds.

Description

[0001] This case is derived from a divisional application for patent application No. 2018104072539, entitled "Technology and Application of Regulating the Jak-Stat Pathway to Promote Cell Differentiation, Dedifferentiation and Rejuvenation". Technical Field

[0002] This invention belongs to the field of cell biology technology, and in particular relates to a method for promoting the differentiation of target cells into nerve cells. Background Technology

[0003] As individuals age, their stem cells also age, leading to malignant transformation or degeneration in multiple organs, including bones, cartilage, heart, muscles, brain, skin, pancreas, liver, kidneys, and gastrointestinal tract. Abnormal immune system function is also associated with aging. In fact, chronic inflammation of tissues and organs is a cause of their degenerative aging. Aged cells often exhibit DNA damage or mutations, telomere shortening, epigenetic, redox, and energy metabolism abnormalities, decreased proliferative capacity, and increased cell death. Aged stem cells lose their ability to differentiate into certain lineages and abnormally differentiate into others. For example, it is well known that bone marrow mesenchymal stem cells from older individuals have decreased osteogenic and chondrogenic abilities but increased adipogenic abilities. Therefore, aged bone marrow filled with adipose tissue is often called "yellow bone marrow." Similarly, neural stem cells in older individuals differentiate less into neurons and more into astrocytes, which is thought to be related to cognitive decline in the elderly.

[0004] Stem cells, particularly mesenchymal stem cells (MSCs), are promising candidates for treating or intervening in the aging process and related diseases due to their accessibility, scalability, pluripotency, growth factor release, and immunomodulatory capabilities. MSCs have been widely used clinically, including in the treatment of graft-versus-host disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), spinal cord injury, lupus erythematosus, arthritis, and various age-related diseases. For allogeneic use, umbilical cord mesenchymal stem cells are widely considered. However, long-term / repeated use of "non-autologous" cells always carries clinical risks. Autologous transplantation is considered safer; unfortunately, as people age, their mesenchymal stem cells also age. Aged mesenchymal stem cells lose many important functions, limiting their clinical application. Induced pluripotent stem cells (iPSs), which are young cells obtained from older individuals, were once considered a good cell source for treatment. However, iPS induction efficiency is low, and the introduction of exogenous genes often leads to genetic mutations, hindering clinical application. Recently, genetic modification has been used as a method for rejuvenation, but this method still carries risks of off-target effects and tumorigenesis. Therefore, developing targeted methods for preparing safe and rejuvenating autologous cells, as well as obtaining safe and reparative autologous cells, is of great significance for preventing, delaying and reversing the human aging process, repairing the structure or function of tissues and organs, and improving health and quality of life. Summary of the Invention

[0005] The purpose of this invention is to provide a technology and its application for regulating the JAK-STAT pathway to induce cell differentiation, dedifferentiation, and rejuvenation, particularly a method for regulating the JAK-STAT signaling pathway to modulate cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging, apoptosis, and to reverse aging and prolong lifespan. Cell products obtained through this method (such as regenerative fibroblasts and rejuvenated mesenchymal stem cells) can be used to prevent, delay, and reverse the human aging process, and to repair the structure or function of tissues and organs. The regenerative fibroblasts prepared according to this patented technology possess both the characteristics of skin fibroblasts and mesenchymal stem cells, and are therefore named regenerative fibroblasts (rFib), induced and rejuvenated mesenchymal stem cells (irMSC), or induced mesenchymal stem cells (iMSC), collectively referred to as rFib in this paper.

[0006] The technical solution of the present invention is as follows: a technology and its application for regulating the JAK-STAT pathway to induce cell differentiation, dedifferentiation, and rejuvenation, characterized in that the method described herein is to quantitatively and / or periodically activate or inhibit the JAK-STAT signaling pathway.

[0007] The method targets genes or proteins that are highly expressed, poorly expressed, or suppressed in the JAK-STAT signaling pathway, including CXCL2 (gene number / Accession: AY577905.1).

[0008] OS1 (Gene ID / Accession: NM_005633.3), STAT5B (Gene ID / Accession: NM_012448.3), JAK1 (Gene ID / Accession: NM_001321857.1), JAK3 (Gene ID / Accession: NM_000215.3), SOCS3 (Gene ID / Accession: NM_003955.4), IL6ST (Gene ID / Accession: NM_001243835.1), STAT1 (Gene ID / Accession: NM_007315.3), STAT2 (Gene ID / Accession: NM_198332.1), STAT 3 (Gene ID / Accession: NM_213662.1), STAT4 (Gene ID / Accession: NM_001243835.1), STAT6 (Gene ID / Accession: NM_001178081.1), STAT5A (Gene ID / Accession: NM_001288720.1), IRF9 (Gene ID / Accession: NM_006084.4), IL6 (Gene ID / Accession: XM_005249745.5), IL6R (Gene ID / Accession: NM_181359.2), IL2 (Gene ID / Accession: NM_000586.3) (e.g., IL2) At least one of the following: A and / or IL2B), PRKCD (Gene ID / Accession: NM_001354679.1), CXCL12 (Gene ID / Accession: NM_000609.6), CXCR4 (Gene ID / Accession: NM_003467.2), JAK2 (Gene ID / Accession: NM_004972.3), IL15RA (Gene ID / Accession: NM_001351095.1), IL20RB (Gene ID / Accession: XM_006713665.4), GHR (Gene ID / Accession: NM_001242406.2), and PRLR (Gene ID / Accession: NM_001204314.2).

[0009] The cells described are the initial target cells for regulating JAK-STAT. These target cells are derived from mammals such as humans, mice, monkeys, and pigs, and include fibroblasts, epithelial cells, adipocytes, blood cells, mesenchymal stem cells, nerve cells, muscle cells, cardiomyocytes, smooth muscle cells, vascular endothelial cells, induced pluripotent stem cells, embryonic stem cells, osteoblasts, chondrocytes, adipocytes, and osteoclasts. The cells prepared by the method described herein are defined as target cells, derived from target cells, including various cell types with different characteristics generated during the regulation of JAK-STAT in target cells. These characteristics include at least one of the following: differentiation, dedifferentiation, transdifferentiation, rejuvenation, senescence, and apoptosis.

[0010] The quantification of the activation or inhibition of the JAK-STAT signaling pathway is that at least one gene or protein target of the JAK-STAT signaling pathway in the target cell is upregulated or downregulated by any one of the following multiples from 1 to 300 times relative to the target cell.

[0011] The timing of activation or inhibition of the JAK-STAT signaling pathway is defined as the high expression, low expression, or non-expression of at least one gene or protein target that regulates the JAK-STAT signaling pathway in the target cell, with a regulation time of 24 hours to 220 days. The resulting target cells or long-term maintenance of at least one gene or protein target that regulates the JAK-STAT signaling pathway are either highly expressed, low expressed, or non-expressed, or restored to the same expression level as the target cells.

[0012] The method described above can activate or inhibit the JAK-STAT signaling pathway by modulating at least one of the following pathways or targets: NOD-like receptor signaling pathway, Focal adhesion, cell cycle, tricarboxylic acid cycle, TGFbeta signaling pathway, WNT signaling pathway, Notch signaling pathway, p53 signaling pathway, insulin signaling pathway, calcium signaling pathway, Interleukin-19, Interleukin-20, Interleukin-22, Interleukin-24, IL7HDAC (histone deacetylase), PKC signaling pathway, RAR pathway, adenylate cyclase signaling pathway, HMT (histone methyltransferase), DNMT (DNA methyltransferase), and histone demethylase inhibitors.

[0013] The cellular targets and signaling pathways that activate or inhibit the JAK-STAT signaling pathway are described.

[0014] The gene or protein targets in the NOD-like receptor signaling pathway are selected from NAIP, IL6, CXCL12, NOD1, TAB3, CARD6, CXCL2, CXCL1, CXCL3, CARD8, CARD9, CASP1, CASP12, and CASP4.

[0015] One or more of the following: CASP5, NFKB1, TMEM173, TNF, NFKBIB, NOD2, PYDC1, PYCARD, TAB1, TAB2, TNF, TLR4, NLRP1, NLRP12, NLRP3, NLRP6, MCU, RIPK3, RHOA, TAK1, BIRC2, ATG16L1, ATG5, ATG12, TANK.

[0016] The gene or protein targets in the Focal adhesion pathway are selected from...

[0017] TNXB,RAPGEF1,ITGB8,SRC,THBS1,ITGA3,VCL,CAPN2,FLT4,FLT1,ITGA3,ITGB1,ITGB3,ITGB5,ITGB6,ITGB7,ITGA1,ITGA10,I TGA11,ITGA2,ITGA2B,ITGA5,ITGA6,ITGA7,ITGA8,,ITGA9,ITGAV,PDRVG,PDGFA,PDGFB,PDGFC,PDGFD,PDGFRA,PDGFRB,BIRC3,

[0018] One or more of BIRC2, BCL2, DOCK1, FN1, HGF, EGF, EGFR, IGF1, IGF1R, VEGFA, VEGFB, VEGFC, and CTNNB1;

[0019] The gene or protein targets used to regulate the cell cycle are selected from...

[0020] MAD2L1, BUB1, ORC1, ORC2, ORC3, ORC4, ORC5, ORC6, ATM, ATR, CCNA1, CCNA2, CCNB1, CCNB2, CCNB3, CCND1, CCND2, SMAD2, SMAD3, SMAD4, E2F2, E2F2, E2F4, E2F5, EP300, FZR1, GADD45A, GADD45B, GADD 45B, STAG1, STAG2, CDC14A, CDC14B, CDC20, CDC25A, CDC25B, MYC, SMC3, CDC16, YWHAH, YWHAB, YWHAQ, YWHAE, YWHAG, YWHAZ (one or more of these).

[0021] The gene or protein targets used to regulate the tricarboxylic acid cycle are selected from...

[0022] One or more of the following: IDH3G, IDH3B, MDH2, SDHB, OGDH, MDH1, OGDHL, SUCLG1, SUCLG2, SUCLA2, SDHA, SDHB, SDHC, PDH A1, PDHB, ACLY

[0023] The gene or protein targets in the TGF beta signaling pathway are selected from...

[0024] ACVR1C,THBS1,FST,TGFB1,TGFBR1,TGFBR2.TGFBR3,BMP4,RUNX3,RUNX2,CREBBP,

[0025] One or more of IFNG, HRAS, FOS, TGFB2, TGFB3, ACVRL1, FOXO3, MTOR, KRAS, CREB1, ATF1, ATF2, ATF4, AKT1, AK T2, AKT3, HNF4A, HNF4G, and PIK3R3.

[0026] The gene or protein targets in the WNT signaling pathway are selected from...

[0027] One or more of PRKCA, WNT7B, PRICKLE1, LRP6, CTNNB1, FZD4, CCND2, PRICK, WNT5A, WNT1, WNT10A, WNT11, WNT9A, WNT9B, WNT3, and WNT4B;

[0028] The gene or protein targets in the Notch signaling pathway are selected from...

[0029] CIR1,KAT2B,MAML2,PSEN2,DVL2,RFNG,SNW1,DLL4,DTX3,DLL3,DLL1,DTX1,DTX2,

[0030] One or more of CREBBP, CTBP1, CTBP2, JAG1, JAG2, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PSEN1, PSEN2;

[0031] The gene or protein targets in the P53 signaling pathway are selected from...

[0032] One or more of CCNG2, SIAH1, BBC3, TP53AIP1, TP53, SETD7, ATF3, CCNA2, CDK2, CCNG1, CHEK1, PRKCDKAT2B, PRL23, PPP2CA.

[0033] The gene or protein targets in the Calcium signaling pathway are selected from RYR1, RYR2, RYR3, ESR1, and AR (androgen).

[0034] One or more of the following: receptor (KDR (kinase insertdomain receptor), VDR (vitamin D receptor), ITPR1, ITPR2, ITPR3, PDE1A, PDE1B, PDE1C, PRKCA, PRKCD, PRKCE, PRKCG.

[0035] The gene or protein targets in the insulin signaling pathway are selected from one or more of the following: RAPGEF1, PHKG1, PYGL, TRIP10, INS, INSR, IRS1, PDPK1, PIK3CA, HRAS, GRB2, PTPN1, and PTPN11.

[0036] The gene or protein target of PKC is selected from...

[0037] One or more of PRKCA, PRKCB, PRKDC, PRKCZ, PRKCE, PRKCG, PRKCD, PRKCH, PRKCI, PRKCQ, PRKD1, SLC9A5, MAPK3, MAPK9, MAPK8, and MAPK1.

[0038] The gene or protein targets in the RAR are selected from...

[0039] One or more of RARA, RARS, RARB, RARG, RXRA, RXRG, FAM120B, NCOA1, and NCOR2

[0040] The gene or protein target that regulates HDAC is selected from one or more of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, and HDAC11.

[0041] The gene or protein target in the adenylate cyclase signaling pathway is selected from one or more of PRKAR1A, ADCY10, ADCYAP1, ADCY1, ADCY2, ADCY6, ADCY3, GNAI1, GNAL, GNAT3, PRKACA, PRKAR2B, PRKACB, PRKAR1B, PRKACG, CDKN1B, PRKAR2A, NCAM1, and CDKN1A.

[0042] The gene or protein target in the HMT is selected from HNMT.

[0043] One or more of DNMT1,KMT2A,EHMT2,EHMT1,KMT2A,DOT1L,EZH2,SETD7,DNMT3B,DNMT3A,SETDB1,S;ETD2;

[0044] The gene or protein target in DNMT is selected from one or more of DNMT1, DNMT3B, DNMT3A, CDKN2A, CDKN2B, EHMT2, EHMT1, DNMT3L, CDH1, PARP1, and MBD2.

[0045] The gene or protein target in the histone demethylase is selected from...

[0046] One or more of KDM1A, KDM4A, KDM5A, KDM5B, KDM2A, KDM5C, KDM4B, KDM4C, KDM5D, KDM4D, KDM1B, HIST1H3A, HIST4H4, HIST2H3C, HAT1, HIST1H4C, HIST1H4F, HIST1H4J, HIST1H2AE, HIST1H2BB, CLOCK, NOCA1;

[0047] The method employs at least one of the following methods: combinations of small molecule compounds, combinations of cytokines or recombinant proteins, gene editing technology, and transgenic technology;

[0048] The aforementioned small molecule compound combination includes one or more of the following compounds:

[0049] Histone deacetylate inhibitors include sodium phenylbutyrate, butyrate, sodium butyrate, VPA, Scriptaid, Apidin, LBH-589 (Panobinostat), MS-275, SAHA (Vorinostat), Trichostatin (TSA), Psammaplin A, Splitomicin, SRT1720, Resveratrol, Sirtinol, APHA, CI-994, Depudecin, FK-228, HC-Toxin, ITF-2357 (Givinostat), Chidamide, and RGFP. 966, PHOB, BG45, NexturastatA, TMP269, CAY10603, MGCD-0103, Niltubacin, PXD-101 (Belinostat), Pyroxamide, Tubacin, EX-527, BATCP, Cambinol, MOCPAC, PTACH, MC1568, NCH51 and TC-H106;

[0050] TGF-β receptor inhibitors: 616452, LY2109761, Pirfenidone, Repsox (E-616452), SB431542, A77-01, A8301, GW788388, ITD-1, SD208, SB525334, LY364947, ASP3029, D4476 and SB505124;

[0051] PKC inhibitors include Go6983, Go6976, and Bisindolylmaleimide I (GF109203X);

[0052] WNT / β-catenin agonists include MAY-262611, CHIR98014, CHIR99021, LiCl, Li2CO3, TD114-2, AZD2858, AZD1080, BIO, Kenpaullone, TWS119, LY2090314, CBM1078, SB216763, and AR-A014418; cAMP agonists include Forskolin, IBMX, Prostaglandin E2 (PGE2), NKH477, 8-pCPT-2′-O-Me-cAMP, GSK256066, Apremilast (CC-10004), Roflumilast, Cilomilast, Rolipram, Milrinone, 8-Bromo-cAMP, Dibutyryl-Camp, and Sp-8-Br-cAMPs;

[0053] RAR agonists include TTNPB, Bexarotene, Ch55, Tamibarotene, Retinol, AM580, ATRA, Vitamin A, Vitamin A derivatives, and 13-cis RA;

[0054] ROCK inhibitors include Y-27632, Y-276322HCl, Thiazovivin, Ripasudil (K-115), Fasudil, GSK429286A, RKI-1447, and PKI-1313;

[0055] JNK inhibitors include SP600125, JNK Inhibitor IX, AS601245, AS602801, and JNK-IN-8;

[0056] DNMT inhibitors include RG108, Thioguanine, 5-Aza-2'-deoxycytidine (Decitabine), SGI-1027, Zebularine, and 5-Azacytidine (AZA);

[0057] HMT inhibitors include EPZ004777, EPZ5676, GSK503, BIX 01294, and SGC 0946; histone demethylase inhibitors include parnate (tranylcypromine), tranylcypromine (2-PCPA)HCl SP2509, 4SC-202, ORY-1001 (RG-6016), GSKJ1, and GSK-LSD1.

[0058] JAK-STAT inhibitors include STAT5-IN-1,JAK3-IN-1,JAK3-IN-7,WP1066,Homoharringtonine,Pyridone 6,Pyridone 6,Artesunate,ruxolitinib,SH-4-54,Baricitinib,Ruxolitinib phosphate,AG-490,Baricitinib phosphate,SAR-20347,CYT387 Mesylate,AS1517499,Peficitinib,Ruxolitinib sulfate,NSC 74859,Stattic,Tofacitinib citrate,Pimozide,Oclacitinib maleate,Ruxolitinib S enantiomer,SB1317,Niclosamide,Scutellarin,Solcitinib,Mogrol,Nifuroxazide,TG101348(SAR302503),AG-1478(Tyrphostin AG-1478)(EGFR inhibitor);

[0059] KX2-391(Src inhibitor)

[0060] PKI-402(PI3Kα / β / γ / δandmTOR inhibitor)

[0061] NSC 74859(S3I-201)(STAT3 inhibitor)

[0062] Fludarabine (STAT-1 inhibitor)

[0063] U0126-EtOH(UO126 EtOH)(MEK1 andMEK2 inhibitor)

[0064] SGI-1776Free Base(Pim1,Pim2 andPim3 Inhibitor)

[0065] Sorafenib (Nexavar) (VEGFR, PDGFR, c-RafandB-Rafine inhibitor)

[0066] PLX-4720(B-RafV600E andc-Raf-1Y340D / Y341D inhibitor)

[0067] The cytokine combination or recombinant protein combination includes PDGFAA, PDGFAB, BMP4, IGF1, bFGF, EGF, VEGF, insulin, ActivinA, TGF-beta1, Noggin, BMP-2, Shh, IL-6, CXCL10, CXCL12, CXCL2, HGF, IFN gamma, IL-2, IL-6R alpha, IL-2Ralpha, TNF-alpha, TNF-beta, TPO, IGF2, IGFBP5, IGFBP6, IGFBP4, IGFBP7, IGFBP9, PDGF-BB, MMP3, GDF11, and TIMP2.

[0068] The gene editing technologies mentioned include using CRISPR / Cas9 gene editing technology and TALEN gene editing technology to upregulate or knock out gene or protein targets in the JAK-STAT signaling pathway, such as STAT5A;

[0069] The aforementioned transgenic technology includes using lentiviruses or retroviruses to overexpress or inhibit gene or protein targets in the JAK-STAT signaling pathway, such as STAT5A;

[0070] A type of repair fibroblast is characterized in that the repair fibroblast is prepared by the method of claims 1 to 8; the JAK-STAT signaling pathway in the repair fibroblast is inhibited; the gene or protein target in the JAK-STAT signaling pathway that is low-expressed or inhibited includes at least one of SOS1, STAT5B, JAK1, JAK3, SOCS3, IL6ST, STAT1, STAT2, STAT3, STAT4, STAT6, STAT5A, IRF9, IL6, IL6R, IL2, IL2A, IL2B, PRKCD, CXCL12, CXCR4, JAK2, IL15RA, IL20RB, GHR, CXCL2 and PRLR.

[0071] The signaling pathways in the repaired fibroblasts also underwent the following changes: the NOD-like receptor signaling pathway was inhibited; and / or the TGF betareceptor signaling pathway was inhibited; and / or the insulin signaling pathway was downregulated; and / or the Wnt signaling pathway was upregulated and / or the Notch signaling pathway was downregulated; and / or at least one of the following: the p53 signaling pathway was downregulated.

[0072] The method for preparing the repair-type fibroblasts is characterized in that the target cells of the repair-type fibroblasts are ordinary fibroblasts, which are derived from connective tissues (such as blood, skin, bone marrow, and heart) of mammals (such as humans, monkeys, mice, and pigs).

[0073] The preparation method involves treating ordinary fibroblasts with a combination of small molecule compounds to ultimately prepare repaired fibroblasts; the combination of small molecule compounds includes at least one of Jak-Stat inhibitors, WNT / β-catenin agonists, histone deacetylase inhibitors, and cAMP agonists.

[0074] The method for preparing repair-type fibroblasts further includes using at least one of the following: RAR agonist, DNMT inhibitor, HMT inhibitor, histone demethylase inhibitor, ascorbate, JNK inhibitor, PKC inhibitor, ROCK inhibitor, and TGF-β inhibitor.

[0075] The method for preparing the repaired fibroblasts involves using a first-stage compound and a second-stage compound in a sequential manner. The first-stage compound is a WNT / β-catenin agonist, a histone deacetylase inhibitor, and a cAMP agonist; or the first-stage compound is a histone deacetylase inhibitor, a TGF-β receptor inhibitor, a WNT / β-catenin agonist, and a cAMP agonist.

[0076] The second-stage compounds include histone deacetylase inhibitors, TGF-β inhibitors, WNT / β-catenin agonists, cAMP agonists, RAR agonists, HMT inhibitors, ascorbate, PKC inhibitors, and ROCK inhibitors.

[0077] The preparation method involves at least one of the following: VPA (0.05–10 mM); CHIR99021 (1–15 μM); Repsox (0.5–10 μM); Forskolin (3–50 μM); Go 6983 (1–20 μM); Y-27632 (1–25 μM); AM580 (0.02–1 μM); EPZ004777 (0.5–15 μM); Vc (0.2 mM); TTNPB (0.2–20 μM); 5-Azacytidine (1–15 μM); SP600125 (1–50 μM); or first treating ordinary fibroblasts with a first-stage compound, wherein the first-stage compound is VPA (0.05–10 mM); CHIR99021 (1–15 μM); Repsox (0.5–10 μM); Forskolin (3–5 ... Forskolin (3–50 μM); Forskolin (3–50 μM); Forskolin (3–50 μM); Forskolin (3–50 μM); Forskolin ( Cells were treated with rskolin at 3–50 μM for 2–10 days; after the first stage treatment, cells were treated with second-stage compounds for 4–20 days. The second-stage compounds were: VPA (0.05–10 mM); CHIR99021 (1–15 μM); Repsox (0.5–10 μM); Forskolin (3–50 μM); Go6983 (1–20 μM); Y-27632 (1–25 μM); AM580 (0.02–1 μM); EPZ004777 (0.5–15 μM); Vc (0.2 mM); and TTNPB (0.2–20 μM).

[0078] The telomere length of the repaired fibroblasts is 1.5 to 12 times longer than that of the initial ordinary fibroblasts, and the length is close to that of the same type of cells from immature individuals. Other types of cells (such as osteoblasts and chondrocytes) prepared from the repaired fibroblasts have longer telomeres and exhibit stronger functional activity compared with the same type of cells from the same animal individual.

[0079] 17. The repaired fibroblasts prepared by any of the methods described in claims 9 to 15, characterized in that the cell products (such as cell secretions, cell lysates) obtained from the preparation of the repaired fibroblasts are used in constructing tissue engineering materials and delaying or reversing the aging of cells, tissues, organs and organisms.

[0080] The aforementioned repair-type fibroblasts are used in constructing tissue engineering materials to delay or reverse the aging of cells, tissues, organs, and the body.

[0081] A super fibroblast, characterized in that the super fibroblast is prepared by knocking out the STAT5 gene in ordinary fibroblasts.

[0082] The method for preparing super fibroblasts is characterized in that the method involves obtaining young and telomere-elongated super fibroblasts within 3 to 100 days after knocking out the STAT5 gene in ordinary fibroblasts.

[0083] The preparation method involves treating mesenchymal stem cells with a combination of small molecule compounds or gene editing to ultimately obtain rejuvenated mesenchymal stem cells. The combination of small molecule compounds includes at least one of Jak-Stat inhibitors, WNT / β-catenin agonists, DNMT inhibitors, TGF-β inhibitors, and cAMP agonists. The gene editing method involves knocking out genes or protein targets (such as STAT5A) in the Jak-Stat signaling pathway.

[0084] The treatment method for the small molecule compound combination is to treat mesenchymal stem cells for 1–28 days with the following compounds: CHIR99021, 1–15 μM; 5-Azacytidine (AZA), 1 μM–15 μM; and / or 5-Azacytidine (AZA), 1 μM–15 μM; Forskolin, 3–50 μM; and / or 5-Azacytidine (AZA), 1 μM–15 μM; Forskolin, 3–50 μM; CHIR99021, 1–15 μM.

[0085] Applications of cell products (such as repair fibroblasts) prepared by any of the methods of claims 1 to 22, or cell culture media / bases of said cell products, lysis buffers of said cell products, kits, drugs, health products, food, cosmetics or medical devices.

[0086] Cell products (such as repair-type fibroblasts) prepared by any of the methods described herein may be used as seed cells for tissue engineering materials, scaffold sources for tissue engineering materials, and in the repair of damaged mammalian tissues and organs, as well as in the repair of aging and degenerated tissues and organs.

[0087] Cell products (such as repair fibroblasts) prepared by any of the methods described herein may be used in medical research or as immunomodulators.

[0088] The application of cell products (such as repair fibroblasts) prepared by any of the methods described herein in the in vitro / in vivo prevention, delay, and reversal of the aging process of mammalian tissues, organs, and bodies.

[0089] The application of any of the methods described herein and the cell products prepared therefrom in the reprogramming of cells, tissues, organs, or organisms, or in the rejuvenation of cells, tissues, organs, or organisms.

[0090] The present invention is characterized by the following: It regulates cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging, apoptosis, and reverses aging and prolongs lifespan by quantitatively and / or time-dependently modulating gene or protein targets in the Jak-Stat signaling pathway. This is achieved through combinations of small molecule compounds, cytokines, or recombinant proteins, gene editing technology, and transgenic technology to quantitatively and / or time-dependently regulate gene or protein targets in the Jak-Stat signaling pathway within cells. These gene or protein targets include CXCL2, SOS1, STAT5B, JAK1, JAK3, SOCS3, IL6ST, STAT1, STAT2, STAT3, STAT4, STAT6, STAT5A, IRF9, IL6, IL6R, and IL2 (e.g., IL2). At least one of IL-1 and / or IL-2B), PRKCD, CXCL12, CXCR4, JAK2, IL-15RA, IL-20RB, GHR, and PRLR; the small molecule compound combination for regulating the Jak-Stat signaling pathway includes at least one of Jak-Stat inhibitors, WNT / β-catenin agonists, histone deacetylases inhibitors and cAMP agonists, RAR agonists, DNMT inhibitors, HMT inhibitors, histone demethylase inhibitors, ascorbate, JNK inhibitors, PKC inhibitors, ROCK inhibitors, and TGF-β inhibitors. The cytokine combination or recombinant protein combination includes PDGFAA, PDGFAB, BMP4,

[0091] The gene editing technologies mentioned include using CRISPR / Cas9 gene editing technology and TALEN gene editing technology to upregulate or knock out gene or protein targets in the JAK-STAT signaling pathway, such as STAT5A. The transgenic technologies mentioned include using lentiviruses or retroviruses to overexpress or inhibit gene or protein targets in the JAK-STAT signaling pathway, such as STAT5A.

[0092] This invention prepares reparative skin fibroblasts by inhibiting gene or protein targets (such as STAT5A, JAK1) of the Jak-Stat signaling pathway in fibroblasts using small molecule compounds. In these reparative skin fibroblasts, the Jak-Stat signaling pathway is inhibited, and the signaling pathway in the reparative fibroblasts also undergoes the following changes: the NOD-like receptor signaling pathway is inhibited; and / or TGF-β2 is inhibited. The betareceptor signaling pathway is inhibited; and / or the insulin signaling pathway is downregulated; and / or the Wnt signaling pathway is upregulated and / or the Notch signaling pathway is downregulated; and / or the p53 signaling pathway is downregulated at least one of the following: the telomere length of the repaired skin fibroblasts is 1.5 to 12 times longer than that of the initial ordinary fibroblasts, and the length is close to the level of the same type of cells in immature individuals; other types of cells (such as osteoblasts and chondrocytes) prepared from the repaired fibroblasts have longer telomeres and exhibit stronger functional activity compared with the same type of cells from the same animal individual; the repaired fibroblasts and cell products (such as cell secretions and cell lysates) prepared from these cells can be used to construct tissue engineering materials to delay or reverse the aging of cells, tissues, organs and the body.

[0093] This invention provides a technique for quantitatively and / or timedly regulating gene or protein targets in the Jak-Stat signaling pathway to modulate cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging, apoptosis, and to reverse aging and prolong lifespan. This technique can promote transdifferentiation between different cell types and can be used to prepare different types of rejuvenated cells (such as reversing the aging of mesenchymal stem cells and preparing super fibroblasts). It can also be used to promote cell aging and apoptosis. The cells and cell products prepared by this technique can be used to prevent, delay, and reverse the aging process of mammalian tissues, organs, and organisms in vitro / in vivo; to reprogram cells, tissues, organs, and organisms; and to serve as seed cells for tissue engineering materials, scaffold sources for tissue engineering materials, and for repairing damaged mammalian tissues and organs, as well as repairing aging and degenerated tissues and organs.

[0094] Mechanism of invention: By quantitatively and / or timedly regulating the expression of gene or protein targets in the Jak-Stat signaling pathway in cells, changes in different metabolic pathways in cells are altered, thereby causing the target cells to change their cellular state and transform into different types of cells or possess different cellular characteristics.

[0095] Compared with existing technologies, the present invention has the following beneficial effects: The repaired fibroblasts provided by the present invention, compared with fibroblasts from the same donor or fibroblasts from donors of the same age, possess rejuvenating characteristics but lack tumorigenicity. These rejuvenating characteristics include epigenetic changes and / or changes in the expression levels of aging-related genes and / or elongation of telomeres and / or accelerated cell proliferation and / or the ability of cells to undergo long-term stable passage. Furthermore, these rejuvenated cells and their cell products can reverse aging in mammals and extend lifespan. The present invention also provides quantitative and / or timed regulation... The Jak-Stat signaling pathway targets genes or proteins to regulate cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging, apoptosis, and to reverse aging and prolong lifespan. It can systematically regulate cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging, and apoptosis. The corresponding cells and cell products can be used in vitro / in vivo to prevent, delay, and reverse the aging process of mammalian tissues, organs, and organisms; to reprogram cells, tissues, organs, and organisms; and as seed cells for tissue engineering materials, scaffold sources for tissue engineering materials, and to repair damaged mammalian tissues and organs, as well as to repair aging and degenerated tissues and organs. Attached Figure Description

[0096] Figure 1 Preparation of regenerative fibroblasts (rFib).

[0097] Figure 2 .rFib is younger than Fib and bMSC.

[0098] Figure 3 Senescent bMSCs have poorer osteogenic and chondrogenic differentiation capabilities than rFib, and rFib is not tumorigenic.

[0099] Figure 4 .rFib has immunomodulatory functions in vitro.

[0100] Figure 5 .rFib cells have the ability to regulate the immune system in vivo.

[0101] Figure 6 .rFib has the ability to repair bone defects regardless of age.

[0102] Figure 7 In vivo cartilage repair experiment.

[0103] Figure 8 Inhibiting the STAT5 gene can rejuvenate skin fibroblasts and enable them to differentiate into multiple lineages.

[0104] Figure 9 Changes in STAT5 and H3K9me after STAT5 is knocked out.

[0105] Figure 10 Detection of rejuvenation and differentiation ability of another 62-year-old (62Y) Fib after STAT5 knockout.

[0106] Figure 11 To rejuvenate MSC cells, cells were treated with different compounds for 3 days and then β-galactosidase staining was performed; 55Y = 55 years old, 82Y = 82 years old, and Y = year represents the age of the cell donor.

[0107] Figure 12 .rFib cells can prolong the lifespan of aged NOD / SCID mice and increase their bone density.

[0108] Figure 13 .rFib cells are distributed in multiple organs of mice and can differentiate into functional cells.

[0109] Figure 14 .rFib cells can increase bone mineral density in aged osteoporotic mice.

[0110] Figure 15 rFib culture medium significantly promotes skin healing. The rFib culture medium group showed almost complete healing 12 days after modeling.

[0111] Figure 16 .rFib can improve lower limb ischemia symptoms in mice.

[0112] Figure 17 Mix Y treatment inhibits the expression of STAT5 and STAT3 genes (AB), downregulates CDKN1A gene, elongates telomeres (CD), and rejuvenates cells.

[0113] Figure 18 Mix Pn treatment downregulated the expression of STAT5 (A), and inhibited the expression of ATF3, CDKN1A, GADD45B, and IL6 (BE), indicating cell rejuvenation.

[0114] Figure 19 Treatment with Mix Y-Mix Pn2 inhibited JAK1 expression in fibroblasts (A) and prolonged telomere length (B).

[0115] Figure 20 A. Tuj1 staining of nerve cells obtained from the transformation of skin fibroblasts into nerve cells;

[0116] B. Identification of Nestin expression levels.

[0117] Figure 21 When small molecule compounds are used, STAT5 gene expression is upregulated during the differentiation of ES into neurons.

[0118] Figure 22 Module for KEGG pathway enrichment. Detailed Implementation

[0119] Example 1: Obtaining and Characterizing Repair-Type Skin Fibroblasts

[0120] 1. Human skin fibroblasts were seeded into 6-well plates and cultured in skin fibroblast culture medium for 24 hours;

[0121] 2. Replace the cell culture medium with rFib induction culture medium containing the small molecule cocktail combination Mix V, and change the medium every 2 days;

[0122] 3. After culturing in rFib induction medium containing the small molecule cocktail combination Mix V for 5 days, change the medium to rFib induction medium containing the small molecule cocktail combination Mix P, and change the medium once every 2 days;

[0123] 4. After culturing in rFib-induced medium containing the small molecule cocktail combination Mix P for 7 days, the medium was replaced with medium containing 10% FBS.

[0124] HG-DMEM containing 10 ng / mL bFGF, 100 ng / mL PDGF-AB, and 10 ng / mL BMP4; or HG-DMEM containing 10% FBS; or cultured using rFib medium; long-term passaged after 3 days of cell treatment; identification began 3 days after cell treatment.

[0125] 5. For long-term passage, rFib is cultured in MSC basal medium and passaged when the cell confluence reaches 90%.

[0126] Skin fibroblast culture medium: 10% FBS + HG-DMEM; or Fibstar-CO medium, brand name HCell, catalog number FMS003C.

[0127] Mix V: high glucose (HG)-DMEM supplementedwith 10% FBS, containing VPA, 0.5mM; CHIR99021, 3μM; Repsox, 1μM; Forskolin, 10μM;

[0128] Mix P: HG-DMEM supplementedwith 10% FBS, containing VPA, 0.5mM; CHIR99021, 3μM; Repsox, 1μM; Forskolin, 10μM; SP600125, 10μM; Go 6983, 5μM; Y-27632, 5μM; AM5800.05μM; EPZ0047775μM; Vc, 0.2mM; TTNPB, 5μM.

[0129] Alternatively, add VPA, 0.5mM; CHIR99021, 3μM to the FibGro medium with brand name HCell and product number FGS0040.

[0130] Repsox, 1μM; Forskolin, 10μM; SP600125, 10μM; Go 6983, 5μM; Y-27632, 5μM; AM5800.05μM;

[0131] EPZ0047775μM; Vc, 0.2mM; TTNPB, 5μM.

[0132] MSC basal culture medium: 10% FBS + LG-DMEM or bone marrow mesenchymal stem cell complete culture medium purchased from Cyagen, catalog number HUXMA-90011, or rFib medium from HCell, catalog number CRM0016-01.

[0133] Note: Unless otherwise specified, the cells used in the examples are derived from human cells.

[0134] Figure 1 Preparation of regenerative fibroblasts (rFib)

[0135] A. Schematic diagram of the process of conversion from skin fibroblasts (Fib) to rFib. Near-senescent Fib (P13 generation) gains enormous growth potential after conversion to rFib (can be expanded up to 19 more generations); the same treatment induces the death of bone marrow mesenchymal stem cells.

[0136] A comparison of the growth curves of B. rFib and its homologous Fib during long-term amplification. The rFib obtained after treatment grows faster than the homologous Fib.

[0137] C. Histochemical analysis of the trilineage differentiation capacity of cells before and after conversion (parental skin cells were derived from P8 generation cells of a 39-year-old volunteer). Osteoblastic, adipocyte-derived, and chondrocyte-derived abilities were assessed on day 0 (Fib, before treatment), day 5, day 12, day 15 (rFib), and after several amplifications following differentiation induction at day 21. Osteoblast (Osteogenesis, identified by Alizarin Red staining), Adipocyte (Adipogenesis, identified by Oil Red staining), and Chondrocyte (Chondrogenesis, identified by Alicanian Blue staining) were identified. Even after multiple passages (P9 generation, P16 generation), rFib cells maintained good trilineage differentiation capacity.

[0138] Dq-RT-PCR analysis was performed on ALP levels 14 days after osteogenic differentiation induction, COL2A1 levels 14 days after chondrogenic differentiation induction, and PPARG levels 21 days after adipogenic differentiation induction. The results showed that rFib cells, similar to young bMSCs, highly expressed genes related to trilineage differentiation after induction. *p<0.05,**p<0.01,***p<0.001, n≥3, statistically significant compared to D0(Fib).

[0139] EG. After treatment with small molecule combination, the expression of JAK1 and STAT5 in cells changed at different time points (decreased after treatment), the expression of telomerase (TERT) (high expression after 5 days), and the change in telomere length (significantly prolonged at 15 days).

[0140] H. Transcriptome analysis of rFib cells showed that their clustering was closer to that of FIB and bMSC, rather than iPSC and ESC, which have characteristics of both skin fibroblasts and mesenchymal stem cells. rFib is a safe cell type that does not have tumorigenicity.

[0141] Figure 2 .rFib is younger than Fib and bMSC.

[0142] Immunofluorescence staining of H3K9me3 and H4K20me3 in A.D0 (P11 generation parental Fib) and D15 (rFib). The senescence marker H4K20me3 in rFib cells was significantly reduced compared to its homologous Fib.

[0143] Immunofluorescence staining and quantitative analysis of γH2AX in B and C.D0 (P11 generation parental Fib) and D15 (rFib). The senescence marker γH2AX in rFib cells was significantly reduced compared to its homologous Fib.

[0144] D. Growth curves of Fib, bMSC, and rFib from two older individuals during long-term expansion. Cells of the same color are from the same individual. The growth rate of rFib was significantly higher than that of bMSC and Fib from the same individual, and also faster than that of young bMSC (33 years old).

[0145] EG. q-RT-PCR detection of several cellular senescence markers (CDKN1A, ATF3, and IL-6). The expression of senescence markers in rFib cells and rFib-induced osteoblasts and chondrocytes was significantly reduced. 12W represents skin cells from a miscarried fetus at 12 weeks of gestation.

[0146] H. The relative telomere lengths of Fib, rFib, bMSC and their differentiated osteoblasts (rFib-OB, bMSC-OB) and chondrocytes (rFib-CH, bMSC-CH) were determined by q-RT-PCR using T / S values.

[0147] In the EH plot, cells from the same individual are labeled with the same color. * indicates a significant difference compared to homologous Fib, # indicates a significant difference compared to the corresponding bMSC, bMSC-OB (osteoblasts differentiated from bMSCs), and bMSC-CH (chondrocytes differentiated from bMSCs). *p<0.05,**p<0.01,***p<0.001,#p<0.05,##p<0.01,###p<0.001, n=3.

[0148] Figure 3 Senescent bMSCs have poorer osteogenic and chondrogenic differentiation capabilities than rFib, and rFib is not tumorigenic.

[0149] A. Alizarin Red staining of bMSCs and rFib from individuals of different ages to assess osteogenic differentiation. The results showed that the osteogenic differentiation capacity of bMSCs from older individuals was significantly reduced, while rFib from the same individuals maintained good osteogenic differentiation capacity.

[0150] B. Oil Red O staining of adipogenic differentiation of bMSCs and rFib from individuals of different ages. The results showed that the adipogenic differentiation capacity of bMSCs from older individuals increased significantly, while the adipogenic differentiation capacity of rFib from older individuals remained consistent with that of younger individuals.

[0151] Quantitative analysis of corresponding osteogenic (ALP) and adipogenic (PPARG) marker genes by CD.q-RT-PCR. The results were consistent with the staining results.

[0152] D. Immunohistochemical staining of COL2A1 and MMP13 in chondrogenic differentiation of bMSCs and rFib from individuals of different ages. In older individuals, COL2A1 expression was low but MMP13 expression was high after chondrogenic differentiation of bMSCs. rFib from older individuals showed similar COL2A1 / MMP13 expression levels to those from younger individuals.

[0153] FG.q-RT-PCR was used to detect the expression of COL2A1 and MMP13 in bMSCs and rFib after chondrogenic differentiation from individuals of different ages, and the results were consistent with the staining results.

[0154] The karyotypes of rFib from generation H.P9 and P13, as well as its homologous generation P6 Fib, were analyzed. The results showed that the karyotype of rFib remained consistent with that of its Fib even after long-term passage.

[0155] In the rFib teratoma formation assay, human embryonic stem cells (hESCs) served as a positive control. Subcutaneous transplantation of hESCs into NOD / SCID mice resulted in the formation of teratomas (with distinct three germ layers), while no tumors were observed in mice transplanted with rFib.

[0156] J. Analysis of telomere length and telomerase expression in cells. The telomeres of rFib cells were significantly longer than those of their homologous Fib cells and remained stable after induction. However, telomerase expression was only transiently high during induction, then returned to low levels, unlike tumor cells (where telomerase is consistently highly expressed). This indicates that rFib cells do not possess tumorigenicity while promoting cell rejuvenation.

[0157] 6.1 In vitro immunomodulation assay

[0158] Cells were treated with mitomycin C for 2.5 hours, digested, and counted. Cells were seeded at 1 × 10⁵ cells / well in 24-well plates. Lymphocytes were stained with carboxyfluorescein succinimide (CFDA-SE) at 37°C for 30 minutes and then seeded at 2 × 10⁵ cells / well. 5 Lymphocytes were seeded at a density of [missing information] in 24-well plates. Lymphocyte proliferation was stimulated with PHA at a final concentration of 2 μg / ml. Five groups were set up: bMSCs + lymphocytes + PHA co-culture group, rFib + lymphocytes + PHA co-culture group, skin fibroblasts + lymphocytes + PHA co-culture group, lymphocytes + PHA positive control group, and lymphocytes alone negative control group. After 5 days of co-culture, lymphocytes from each well were collected and washed three times with PBS. Lymphocyte proliferation was detected by flow cytometry using anti-CD3, CD4, and CD8 antibodies. All antibodies were obtained from BDbiosciences.

[0159] Figure 4 .rFib has immunomodulatory functions in vitro.

[0160] A. Parental Fib, rFib, and bMSCs were cultured with T cells using a mixed lymphocyte reaction method. PBMCs derived from healthy volunteers were labeled with CFSE. **p<0.01,***p<0.001, n=3, statistical significance compared to the "T+PHA" group. The results indicate that rFib has the ability to inhibit T cell proliferation, while its homologous Fib cells do not, suggesting that rFib has immunomodulatory capabilities.

[0161] BC.rFib has the ability to regulate CD4+ immune cell subtypes.

[0162] DE.rFib has the ability to regulate CD8+ immune cell subtypes.

[0163] 6.2 In vivo immune regulation experiment

[0164] Press 1×10 6 The culture medium of bMSCs, rFib, and skin fibroblasts cultured at a density of 10 cm culture dish for 48 h was filtered through a 0.22 μm Millipore membrane to remove cells or cell debris, and then concentrated 100 times using an ultrafiltration centrifuge tube.

[0165] Acute liver injury was induced in 8–12 week old C57BL / 6 mice by tail vein injection of concanavalin A dissolved in PBS at a dose of 25 mg / kg body weight, with PBS-only injection serving as a control (Han et al., 2014). Six mice were in each group. Thirty minutes later, either concentrated culture medium or PBS was injected via tail vein. Eight and a half hours after tail vein injection, the mice were sacrificed. Blood and liver samples were collected. Liver was stained with hematoxylin and eosin (HE), CD3+ T cells were analyzed by flow cytometry, and ALT / AST ratios were measured in the blood.

[0166] Serum ALT / AST were quantified using an ELISA kit (Shanghai Enzyme Linked Laboratory) according to the manufacturer's instructions. Three independent replicates were tested in each group, and the data are presented as mean ± SD.

[0167] Figure 5 .rFib cells have the ability to regulate the immune system in vivo.

[0168] A. Acute liver injury was induced in C57BL / 6 mice using concanavalin A, followed by treatment with concentrated rFib cell culture medium. It was clearly observed that the livers of mice treated with rFib culture medium showed no obvious abnormalities (hemorrhage, necrosis, etc.).

[0169] B. The absolute number of T lymphocytes in liver tissue was measured 8.5 hours after tail vein injection of cell culture medium. The results showed that the rFib culture medium had significant immunomodulatory capabilities, similar to bMSCs.

[0170] Mice treated with CD.rFib culture medium showed near-normal levels of blood ALT and AST, with no obvious signs of liver damage.

[0171] 6.3 PCR of common genes

[0172] Total RNA extraction was performed according to the TRIzol kit (Takara Bio) instructions. RNA (1.0 μg) was reverse transcribed into cDNA using the Primescript RT kit (Takara Bio). For q-RT-PCR, cDNA was used as a template with specific primers and SYBR Green using SYBR Premix EX Taq™ II (Takara Bio). Cycling conditions were performed according to the manufacturer's (Takara) instructions. Relative expression levels were normalized using an internal control (ACTIN). For gPCR, genomic DNA was used as a template with human-specific primers ACTIN using Premix Taq (Takara Bio).

[0173] Example 2: rFib cells possess bone defect repair capabilities

[0174] Femoral defect models were established using 8- to 10-week-old NOD / SCID mice weighing 20–24 g, with approval from the ethics committee. Five animals were used in each group. The modeling method was as follows: Under anesthesia with sodium pentobarbital, the skin and subcutaneous tissue were incised, and sufficient mid-shaft of the femur was exposed by blunt dissection between the rectus femoris and semitendinosus muscles. The surgery was performed at the proximal junction of the right femur. A continuous bone defect of 4 mm × 1 mm was constructed. Skin fibroblasts (Fib), bone marrow mesenchymal stem cells (bMSCs), and rFib were stained with Hoechst 33342 (Thermo, NucBlue live cell), mixed with Matrigel, and transplanted into the defect site at 5 × 10⁵ cells / mouse.

[0175] Twenty-eight days after transplantation, mice were euthanized by injecting a lethal dose of sodium pentobarbital. The thighs of the mice were bluntly dissected, fixed with 4% PFA, and examined using μCT imaging (SkyScan 1272, Bruker microCT). The acquired data were then analyzed.

[0176] Figure 6 .rFib has the ability to repair bone defects regardless of age.

[0177] A. Schematic diagram of surgical procedure in a mouse model of midshaft femoral defect.

[0178] B. Femoral samples from different groups of mice and H&E staining. Young bMSCs (31 years old) and rFib (39 years old) both showed good bone defect repair capabilities.

[0179] C. rFib cells, labeled with Hoechst33342, exhibited autofluorescence in blue. Sections of the repair site showed that rFib cells formed new bone at the defect site, with their number and location corresponding one-to-one with newly formed osteoblasts.

[0180] D. Micro-CT analysis of different experimental groups: Even rFib from elderly individuals (62 years old) can repair bone defects, while the bone defect repair capacity of rFib from elderly individuals (62 years old) is very weak.

[0181] E. Analysis of micro-CT data from different experimental groups: The repair capacity of rFib from young (39 years old) and old (62 years old) individuals was similar, indicating that the bone defect repair capacity of rFib is not limited by age.

[0182] Example 3: rFib cells possess cartilage defect repair capabilities

[0183] Articular cartilage defect model and cell transplantation

[0184] NOD / SCID mice, aged 8 to 10 weeks and weighing 20–24 g, were used. A modified articular cartilage model was used to evaluate the efficacy of rFib (Cheng et al., 2014). Articular cartilage defects (1.5 mm × 1 mm) were constructed in the trochlear groove of the distal femur using a biopsy perforator. Cells ((2.5 × 10⁻⁶) 5 Each molecule was labeled with Hoechst 33342 in 35 μl Matrigel and implanted into the defect site. Cell-free Matrigel was implanted as a control.

[0185] Figure 7 In vivo cartilage repair experiment

[0186] AB. Safranin-Fibr staining of cartilage tissue samples and 10μm sections. In Safranin-Fibr staining, red represents cartilage and green represents bone tissue. Young rFib (39 years old) and bMSC (31 years old) can repair cartilage defects. Older bMSC (62 years old), although capable of generating new tissue, do not produce new cartilage. However, rFib from an older individual (62 years old) can still form cartilage tissue.

[0187] Figure C shows the Pineda score for cartilage repair. The results indicate that rFib from older individuals (62 years old) and rFib from younger individuals (39 years old) have similar cartilage repair capabilities.

[0188] Image D, slices show that Hoechst33342-labeled rFib formed new cartilage tissue. The newly generated cartilage tissue has a similar structure and morphology to normal cartilage, with no abnormal tissue formation.

[0189] Examples 4-12: Different combinations of small molecule compounds were used to treat cells for different durations to obtain repaired fibroblasts. The identification method was the same as in Example 1. The combinations are shown in Table 1 below.

[0190]

[0191]

[0192] Table 1. Example combinations and processing days

[0193] Example 13, Preparation of Super Fibroblasts

[0194] 1. Constructing a vector plasmid for knocking out the stat5a gene in crispr / cas9: The following plasmids were purchased from Cyagen.

[0195] pLV[2gRNA]-EGFP:T2A:Puro-U6>hSTAT5A[gRNA#4]-U6>hSTAT5A[gRNA#10]

[0196] pLV[Exp]-CBh>hCas9:T2A:Hygro

[0197] 2. Transfect the virus according to the product instructions. On the first day after transfection, remove the virus-containing culture medium and replace it with fresh complete culture medium. Incubate at 37°C with 5% CO2. 2 Incubate in an incubator.

[0198] 3. On the second day after transfection, the genes carried by the lentivirus begin to be expressed. Cells can continue to be cultured to further accumulate expression products or change cell phenotype.

[0199] 4. After cell expansion, the virus-transfected cells were purified using antibiotics, and the cells were further expanded and cultured in HG-DMEM containing 10% FBS; the cells were cultured continuously for 150 days.

[0200] Figure 8 Inhibiting the STAT5 gene can rejuvenate skin fibroblasts and enable them to differentiate into multiple lineages.

[0201] A. Two representative modules of the KEGG pathway enrichment of rFib were screened from 12,036 genes identified by WGCNA, and cassette diagrams showing changes in the expression of two gene modules in several induction samples.

[0202] B. After knocking out the STAT5 gene, the cells were cultured for another 40 days. Immunohistochemical staining showed that Fib knocked out STAT5 (Fib-STAT5-KO).

[0203] H4K20me3 (an aging marker, the higher the level, the older the person) was significantly reduced compared to the control group.

[0204] C. Identification of aging marker genes (ATF3, GADD45B, IL6, CDKN1A, highly expressed in senescent cells). Results showed that knockout...

[0205] The expression of STAT5, a Fib cell senescence marker gene, was significantly reduced.

[0206] D.STAT5 knockout FIBs showed osteogenic (Alizarin Red S) and chondrogenic (Alcian Blue) abilities.

[0207] EG. After knocking out the STAT5 gene, changes in the expression of JAK1 and STAT5 in cells (decreased after knockout), the expression of telomerase (TERT) (high expression after 43 days), and the changes in telomere length (significantly prolonged at 54 days).

[0208] H. A schematic diagram illustrating the regulation of the Jak-Stat signaling pathway in fibroblasts to rejuvenate cells and the body.

[0209] Figure 9 Changes in STAT5 and H3K9me after STAT5 knockout

[0210] After A.Fib knocks out STAT5, STAT5A is no longer expressed.

[0211] B. Knocking out STAT5 has no effect on H3K9me.

[0212] Figure 10 Detection of rejuvenation and differentiation ability of another 62-year-old (62-year-old) Fib STAT5 knockout strain.

[0213] A. Detection of aging-related genes.

[0214] B. Detection of osteogenic and chondrogenic differentiation.

[0215] CD. Detection of telomerase and telomere length

[0216] Detection of E.STAT5 expression

[0217] F. Detection of STAT5 in the same cell line treated with the Mix V+Mix P system.

[0218] Example 14, Rejuvenation of MSC Cells

[0219] 1. Bone marrow mesenchymal stem cells from different individuals were cultured using low-glucose-DMEM containing 10% FBS;

[0220] 2. After treating cells with different combinations of compounds for 3 days, the cells were cultured in low-glucose-DMEM medium containing 10% FBS for another 3 days, followed by β-galactosidase staining.

[0221]

[0222] Table 2, Treatment with different compound combinations

[0223] Figure 11 To rejuvenate MSC cells, cells were treated with different compounds for 3 days and then β-galactosidase staining was performed; 55Y = 55 years old, 82Y = 82 years old, and Y = year represents the age of the cell donor.

[0224] Example 15: Intravenous injection of rFib prolongs the lifespan of aging mice.

[0225] A P9 generation Fib, a P13 generation rFib from a 39-year-old individual, and an rFib from a 62-year-old individual were labeled with Hoechst 33342 and dissolved in 200 μL DMEM. Cells (10 6 (200 μL DMEM was injected per mouse) via the tail vein into naturally aging NOD / SCID mice (43 weeks old; the average lifespan of these mice is 36–38 weeks, and 43 weeks old is approximately equivalent to 86 years in humans). In the solvent group, only 200 μL of DMEM was injected. Samples were collected for analysis after the mice died naturally.

[0226] Figure 12 .rFib cells can prolong the lifespan of aged NOD / SCID mice and increase their bone mineral density.

[0227] A. Survival curves of aging mice; mice injected with rFib cells, regardless of whether the cells were derived from young individuals (39 years old) or old individuals (62 years old), showed an effective extension of lifespan. Even young Fib cells did not have a lifespan-extending effect, and their survival curves were not significantly different from those of the solvent group.

[0228] B. Morphological observation of the two groups of mice. The 43-week-old mice were obviously aged (disheveled and dull fur, hunchback). The condition of the mice was significantly improved 4 weeks after injection of rFib cells. The mice injected with DMEM were even more aged 4 weeks later.

[0229] CE. These are gross anatomical photographs, H&E staining of gastric mucosal sections, and micro-CT analysis of the third lumbar vertebra in four mice: 10-week-old, 25-week-old, 47-week-old (DMEM injected at 43 weeks, died at 47 weeks), and 49-week-old (rFib injected at 43 weeks, died at 49 weeks). The images clearly show that the gastrointestinal tract of the mice injected with rFib cells is similar to that of young mice (10-week and 25-week), with the thickness and density of the gastric mucosa and the trabecular structure of the lumbar vertebrae resembling that of 25-week-old mice. In contrast, the aged mice in the DMEM-injected group showed significant gastrointestinal lesions, with short and sparse gastric mucosa and severe loss and breakage of the lumbar trabeculae.

[0230] F. Micro-CT data analysis: Aged mice injected with rFib showed bone mineral density (BMD) and relative bone volume (BV / TV) closer to those of 25-week-old mice. Trabecular bone number (Tb.N) and trabecular bone separation (Tb.Sp) were also improved compared to mice injected with DMEM (n=5 per group, *p<0.05, **p<0.01, ***p<0.001, n=5).

[0231] G.p16 Ink4a Expression analysis showed no significant difference between aged mice injected with rFib and 25-week-old and 10-week-old mice, while aged mice injected with DMEM showed a significant difference in p16 expression. Ink4a Expression increased significantly;

[0232] H. Osteogenesis (ALP) and osteoclastogenesis (TRAP) staining analysis showed that aged mice injected with rFib cells had significantly increased osteogenic expression and decreased osteoclastogenesis expression compared to DMEM-injected mice.

[0233] IJ. Quantitative analysis of osteogenic (I) and osteoclast (J) showed that the expression of osteogenic and osteoclast in aged mice injected with rFib cells was close to that in 25-week-old mice;

[0234] K. Immunohistochemical staining showed that human antibody (hCD29, green) was positive in the bones of mice injected with rFib, and osteogenic marker Osteocalcin (red) was expressed, indicating that rFib differentiated into osteoblasts in NOD / SCID mice.

[0235] In LM.rFib culture medium, the secretion of GDF11 (a protein with anti-aging function) and PDGFA (a protein that promotes osteogenic formation) was significantly higher than that of its homolog Fib. The anti-aging and bone density-enhancing functions of rFib may be related to paracrine effects.

[0236] Figure 13 .rFib cells are distributed in multiple organs of mice and can differentiate into functional cells.

[0237] AB.rFib cells were distributed in the stomach, spleen, lungs, and liver of mice (detected by fluorescence and PCR methods).

[0238] C.rFib cells were distributed in the skeletons of mice (detected by PCR, 1# to 10# represent mouse numbers, 1# to 5# represent mice injected with rFib cells, and 6# to 10# represent mice injected with DMEM).

[0239] D. Immunohistochemical staining showed that human antibody (hCD29, green) was positive in the bones of mice injected with rFib, and osteogenic marker Osteocalcin (red) was expressed, indicating that rFib differentiated into osteoblasts in NOD / SCID mice.

[0240] The other paracrine secretions of E.rFib (BFGF, HGF, VEGF) are the same as or not significantly different from those of Fib, and are not the main mechanism by which it exerts its anti-aging function.

[0241] Example 16: Intravenous injection of rFib can increase bone mineral density in aged osteoporotic animals.

[0242] Figure 14 .rFib cells can increase bone mineral density in aged osteoporotic mice.

[0243] A. Human-derived cells were used to intervene in age-related osteoporosis (28-week-old NOD / SCID mice). The experimental group received 1*10⁶ rFib cells via tail vein injection in 200 μL of DMEM, while the control group received only DMEM. The treatment was administered weekly for 3 weeks. Mice were sacrificed 28 days after the first injection for lumbar vertebral bone mineral density measurement. Micro-CT results showed that the trabecular structure of the third lumbar vertebra in the experimental group was more compact.

[0244] B. Micro-CT data analysis of the experimental group mice showed that their bone mineral density (BMD), relative bone volume (BV / TV), and trabecular bone number (Tb.N) were all improved.

[0245] Example 17: The culture medium of rFib cells can promote the healing of skin wounds in animals.

[0246] An 8cm wide full-cortical defect was created on the back of C57 mice. The Control group received no treatment, while the rFib Medium group was treated with rFib culture medium applied daily.

[0247] Figure 15 rFib culture medium significantly promotes skin healing. The rFib culture medium group showed almost complete healing 12 days after modeling.

[0248] Example 18: rFib can improve lower limb ischemia in mice.

[0249] A lower limb ischemia model was established in NOD / SCID mice by ligating the unilateral femoral artery. After confirming the successful model using laser Doppler ultrasound, 1×10-1 cells were injected once into the femoral artery ligation site and its distal and proximal ends. 6 Blood flow was measured using laser Doppler ultrasound 7 and 14 days after cell injection.

[0250] Figure 16 .rFib can improve lower limb ischemia symptoms in mice.

[0251] A. Laser Doppler ultrasound was used to detect blood flow in the lower limbs of mice. The results showed that rFib and bMSCs significantly improved ischemia in the lower limbs of mice.

[0252] B. Gross photographs of mouse hind limbs 7 days after ligation. Control indicates the normal hind limb, and Ischemic indicates the ligated hind limb. The results showed that the necrosis of the ligated hind limb was significantly less in the rFib group and the bMSC group.

[0253] Example 19: Preparation of rFib cells by different compound combinations

[0254] 1. Skin fibroblasts were seeded into 6-well plates and cultured in skin fibroblast culture medium for 24 hours;

[0255] 2. Replace the cell culture medium with rFib-induced culture medium containing the small molecule compound combination MixY, changing the medium every 2 days, and treat the cells for 10 days;

[0256] 3. After treatment with the small molecule compound combination Mix Y, the culture medium was changed to HG-DMEM with 10% FBS added, and the cells were cultured for another 3 days before identification began; or rFib medium was used for culture.

[0257] 5. For long-term passage, rFib is cultured in MSC basal medium and passaged when the cell confluence reaches 90%.

[0258] Skin fibroblast culture medium: 10% FBS + HG-DMEM; or Fibstar-CO medium, brand name HCell, catalog number FMS003C.

[0259] Mix Y: high glucose (HG)-DMEM supplemented with 10% FBS, containing Y-27632, 5μM; Vc, 0.2mM; EPZ004777, 5μM; Forskolin, 10μM; Repsox, 1μM.

[0260] Alternatively, add Y-27632, 5μM; Vc, 0.2mM; EPZ004777, 5μM; Forskolin, 10μM; Repsox, 1μM to the FibGro medium of brand HCell, product number FGS0040.

[0261] MSC basal culture medium: 10% FBS + LG-DMEM or bone marrow mesenchymal stem cell complete culture medium purchased from Cyagen, catalog number HUXMA-90011, or rFib medium from HCell, catalog number CRM0016-01.

[0262] Figure 17 Mix Y treatment inhibits the expression of STAT5 and STAT3 genes in cells (AB), downregulates CDKN1A gene, elongates telomeres (CD), and rejuvenates cells.

[0263] Example 20: Preparation of rFib cells by different compound combinations

[0264] 1. Skin fibroblasts were seeded into 6-well plates and cultured in skin fibroblast culture medium for 24 hours;

[0265] 2. Replace the cell culture medium with rFib-induced culture medium containing the small molecule compound combination Mix Pn, changing the medium every 2 days, and treat the cells for 7 days;

[0266] 3. After treatment with the small molecule compound combination Mix Pn, the culture medium was replaced with HG-DMEM containing 10% FBS, and the cells were cultured for another 3 days before identification; or rFib medium was used for culture.

[0267] 5. For long-term passage, rFib is cultured in MSC basal medium and passaged when the cell confluence reaches 90%.

[0268] Skin fibroblast culture medium: 10% FBS + HG-DMEM; or Fibstar-CO medium, brand name HCell, catalog number FMS003C.

[0269] Mix Pn: HG-DMEM supplementedwith 10% FBS, containing VPA, 0.5mM; CHIR99021, 3μM; Repsox, 1μM; Forskolin, 10μM; Go 6983, 5μM; Y-27632, 5μM; AM5800.05μM; EPZ004777, 5μM; Vc, 0.2mM;

[0270] TTNPB, 5μM; .5-Aza-2'-deoxycytidine, 10μM.

[0271] Alternatively, add the following to the HCell FGS0040 FibGro medium: VPA, 0.5 mM; CHIR99021, 3 μM; Repsox, 1 μM; Forskolin, 10 μM; Go 6983, 5 μM; Y-27632, 5 μM; AM580, 0.05 μM; EPZ004777, 5 μM; Vc, 0.2 mM; TTNPB, 5 μM; .5-Aza-2'-deoxycytidine, 10 μM.

[0272] MSC basal culture medium: 10% FBS + LG-DMEM or bone marrow mesenchymal stem cell complete culture medium purchased from Cyagen, catalog number HUXMA-90011, or rFib medium from HCell, catalog number CRM0016-01.

[0273] Figure 18 Mix Pn treatment downregulated the expression of STAT5 (A), and inhibited the expression of ATF3, CDKN1A, GADD45B and IL6 (BE), indicating cell rejuvenation.

[0274] Example 21: Preparation of rFib cells with different compound combinations

[0275] 1. Skin fibroblasts were seeded into 6-well plates and cultured in skin fibroblast culture medium for 24 hours;

[0276] 2. Replace the cell culture medium with rFib induction culture medium containing the small molecule cocktail combination Mix Y, and change the medium every 2 days;

[0277] 3. After culturing in rFib-induced culture medium containing the small molecule cocktail combination Mix Y for 9 days, the culture medium was replaced with HG-DMEM containing 10% FBS for 3 to 7 days.

[0278] 4. After step 3, add rFib induction culture medium containing the small molecule compound combination Mix Pn2, and change the medium once every 2 days;

[0279] 5. After culturing in rFib-induced medium containing the small molecule compound combination Mix Pn2 for 7 days, the medium was replaced with medium containing 10% FBS.

[0280] Cells were treated with HG-DMEM containing 10 ng / ml bFGF, 100 ng / ml PDGF-AB, and 10 ng / ml BMP4 for 3 days; or with HG-DMEM containing 10% FBS for 3 days; or cultured using rFib medium; identification was performed after 3 days.

[0281] 6. For long-term passage, rFib is cultured in MSC basal medium and passaged when the cell confluence reaches 90%.

[0282] Skin fibroblast culture medium: 10% FBS + HG-DMEM; or Fibstar-CO medium, brand name HCell, catalog number FMS003C.

[0283] Mix Y: high glucose (HG)-DMEM supplemented with 10% FBS, containing Y-27632, 5μM; Vc, 0.2mM; EPZ004777, 5μM; Forskolin, 10μM; Repsox, 1μM.

[0284] Alternatively, add Y-27632, 5μM; Vc, 0.2mM; EPZ004777, 5μM; Forskolin, 10μM; Repsox, 1μM to the FibGro medium of brand HCell, product number FGS0040.

[0285] Mix Pn: HG-DMEM supplementedwith 10% FBS, containing VPA, 0.5mM; CHIR99021, 3μM; Repsox, 1μM; Forskolin, 10μM; Go 6983, 5μM; Y-27632, 5μM; AM5800.05μM; EPZ004777, 5μM; Vc, 0.2mM;

[0286] TTNPB, 5μM.

[0287] Alternatively, add the following to the FibGro medium (brand: HCell, product number: FGS0040): VPA, 0.5mM; CHIR99021, 3μM; Repsox, 1μM; Forskolin, 10μM; Go 6983, 5μM; Y-27632, 5μM; AM580, 0.05μM; EPZ004777, 5μM; Vc, 0.2mM; TTNPB, 5μM.

[0288] MSC basal culture medium: 10% FBS + LG-DMEM or bone marrow mesenchymal stem cell complete culture medium purchased from Cyagen, catalog number HUXMA-90011, or rFib medium from HCell, catalog number CRM0016-01.

[0289] Figure 19 Treatment with Mix Y-Mix Pn2 inhibited JAK1 expression in fibroblasts (A) and prolonged telomere length (B).

[0290] Example 22: Transdifferentiating skin fibroblasts into nerve cells

[0291] 1. Skin fibroblasts were seeded into 6-well plates and cultured in skin fibroblast culture medium for 24 hours;

[0292] 2. Replace the cell culture medium with a neural induction culture medium containing the small molecule compound combination MixNeu, and change the medium every 2 days;

[0293] 3. After culturing cells in a neural induction culture medium containing the small molecule compound combination MixNeu for 5–12 days, the elongated spindle-shaped cell morphology can be observed to change into the morphology of nerve cells. The culture medium is then replaced with nerve cell culture medium, and the cells are continuously passaged.

[0294] 4. Identification of transdifferentiated neural cells by immunofluorescence and quantitative PCR.

[0295] Skin fibroblast culture medium: 10% FBS + HG-DMEM; or Fibstar-CO medium, brand name HCell, catalog number FMS003C.

[0296] MixNeu:HG-DMEM supplementedwith 10% FBS, containing A8301, 0.5μM; bFGF, 10ng / mL; EPZ004777, 5μM; RG108, 10μM; parnate, 2μM; CHIR99021, 10μM; Forskolin, 50μM; VPA, 0.5mM; AM5800.05μM; BIX 01294, 1μM.

[0297] Neural cell culture medium: DMEM / F12, 5 mL; Neurobasal, 5 mL; N2, 1 / 100; B27, 1 / 50; cAMP, 100 μM;

[0298] BDNF, 20ng / mL; GDNF, 20ng / Ml; KOSR, 10% (v / v).

[0299] Figure 20 They transform from skin fibroblasts into nerve cells;

[0300] A. Tuj1 staining of nerve cells obtained from skin fibroblast transformation;

[0301] B. Identification of Nestin expression levels.

[0302] Example 23: Differentiation of embryonic stem cells into nerve cells

[0303] 1. After digesting the adherent embryonic stem cells, they are cultured in suspension in a neural induction medium;

[0304] 2. After culturing in the neural induction medium for 10-15 days, cell spheres can be observed to adhere to the plate one after another. The suspended cell spheres are then seeded into 6-well plates treated with Matrigel. The cells adhere and are cultured for another 5-7 days in the neural induction medium.

[0305] 3. After the cells adhere to the culture medium, replace the neural induction medium with neural cell culture medium;

[0306] 4. Identify the induced cells using immunofluorescence staining and quantitative PCR to detect neural markers.

[0307] Neural induction solution: DMEM / F12 supplemented with 10% KOSR, including bFGF, 10 ng / mL; Y-27632, 5 μM;

[0308] VPA, 0.5mM; EPZ004777, 5μM; Forskolin, 10μM; Repsox, 1μM.

[0309] Neural cell culture medium: DMEM / F12, 5 mL; Neurobasal, 5 mL; N2, 1 / 100; B27, 1 / 50; cAMP, 100 μM;

[0310] BDNF, 20ng / mL; GDNF, 20ng / Ml; KOSR, 10% (v / v).

[0311] Figure 21 When small molecule compounds are used, STAT5 gene expression is upregulated during the differentiation of ES into neural cells.

[0312] Example 24, Characterization of signaling pathways in rFib cells prepared as in Example 1

[0313] As shown in Example 1, the transcriptome of rFib cells from different individuals was sequenced, and 12,036 genes in each cell sample were analyzed by WGCNA to obtain 12 clustering modules.

[0314] Figure 22 KEGG pathway enrichment module

[0315] The bar chart shows the 12 modules enriched by the KEGG pathway, with representative genes for each pathway displayed in order of gene members. The box plot shows the distribution of average gene expression levels within each module.

Claims

1. A method for promoting the differentiation of skin fibroblasts into nerve cells, characterized in that, Includes the following steps: (1) After culturing skin fibroblasts in skin fibroblast culture medium, the skin fibroblast culture medium was replaced with neural induction culture medium containing the small molecule compound combination Mix Neu. The medium was changed every 2 days and cultured for 5 to 12 days. (2) Replace the nerve induction culture medium containing the small molecule compound combination Mix Neu with nerve cell culture medium, and continuously passage the cells to obtain nerve cells; The skin fibroblast culture medium is 10% FBS + HG-DMEM, or Fibstar-CO medium of brand HCell, catalog number FMS003C; The Mix Neu was HG-DMEM supplemented with 10% FBS, containing A8301 0.5μM, bFGF 10ng / mL, EPZ004777 5μM, RG108 10μM, parnate 2μM, CHIR99021 10μM, Forskolin 50μM, VPA 0.5mM, AM580 0.05μM, and BIX 01294 1μM. The neural cell culture medium consisted of: DMEM / F12 5 mL, Neurobasal 5 mL, N2 1 / 100, B27 1 / 50, cAMP 100 μM, BDNF 20 ng / mL, GDNF 20 ng / mL, and KOSR 10% (v / v).

2. The method according to claim 1, characterized in that, The skin fibroblasts mentioned are human skin fibroblasts.

3. A method for promoting the differentiation of embryonic stem cells into neural cells, characterized in that, Includes the following steps: (1) After digesting adherent embryonic stem cells, they were cultured in suspension in a neural induction solution; (2) After culturing in the neural induction medium for 10-15 days, the suspended cell spheres were seeded onto a 6-well plate treated with Matrigel. The cells adhered to the plate and were cultured in the neural induction medium for another 5-7 days. (3) After the cells adhere to the wall, the nerve induction medium is replaced with nerve cell culture medium and cultured for a longer period to obtain nerve cells; The neural induction solution is DMEM / F12 supplemented with 10% KOSR, containing bFGF 10ng / mL, Y-27632 5μM, VPA 0.5mM, EPZ004777 5μM, Forskolin 10μM and Repsox 1μM; The neural cell culture medium consisted of: DMEM / F12 5 mL, Neurobasal 5 mL, N2 1 / 100, B27 1 / 50, cAMP 100 μM, BDNF 20 ng / mL, GDNF 20 ng / mL, and KOSR 10% (v / v).

4. The method according to claim 3, characterized in that, The embryonic stem cells mentioned are human embryonic stem cells.

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