Methods for producing rejuvenated reparative fibroblasts and uses thereof

Abstract

A method for producing rejuvenated repair fibroblasts and its use is provided, in which the Jak-Stat signaling pathway in the repair fibroblasts is inhibited, resulting in rejuvenated cells. In this method, the corresponding signaling pathway in normal fibroblasts is inhibited by a combination of low molecular weight compounds, cytokines, or recombinant proteins, and the method can be applied to the reprogramming or rejuvenation of cells, tissues, organs, and organisms. [Selected Figure] Figure 1

Description

[Technical field]

[0001] The present invention belongs to the technical field of cell biology and relates to a method for producing rejuvenated repair fibroblasts and their use, in particular to a method for regulating cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging and apoptosis by controlling the JAK-STAT signaling pathway, thereby extending the life span of an organism, and the production and use of the cell product. [Background technology]

[0002] Stem cells are considered the "holy grail" of regenerative medicine and anti-aging. As an individual begins to age, the stem cells in the body also age, causing the deterioration or degeneration of multiple organs, including bone, cartilage, heart, muscle, brain, skin, pancreas, liver, kidneys, gastrointestinal tract, etc. Abnormal immune system function is also related to aging. In fact, chronic inflammation of tissues and organs is the cause of their degeneration and aging. Aged cells usually have problems such as DNA damage or mutation, shortening of telomeres, epigenetics, redox, and energy metabolism abnormalities, reduced proliferation ability, and increased cell death. Aged stem cells lose the ability to differentiate into certain cell types and abnormally differentiate into other cell types. For example, as is known, bone marrow mesenchymal stem cells derived from aged individuals have a reduced ability to differentiate into cartilage and an increased ability to form adipocytes. Thus, aged bone marrow filled with adipose tissue is also called "yellow bone marrow". Similarly, neural stem cells in aged individuals have a reduced differentiation into neurons and an increased differentiation into astrocytes. This is thought to be related to the decline in cognitive ability in the elderly.

[0003] Stem cells, especially mesenchymal stem cells, have become promising candidates to treat or intervene in the aging process and related diseases due to their easy availability, scalability and versatility, growth factor release, and immune-modulating ability. Mesenchymal stem cells are widely used clinically to treat many types of diseases, such as graft-versus-host disease, multiple sclerosis, amyotrophic lateral sclerosis, spinal cord injury, lupus erythematosus, arthritis, and aging. For allogeneic use, umbilical cord mesenchymal stem cells are widely considered. However, there are always clinical risks in the long-term / repeated use of "non-self" cells. Although autologous transplantation is considered safer, unfortunately, mesenchymal stem cells also senesce with age. Aged mesenchymal stem cells lose many important functions, limiting their clinical application. Induced pluripotent stem cells (iPS) are young cells that can be obtained from elderly people and were considered to be a suitable cell source for treatment. However, the low induction efficiency of iPS and the need for the introduction of foreign genes often lead to genetic mutations, which is not favorable for clinical application. Recently, gene modification has been used as a method of rejuvenation, but this method still has the risk of off-target and tumor formation.Therefore, the development of a method for producing self-rejuvenating and safe cells, and the acquisition of self-rejuvenating and safe repair cells are of great importance for preventing, delaying and reversing the progression of human aging, repairing tissue, organ structure or function, and improving health and quality of life. Summary of the Invention

[0004] The present invention aims to provide a method for producing rejuvenated rejuvenated fibroblasts and their uses, in particular a method for regulating cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, senescence, and apoptosis by controlling the JAK-STAT signaling pathway, thereby reversing senescence and extending the life span of an organism. The cell products (e.g., rejuvenated fibroblasts, rejuvenated mesenchymal stem cells) obtained by this method can be applied to prevent, delay, and reverse the progression of human aging, and to repair tissue, organ structure, or function. The rejuvenated fibroblasts produced by the technology of the present invention have the properties of both dermal fibroblasts and mesenchymal stem cells, and are therefore named regenerative fibroblasts (rFib), which may also be named induced and rejuvenated mesenchymal stem cells (irMSC) or induced mesenchymal stem cells (iMSC), and are referred to as rFib in the present specification.

[0005] The technical means of the present invention are as follows: A method for producing rejuvenated and repaired fibroblasts that activate or inhibit the JAK-STAT signaling pathway in a quantitative and / or timed manner, and the use thereof.

[0006] In the method, the gene or protein targets that are highly expressed or highly expressed and low expressed or suppressed in the JAK-STAT signaling pathway are CXCL2 (gene number / Accession: AY577905.1), SOS1 (gene number / Accession: NM_005633.3), STAT5B (gene number / Accession: NM_012448.3), JAK1 (gene number / Accession: NM_001321857.1), JAK3 (gene number / Accession: NM_000215.3), and the like. ), SOCS3 (Gene No. / Accession:NM_003955.4), IL6ST (Gene No. / Accession:NM_001243835.1), STAT1 (Gene No. / Accession:NM_007315.3), STAT2 (Gene No. / Accession:NM_198332.1), STAT3 (Gene No. / Accession:NM_213662.1), STAT4 (Gene No. / Accession:NM_001243835.1), STAT6 (Gene No. / Accession:NM_0011 78081.1), STAT5A (Gene No. / Accession:NM_001288720.1), IRF9 (Gene No. / Accession:NM_006084.4), IL6 (Gene No. / Accession:XM_005249745.5), IL6R (Gene No. / Accession:NM_181359.2), IL2 (Gene No. / Accession:NM_000586.3) (e.g., IL2A and / or IL2B), PRKCD (Gene No. / Accession:NM_001354679.1), CXCL1 2 (Gene No. / Accession:NM_000609.6), CXCR4 (Gene No. / Accession:NM_003467.2), JAK2 (Gene No. / Accession:NM_004972.3), IL15RA (Gene No. / Accession:NM_001351095.1), IL20RB (Gene No. / Accession:XM_006713665.4), GHR (Gene No. / Accession:NM_001242406.2) and PRLR (Gene No. / Accession:NM_001204314).2) contains at least one of the following.

[0007] The cells are initial target cells that regulate JAK-STAT, and the target cells are derived from mammals, including humans, mice, monkeys, and pigs, and the target cells include fibroblasts, epithelial cells, adipocytes, blood cells, mesenchymal stem cells, nerve cells, muscle cells, cardiac muscle cells, smooth muscle cells, vascular endothelial cells, induced pluripotent stem cells, embryonic stem cells, osteoblasts, chondrocytes, adipocytes, and osteoclasts; The cells produced by the method are defined herein as target cells, and include various types of cells that are derived from target cells and have different characteristics produced during the process of regulating JAK-STAT in the target cells, said characteristics including at least one of differentiation, reverse differentiation, transdifferentiation, rejuvenation, senescence, and apoptosis.

[0008] Quantification of activation or inhibition of the JAK-STAT signaling pathway is achieved by upregulating or downregulating at least one gene or protein target of the JAK-STAT signaling pathway in a target cell by any one of 1 to 300 fold relative to the target cell.

[0009] The timing of activation or inhibition of the JAK-STAT signaling pathway is controlled for 24 hours to 220 days so that at least one gene or protein target of the JAK-STAT signaling pathway in the target cell is highly expressed, low expressed, or not expressed, and the target cell obtained thereby maintains high, low or non-expression of at least one gene or protein target of the JAK-STAT signaling pathway for a long period of time, or restores the expression level to the same as that of the target cell.

[0010] In the method, the JAK-STAT signaling pathway can be activated or suppressed by regulating at least one of the pathways or targets selected from the group consisting of NOD-like receptor signaling pathway, focal adhesion, cell cycle, citric acid cycle, TGF-β 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, IL7 HDAC (histone deacetylase), PKC signaling pathway, RAR pathway, adenylate cyclase signaling pathway, HMT (histone methyltransferase), DNMT (DNA methyltransferase), and histone demethylase inhibitors.

[0011] Activating or inhibiting cellular targets and cell signaling pathways of the JAK-STAT signaling pathway; The gene or protein target in the NOD-like receptor signaling pathway is one or more selected from the group consisting of NAIP, IL6, CXCL12, NOD1, TAB3, CARD6, CXCL2, CXCL1, CXCL3, CARD8, CARD9, CASP1, CASP12, CASP4, CASP5, NFKB1, TMEM173, TNF, NFKBIB, NOD2, PYDC1, PYCARD, TAB1, TAB2, TNF, TLR4, NLRP1, NLRP12, NLRP3, NLRP6, MCU, RIPK3, RHOA, TAK1, BIRC2, ATG16L1, ATG5, ATG12, and TANK; the gene or protein target in the focal adhesion pathway is one or more selected from the group consisting of TNXB, RAPGEF1, ITGB8, SRC, THBS1, ITGA3, VCL, CAPN2, FLT4, FLT1, ITGA3, ITGB1, ITGB3, ITGB5, ITGB6, ITGB7, ITGA1, ITGA10, ITGA11, ITGA2, ITGA2B, ITGA5, ITGA6, ITGA7, ITGA8, ITGA9, ITGAV, PDRVG, PDGFA, PDGFB, PDGFC, PDGFD, PDGFRA, PDGFRB, BIRC3, BIRC2, BCL2, DOCK1, FN1, HGF, EGF, EGFR, IGF1, IGF1R, VEGFA, VEGFB, VEGFC, CTNNB1; The gene or protein target for controlling the cell cycle is one or more selected from the group consisting of 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, GADD45B, STAG1, STAG2, CDC14A, CDC14B, CDC20, CDC25A, CDC25B, MYC, SMC3, CDC16, YWHAH, YWHAB, YWHAQ, YWHAE, YWHAG, and YWHAZ; The gene or protein target for controlling the citric acid cycle is one or more selected from the group consisting of IDH3G, IDH3B, MDH2, SDHB, OGDH, MDH1, OGDHL, SUCLG1, SUCLG2, SUCLA2, SDHA, SDHB, SDHC, PDHA1, PDHB, and ACLY; The gene or protein target in the TGF-β signaling pathway is one or more selected from the group consisting of ACVR1C, THBS1, FST, TGFB1, TGFBR1, TGFBR2.TGFBR3, BMP4, RUNX3, RUNX2, CREBBP, IFNG, HRAS, FOS, TGFB2, TGFB3, ACVRL1, FOXO3, MTOR, KRAS, CREB1, ATF1, ATF2, ATF4, AKT1, AKT2, AKT3, HNF4A, HNF4G, and PIK3R3; the gene or protein target in the WNT signaling pathway is one or more selected from the group consisting of PRKCA, WNT7B, PRICKLE1, LRP6, CTNNB1, FZD4, CCND2, PRICK, WNT5A, WNT1, WNT10A, WNT11, WNT9A, WNT9B, WNT3, and WNT4B; the gene or protein target in the Notch signaling pathway is one or more selected from the group consisting of CIR1, KAT2B, MAML2, PSEN2, DVL2, RFNG, SNW1, DLL4, DTX3, DLL3, DLL1, DTX1, DTX2, CREBBP, CTBP1, CTBP2, JAG1, JAG2, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PSEN1, and PSEN2; The gene or protein target in the P53 signaling pathway is one or more selected from the group consisting of CCNG2, SIAH1, BBC3, TP53AIP1, TP53, SETD7, ATF3, CCNA2, CDK2, CCNG1, CHEK1, PRKC DKAT2B, PRL23, and PPP2CA; the gene or protein target in the calcium signaling pathway is one or more selected from the group consisting of RYR1, RYR2, RYR3, ESR1, AR (androgen receptor), KDR (kinase insert domain receptor), VDR (vitamin D receptor), ITPR1, ITPR2, ITPR3, PDE1A, PDE1B, PDE1C, PRKCA, PRKCD, PRKCE, and PRKCG; the gene or protein target in the insulin signaling pathway is one or more selected from the group consisting of RAPGEF1, PHKG1, PYGL, TRIP10, INS, INSR, IRS1, PDPK1, PIK3CA, HRAS, GRB2, PTPN1, and PTPN11; The gene or protein target of PKC is one or more selected from the group consisting of PRKCA, PRKCB, PRKDC, PRKCZ, PRKCE, PRKCG, PRKCD, PRKCH, PRKCI, PRKCQ, PRKD1, SLC9A5, MAPK3, MAPK9, MAPK8, and MAPK1; The gene or protein target in the RAR is one or more selected from the group consisting of RARA, RARS, RARB, RARG, RXRA, RXRG, FAM120B, NCOA1, and NCOR2; The gene or protein target that controls HDAC is one or more selected from the group consisting of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, and HDAC11; the gene or protein target in the adenylate cyclase signaling pathway is one or more selected from the group consisting of PRKAR1A, ADCY10, ADCYAP1, ADCY1, ADCY2, ADCY6, ADCY3, GNAI1, GNAL, GNAT3, PRKACA, PRKAR2B, PRKACB, PRKAR1B, PRKACG, CDKN1B, PRKAR2A, NCAM1, and CDKN1A; The gene or protein target in the HMT is one or more selected from the group consisting of HNMT, DNMT1, KMT2A, EHMT2, EHMT1, KMT2A, DOT1L, EZH2, SETD7, DNMT3B, DNMT3A, SETDB1, and SETD2; The gene or protein target in the DNMT is one or more selected from the group consisting of DNMT1, DNMT3B, DNMT3A, CDKN2A, CDKN2B, EHMT2, EHMT1, DNMT3L, CDH1, PARP1, and MBD2; The gene or protein target in the histone demethylase is one or more selected from the group consisting of KDM1A, KDM4A, KDM5A, KDM5B, KDM2A, KDM5C, KDM4B, KDM4C, KDM5D, KDM4D, KDM1B, HISTIH3A, HIST4H4, HIST2H3C, HAT1, HIST1H4C, HIST1H4F, HIST1H4J, HIST1H2AE, HIST1H2BB, CLOCK, and NOCA1.

[0012] The method includes at least one of a combination of small molecule compounds, a combination of cytokines, or a combination of recombinant proteins, a gene editing technique, and a gene recombination technique; The combination of low molecular weight compounds is Histone deacetylase inhibitors: sodium phenylbutyrate, butyrate, sodium butyrate, VPA, scriptaid, apicidin, LBH-589 (panobinostat), MS-275, SAHA (vorinostat), trichostatin (TSA), psammaplin A, splitomycin, SRT1720, resveratrol, sirtinol, APHA, CI-994, depudecin, FK-228, HC toxin, ITF-2357 (givinostat), chidamide, RGFP 966, PHOB, BG45, Nextulastat A, TMP269, CAY10603, MGCD-0103, Niltubacin, PXD-101 (Belinstat), Pyroxamide, Tubacin, EX-527, BATCP, Cambinol, MOCPAC, PTACH, MC1568, NCH51 and TC-H106 TGF-β inhibitors: 616452, LY2109761, Pirfenidone, Repsox (E-616452), SB431542, A77-01, A8301, GW788388, ITD-1, SD208, SB525334, LY364947, ASP3029, D4476 and SB505124 PKC inhibitors: Go6983, Go6976 and bicindolylmaleimide I (GF109203X) WNT / β-catenin agonists: MAY-262611, CHIR98014, CHIR99021, LiCl, Li2CO3, TD114-2, AZD2858, AZD1080, BIO, Kenpaullone, TWS119, LY2090314, CBM1078, SB216763 and AR-A014418 cAMP agonists: 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, Sp-8-Br-cAMPs RAR agonists: TTNPB, bexarotene, Ch55, tamibarotene, retinol, AM580, ATRA, vitamin A, vitamin A derivatives and 13-cis RA ROCK inhibitors: Y-27632, Y-27632 2HCl, thiazovivin, ripasudil (K-115), fasudil, GSK429286A, RKI-1447 and PKI-1313 JNK inhibitors: SP600125, JNK inhibitor IX, AS601245, AS602801 and JNK-IN-8 DNMT inhibitors: RG108, Thioguanine, 5-Aza-2'-Deoxycytidine (Decitabine), SGI-1027, Zebularine and 5-Azacytidine (AZA) HMT inhibitors: EPZ004777, EPZ5676, GSK503, BIX 01294 and SGC 0946 Histone demethylase inhibitors: Parnate (tranylcypromine), Tranylcypromine (2-PCPA) HCl SP2509, 4SC-202, ORY-1001 (RG-6016), GSKJ1 and GSK-LSD1 JAK-STAT inhibitors: 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) KX2-391 (Src inhibitor), PKI-402 (PI3K α / β / γ / δ and mTOR inhibitor), NSC 74859 (S3I-201) (STAT3 inhibitor), Fludarabine (Fludara) (STAT-1 inhibitor), U0126-EtOH (UO126 EtOH) (MEK1 and MEK2 inhibitor), SGI-1776 free base (Pim1, Pim2 and Pim3 inhibitor), Sorafenib (Nexavar) (VEGFR, PDGFR, c-Raf and B-Raf inhibitor), PLX-4720 (B-Raf V600E and c-Raf-1 Y340D / Y341D inhibitor) One or more of the following are included: The cytokine combination or recombinant protein combination includes PDGFAA, PDGFAB, BMP4, IGF1, bFGF, EGF, VEGF, insulin, activin A, TGF-beta1, Noggin, BMP-2, Shh, IL-6, CXCL10, CXCL12, CXCL2, HGF, IFN gamma, IL-2, IL-6 R alpha, IL-2Ralpha, TNF-alpha, TNF-beta, TPO, IGF2, IGFBP5, IGFBP6, IGFBP4, IGFBP7, IGFBP9, PDGF-BB, MMP3, GDF11, TIMP2; The gene editing technology includes upregulating or knocking out gene or protein targets in the JAK-STAT signaling pathway, such as STAT5A, by crispr / cas9 gene editing technology and TALEN gene editing technology; The genetic engineering techniques include overexpressing or suppressing gene or protein targets in the JAK-STAT signaling pathway, such as STAT5A, by lentivirus or retrovirus.

[0013] A repair type fibroblast produced by the above-mentioned method, wherein the JAK-STAT signaling pathway in the repair type fibroblast is inhibited, and the gene or protein target in the JAK-STAT signaling pathway that is under-expressed or whose expression is 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.

[0014] The following signaling pathways in the repair fibroblasts are altered: NOD-like receptor signaling pathway is inhibited, and / or TGF-β receptor signaling pathway is inhibited, and / or insulin signaling pathway is downregulated, and / or wnt signaling pathway is upregulated, and / or notch signaling pathway is downregulated, and / or p53 signaling pathway is downregulated.

[0015] A method for producing the repair fibroblasts, wherein the target cells for the repair fibroblasts are normal fibroblasts, and the normal fibroblasts are derived from connective tissue (e.g., blood, skin, bone marrow, heart) of a mammal (e.g., human, monkey, mouse, pig).

[0016] In the method, normal fibroblasts are treated with a combination of small molecular compounds to obtain repair fibroblasts, the combination of small molecular compounds including at least one of a Jak-Stat inhibitor, a WNT / β-catenin agonist, a histone deacetylase inhibitor, and a cAMP agonist.

[0017] The method for producing the repair fibroblast further includes using at least one of a RAR agonist, a DNMT inhibitor, an HMT inhibitor, a histone demethylase inhibitor, an ascorbate (ascorbic acid), a JNK inhibitor, a PKC inhibitor, a ROCK inhibitor, and a TGF-β inhibitor.

[0018] In the method for producing repair fibroblasts, a first step compound and a second step compound are used stepwise in a chronological order, the first step compound is a WNT / β-catenin agonist, a histone deacetylase inhibitor and a cAMP agonist, or the first step compound is a histone deacetylase inhibitor, a TGF-β receptor inhibitor, a WNT / β-catenin agonist and a cAMP agonist, and the second step compound is a histone deacetylase inhibitor, a TGF-β inhibitor, a WNT / β-catenin agonist, a cAMP agonist, a RAR agonist, an HMT inhibitor, an ascorbate (ascorbic acid), a PKC inhibitor, a PKC inhibitor and a ROCK inhibitor.

[0019] In the above-mentioned preparation method, at least one of 0.05-10 mM VPA, 1-15 μM CHIR99021, 0.5-10 μM Repsox, 3-50 μM Forskolin, 1-20 μM Go6983, 1-25 μM Y-27632, 0.02-1 μM AM580, 0.5-15 μM EPZ004777, 0.2 mM Vc, 0.2-20 μM TTNPB, 1-15 μM 5-azacytidine, and 1-50 μM SP600125 is used. Alternatively, normal fibroblasts are first treated with the first step compound for 2-10 days. The first stage compound includes 0.05-10 mM VPA, 1-15 μM CHIR99021, 0.5-10 μM Repsox, and 3-50 μM forskolin. After the first stage treatment, the second stage compound is treated for 4-20 days. The second stage compound includes 0.05-10 mM VPA, 1-15 μM CHIR99021, 0.5-10 μM Repsox, 3-50 μM forskolin, 1-20 μM Go6983, 1-25 μM Y-27632, 0.02-1 μM AM580, 0.5-15 μM EPZ004777, 0.2 mM Vc, and 0.2-20 μM TTNPB.

[0020] The telomere length of the reparative fibroblasts is 1.5 to 12 times longer than that of initial normal fibroblasts, and is close to the level of allogeneic cells from minors. Other types of cells (e.g., osteoblasts, chondrocytes) produced from the reparative fibroblasts have longer telomeres and exhibit stronger functional activity than allogeneic cells derived from the same animal.

[0021] Cell products (e.g., cell secretions, cell lysates) produced from the reparative fibroblasts produced by the above-described methods can be applied to the construction of tissue engineering materials, and to the retardation or reversal of cell, tissue, organ and organism aging.

[0022] Use of said reparative fibroblasts in the construction of tissue engineering materials, slowing or reversing aging of cells, tissues, organs and organisms.

[0023] Superfibroblasts are produced by knocking out the STAT5 gene in normal fibroblasts.

[0024] In the method for producing super fibroblasts, super fibroblasts that have been rejuvenated and have extended telomeres are obtained within 3 to 100 days after knocking out the STAT5 gene from normal fibroblasts.

[0025] The method is to treat mesenchymal stem cells with a combination of small molecule compounds or gene editing to finally produce rejuvenated mesenchymal stem cells. The combination of small molecule compounds includes at least one of a Jak-Stat inhibitor, a WNT / β-catenin agonist, a DNMT inhibitor, a TGF-β inhibitor, and a cAMP agonist. The gene editing treatment is to knock out a gene or protein target (e.g., STAT5A) of the Jak-Stat signaling pathway.

[0026] The treatment with the combination of low molecular weight compounds involves treating mesenchymal stem cells with 1 to 15 μM CHIR99021, 1 μM to 15 μM 5-azacytidine (AZA); and / or 1 μM to 15 μM 5-azacytidine (AZA), 3 to 50 μM forskolin; and / or 1 μM to 15 μM 5-azacytidine (AZA), 3 to 50 μM forskolin, and 1 μM to 15 μM CHIR99021 for 1 to 28 days.

[0027] Use of a cell product (e.g., reparative fibroblasts) produced by said method or a cell culture medium / media of said cell product, a lysate of the cell product, a kit, a drug, a health product, a food, a cosmetic or a medical device.

[0028] Use of cell products (e.g., reparative fibroblasts) produced by the method in repairing mammalian tissues, organ damage, and aged, degenerated tissues and organs as a source of seed cells for tissue engineering materials and scaffolds for tissue engineering materials.

[0029] Use of cell products (eg, reparative fibroblasts) produced by the methods in medical research or as immunomodulators.

[0030] Use of cell products (eg, reparative fibroblasts) produced by said methods in preventing, slowing, or reversing the aging process in mammalian tissues, organs, or organisms in vitro / in vivo.

[0031] Use of a cellular product produced by said method in the reprogramming of a cell, tissue, organ or organism, or in the rejuvenation of a cell, tissue, organ or organism.

[0032] In the present invention, the gene or protein target in the Jak-Stat signaling pathway is quantitatively and / or time-controlled to regulate cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging, and apoptosis, reverse aging, and extend the life span of the organism. The gene or protein target in the Jak-Stat signaling pathway in the cell is quantitatively and / or time-controlled by a combination of small molecule compounds, a combination of cytokines, or a combination of recombinant proteins, gene editing technology, or gene recombination technology. The gene or protein target includes at least one of CXCL2, SOS1, STAT5B, JAK1, JAK3, SOCS3, IL6ST, STAT1, STAT2, STAT3, STAT4, STAT6, STAT5A, IRF9, IL6, IL6R, IL2 (e.g., IL2A and / or IL2B), PRKCD, CXCL12, CXCR4, JAK2, IL15RA, IL20RB, GHR, and PRLR. The combination of small molecule compounds that control the Jak-Stat signaling pathway includes at least one of a Jak-Stat inhibitor, a WNT / β-catenin agonist, a histone deacetylase inhibitor, and a cAMP agonist, a RAR agonist, a DNMT inhibitor, an HMT inhibitor, a histone demethylase inhibitor, an ascorbate (ascorbic acid), a JNK inhibitor, a PKC inhibitor, a ROCK inhibitor, and a TGF-β inhibitor. The cytokine combinations or recombinant protein combinations include PDGFAA, PDGFAB, BMP4, IGF1, bFGF, EGF, VEGF, insulin, activin A, TGF-beta1, Noggin, BMP-2, Shh, IL-6, CXCL10, CXCL12, CXCL2, HGF, IFN gamma, IL-2, IL-6 R alpha, IL-2Ralpha, TNF-alpha, TNF-beta, TPO, IGF2, IGFBP5, IGFBP6, IGFBP4, IGFBP7, IGFBP9, PDGF-BB, MMP3, GDF11, TIMP2.The gene editing technology uses crispr / cas9 gene editing technology and TALEN gene editing technology to upregulate or knock out a gene or protein target in the JAK-STAT signaling pathway, such as STAT5A. The gene recombination technology uses lentivirus or retrovirus to overexpress or suppress a gene or protein target in the JAK-STAT signaling pathway, such as STAT5A.

[0033] In the present invention, a low molecular weight compound is used to suppress gene or protein targets (e.g., STAT5A, JAK1) of the Jak-Stat signaling pathway in fibroblasts, thereby producing repaired dermal fibroblasts. The Jak-Stat signaling pathway in the repaired dermal fibroblasts is suppressed. The following changes occur in the signaling pathways in the repaired fibroblasts: the NOD-like receptor signaling pathway is suppressed, and / or the TGF-β receptor signaling pathway is suppressed, 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. The telomere length of the repaired fibroblasts is 1.5 to 12 times longer than that of initial normal fibroblasts, and is close to the level of allogeneic cells from minors. Another type of cell (e.g., osteoblasts, chondrocytes) produced from the repaired fibroblasts has elongated telomeres and exhibits stronger functional activity than allogeneic cells derived from the same animal. The reparative fibroblasts and cell products produced by the cells (eg, cell secretions, cell lysates) can be applied to the construction of tissue engineering materials, and to slow or reverse aging of cells, tissues, organs and organisms.

[0034] The technology of regulating cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, senescence, apoptosis, reversing senescence, and extending the life span of an organism by quantitatively and / or periodically controlling gene or protein targets in the Jak-Stat signaling pathway provided by the present invention can promote transdifferentiation between different types of cells, can be applied to produce different types of rejuvenated cells (e.g., reverse senescence of mesenchymal stem cells and produce super fibroblasts), and can be applied to promote cellular senescence and apoptosis. The cells and cell products produced by the technology can be used to prevent, delay, or reverse the aging process of mammalian tissues, organs, and organisms in vitro / in vivo, can be used for reprogramming cells, tissues, organs, and organisms, and can be used as a source of seed cells for tissue engineering materials and scaffolds for tissue engineering materials to repair damaged mammalian tissues, organs, and aged or degenerated tissues and organs.

[0035] Mechanism of the invention By controlling the expression of gene or protein targets of the Jak-Stat signaling pathway in cells, the cellular state of the target cells is altered, transforming them into other cells of a different type or with different cellular properties, by quantitatively and / or timely regulating changes in different metabolic pathways of the cells.

[0036] Compared with the prior art, the present invention has the following beneficial effects: The repaired fibroblasts provided by the present invention have rejuvenation characteristics but no tumorigenicity compared to fibroblasts derived from the same individual or donor of the same age group. The rejuvenation characteristics include, for example, changes in epigenetics and / or changes in the expression levels of aging-related genes and / or lengthening of cell telomeres and / or increased cell proliferation rate and / or the ability of long-term stable cell passage. The rejuvenated cells and their cell products can reverse aging and extend the lifespan of mammalian organisms. The technology provided by the present invention for regulating cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging, and apoptosis, reversing aging, and extending the lifespan of an organism by quantitatively and / or periodically controlling gene or protein targets in the Jak-Stat signaling pathway can systematically regulate cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging, and apoptosis, and the cells and cell products produced by this technology can be applied to prevent, delay, and reverse the progression of aging in mammalian tissues, organs, and organisms in vitro / in vivo, and can be applied to reprogramming cells, tissues, organs, and organisms, and can be applied to repair damaged mammalian tissues, organs, and aged or degenerated tissues and organs as seed cells for tissue engineering materials and sources of scaffolds for tissue engineering materials. [Brief description of the drawings]

[0037] [Figure 1] 1 shows the production of regenerative dermal fibroblasts (rFib). [Diagram 2] rFib shows rejuvenating characteristics compared to Fib and bMSC. [Diagram 3] The osteogenic and chondrogenic differentiation abilities of aged bMSCs are worse than those of rFibs, indicating that rFibs are not tumorigenic. [Figure 4] rFibs are shown to have immunomodulatory functions in vitro. [Diagram 5] We show that rFib cells have immune-modulating functions in vivo. [Figure 6] This indicates that rFib has the ability to repair bone defects regardless of age. [Figure 7] 1 shows an in vivo cartilage repair experiment. [Figure 8] We show that suppression of the STAT5 gene rejuvenates dermal fibroblasts and acquires the ability to undergo multidirectional differentiation. [Figure 9] This shows the changes in STAT5 and H3K9me after STAT5 knockout. [Figure 10] 1 shows detection of rejuvenation and differentiation capacity after STAT5 knockout in another line of 62-year-old (62Y) Fib. [Figure 11] Figure 2 shows the rejuvenation of MSC cells. After treating the cells with different compounds for 3 days, β-galactosidase staining is performed. 55Y=55 years, 82Y=82 years; Y=year indicates the age of the cell donor. [Figure 12] We show that rFib cells can extend the lifespan and increase bone density in aged NOD / SCID mice. [Figure 13] We show that rFib cells distribute to multiple organs in mice and can differentiate into functional cells. [Figure 14] We show that rFib cells can improve bone density in aged osteoporotic mice. [Figure 15] This indicates that rFib culture medium can significantly promote skin healing. The rFib culture medium group was almost completely healed 12 days after model construction. [Figure 16] We show that rFib can ameliorate lower limb ischemia symptoms in mice. [Figure 17] Mix Y treatment suppresses the expression of cellular STAT5 and STAT3 genes (AB), downregulates CDKN1A gene, and lengthens telomeres (CD), indicating cell rejuvenation. [Figure 18] Mix Pn treatment down-regulates the expression of cellular STAT5 (A), and suppresses the expression of ATF3, CDKN1A, GADD45B and IL6 (BE), indicating cell rejuvenation. [Figure 19]Mix Y-Mix Pn2 treatment suppresses fibroblast JAK1 expression (A) and increases telomere length (B). [Figure 20] 20 shows the conversion of skin fibroblasts to nerve cells. Fig. 20A shows Tuj1 staining of nerve cells converted from skin fibroblasts, and Fig. 20B shows the measurement of the expression level of Nestin. [Figure 21] We show that the expression of the STAT5 gene is upregulated during the differentiation process from ES cells to neurons by the action of a small molecule compound. [Figure 22] Modules containing many KEGG pathways are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Example 1: Obtaining repair-type dermal fibroblasts and identifying their characteristics

[0039] 1. Human dermal fibroblasts were seeded into 6-well plates and cultured in dermal fibroblast culture medium for 24 hours.

[0040] 2. The cell culture medium was replaced with Fib-inducing medium containing the small molecule cocktail Mix V, and the medium was replaced every 2 days.

[0041] 3. After culturing in rFib induction medium containing small molecule cocktail combination Mix V for 5 days, the medium was replaced with rFib induction medium containing small molecule cocktail combination Mix P, and the liquid was replaced every 2 days.

[0042] 4. After 7 days of culture in rFib induction medium containing small molecule cocktail combination Mix P, the medium was replaced with HG-DMEM supplemented with 10% FBS, 10ng / mL bFGF, 100ng / mL PDGF-AB and 10ng / mL BMP4, or HG-DMEM supplemented with 10% FBS, or cultured in rFib medium (medium). After 3 days of treatment, the cells were subcultured for a long period. After 3 days of treatment, the cells were identified.

[0043] 5. During long-term passage, rFibs were cultured in MSC basal medium and passaged when the cell confluence reached 90%.

[0044] Dermal fibroblast culture medium: 10% FBS + HG-DMEM; or Fibstar-CO medium, brand: HCell, product number: FMS003C.

[0045] Mix V: High glucose (HG)-DMEM supplemented with 10% FBS (containing 0.5 mM VPA, 3 μM CHIR99021, 1 μM Repsox, and 10 μM Forskolin)

[0046] Mix P: HG-DMEM supplemented with 10% FBS (containing 0.5 mM VPA, 3 μM CHIR99021, 1 μM Repsox, 10 μM Forskolin, 10 μM SP600125, 5 μM Go 6983, 5 μM Y-27632, 0.05 μM AM580, 5 μM EPZ004777, 0.2 mM Vc, and 5 μM TTNPB)

[0047] Alternatively, FibGro medium (brand HCell, product number FGS0040) was supplemented with 0.5 mM VPA, 3 μM CHIR99021, 1 μM Repsox, 10 μM forskolin, 10 μM SP600125, 5 μM Go 6983, 5 μM Y-27632, 0.05 μM AM580, 5 μM EPZ004777, 0.2 mM Vc, and 5 μM TTNPB.

[0048] MSC basal medium: 10% FBS+LG-DMEM; Bone marrow mesenchymal stem cell complete medium purchased from Cyagen, product number HUXMA-90011; or rFib medium, brand HCell, product number CRM0016-01.

[0049] Note: Unless otherwise specified, the cells used in the examples are of human origin.

[0050] Figure 1: Production of regenerative dermal fibroblasts (rFib)

[0051] A is a schematic diagram of the process of converting dermal fibroblasts (Fibs) to rFibs. After conversion of nearly senescent Fibs (passage P13) to rFibs, they gained enormous proliferation potential (and could be expanded for a further 19 passages). The same treatment process induced the death of bone marrow mesenchymal stem cells.

[0052] B is a comparison of the growth curves of rFib and its homologous Fib during long-term amplification. The growth rate of the treated rFib was faster than that of the homologous Fib.

[0053] C shows histochemical analysis of the trilineage differentiation (differentiation into fat, bone, and cartilage) ability of cells before and after conversion (parental skin cells were derived from P8 passage cells of one 39-year-old volunteer). Samples were amplified multiple times on day 0 (Fib, before treatment), day 5, day 12, and day 15 (rFib), and after 21 days of differentiation induction, their ability to form bone and lipo-cartilage was detected. Osteoblast (bone-forming, identified by Alizarin Red staining), Adipocyte (adipocyte, identified by Oil Red staining), and Chondrocyte (cartilage-forming, identified by Alcian Blue staining). After multiple passages (P9 passage, P16 passage), rFib cells still maintained good trilineage differentiation (differentiation into fat, bone, and cartilage) ability.

[0054] D shows the results of q-RT-PCR analysis of ALP levels 14 days after osteogenic differentiation, COL2A1 levels 14 days after chondrogenic differentiation, and PPARG levels 21 days after adipogenic differentiation. As can be seen, rFib cells, like young bMSCs, highly express genes related to trilineage differentiation after induction. Compared to D0(Fib), *p<0.05, **p<0.01, ***p<0.001, n≧3.

[0055] EG shows changes in the expression of JAK1, STAT5 (decreased after treatment), telomerase (TERT) expression (high expression after 5 days), and telomere length (significantly lengthened at 15 days) in cells at different time points after treatment with a combination of small molecules.

[0056] H shows transcriptome analysis of rFib cells. Its aggregation state is close to that of Fib and bMSC, and unlike iPSC and ESC, it has both the characteristics of dermal fibroblasts and mesenchymal stem cells. rFib are safe and non-tumorigenic cells.

[0057] Figure 2: rFib has rejuvenated characteristics compared to Fib and bMSC

[0058] A shows immunofluorescence staining of H3K9me3 and H4K20me3 at D0 (parental Fib at passage P11) and D15 (rFib). The senescence marker H4K20me3 in rFib cells was significantly decreased compared to its homologous Fib.

[0059] B and C show immunofluorescence staining and quantitative analysis of γH2AX at D0 (parental Fib at passage P11) and D15 (rFib). The senescence marker γH2AX in rFib cells was significantly decreased compared to its homologous Fib.

[0060] D shows the proliferation curves during long-term expansion of Fib, bMSC, and rFib derived from two old individuals. The same color indicates cells derived from the same individual. The proliferation rate of rFib was faster than bMSC and Fib derived from the same individual, and faster than young bMSC (33 years old).

[0061] EG shows q-RT-PCR detection of multiple cellular senescence markers (CDKN1A, ATF3, and IL-6). The expression of senescence markers in rFib cells and osteoblasts and chondrocytes induced by rFib cells was significantly reduced. Here, 12W indicates skin cells derived from a fetus aborted at 12 weeks of pregnancy.

[0062] H. Using q-RT-PCR, we measured the relative telomere length of Fib, rFib, bMSC, and their differentiated osteoblasts (rFib-OB, bMSC-OB), and chondroblasts (rFib-CH, bMSC-CH) using the T / S value.

[0063] In EH figures, cells derived from the same individual are shown in the same color. * indicates significant difference compared to the homologous Fib, and # indicates significant difference compared to the corresponding bMSC, bMSC-OB (bMSC differentiated osteoblasts), and bMSC-CH (bMSC differentiated chondroblasts). *p<0.05, **p<0.01, ***p<0.001, #p<0.05, ##p<0.01, ###p<0.001, n=3.

[0064] Figure 3: Aged bMSC osteogenic and chondrogenic differentiation capacity is worse than rFib, and rFib has no tumorigenicity.

[0065] A shows Alizarin Red staining of osteogenic differentiation of bMSCs and rFibs derived from individuals of different ages. As can be seen from the results, the osteogenic differentiation ability of bMSCs derived from aged individuals was significantly reduced, while rFibs from the same individuals retained good osteogenic differentiation ability.

[0066] B shows Oil Red O staining of adipogenic differentiation of bMSCs and rFibs derived from individuals of different ages. As can be seen, the adipogenic differentiation capacity of bMSCs derived from old individuals was significantly enhanced, while rFibs derived from old individuals retained the adipogenic differentiation capacity consistent with young individuals.

[0067] CD shows the results of quantitative analysis of osteogenesis (ALP) and adipogenesis (PPARG) marker genes in the corresponding cells by q-RT-PCR. The results were consistent with the staining results.

[0068] D shows COL2A1 and MMP13 immunohistochemical staining during chondrogenic differentiation of bMSCs and rFibs derived from individuals of different ages. After chondrogenic differentiation, old bMSCs showed low expression of COL2A1 and high expression of MMP13, while rFibs derived from old individuals showed similar expression of COL2A1 / MMP13 to that of young individuals.

[0069] FG shows the results of q-RT-PCR detection of the expression of COL2A1 and MMP13 after chondrogenic differentiation of bMSCs and rFibs derived from individuals of different ages. The results were consistent with the staining results.

[0070] H shows the karyotype detection of rFibs at passages P9 and P13, and their homologous passage P6 Fibs. As can be seen from the results, the karyotype of rFibs was consistent with that of Fibs even after long-term passage.

[0071] I shows the rFib teratoma formation test. Human embryonic stem cells (hESCs) were used as a positive control. Subcutaneous transplantation of hESCs into NOD / SCID mice resulted in the formation of teratomas (with clear triple germ layer structure), whereas transplantation into rFib mice did not result in tumor formation.

[0072] J shows the analysis of telomere length and telomerase expression in cells. rFib cells have significantly longer telomeres than their homologous Fib cells, and they are still stable after induction is completed, but telomerase is transiently highly expressed during the induction process and then restored to a low expression level, which is different from tumor cells (telomerase is continuously highly expressed in tumor cells). This indicates that rFib cells are rejuvenated but not tumorigenic.

[0073] 6.1 In vitro immunomodulation studies

[0074] Treat with mitomycin C for 2.5 hours, digest and count, and cells are diluted to 1 × 10 5 Lymphocytes were stained with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) for 30 min at 37°C and 2 × 10 5The cells were seeded in a 24-well plate at a density of 100 μg / ml. Lymphocyte proliferation was stimulated with PHA at a final concentration of 2 μg / ml. Five groups were set up: bMSC+lymphocyte+PHA coculture group, rFib+lymphocyte+PHA coculture group, dermal fibroblast+lymphocyte+PHA coculture group, lymphocyte+PHA positive control group, and lymphocyte only negative control group. After 5 days of coculture, lymphocytes in each well were harvested and rinsed three times with PBS. Lymphocyte proliferation was detected by flow cytometer using anti-CD3, CD4, and CD8 antibodies. All antibodies were purchased from BD biosciences.

[0075] Figure 4: rFibs have immunomodulatory functions in vitro.

[0076] A shows that parental Fib, rFib and bMSC were cultured with T cells in mixed lymphocyte reaction, respectively. PBMCs derived from healthy volunteers were labeled with CFSE. Compared with "T+PHA" group, **p<0.01, ***p<0.001, n=3. As can be seen from the results, rFib has the ability to suppress the proliferation of T cells, while its homologous Fib cells have no ability to suppress the proliferation of T cells, indicating that rFib has immune-modulating ability.

[0077] BC indicates that rFibs have regulatory potential on CD4+ immune cell subtypes.

[0078] DE indicates that rFibs have regulatory potential on CD8+ immune cell subtypes.

[0079] 6.2 In vivo immunomodulation experiments

[0080] 1×10 6 After culturing bMSCs, rFibs, and dermal fibroblasts in 10-cm culture dishes at a density of 100 μm for 48 hours, the culture medium was filtered through a 0.22 μm filter membrane (Millipore) to remove cells or cell debris, and then concentrated 100-fold using an ultrafiltration centrifuge tube.

[0081] Acute liver injury was induced in 8-12 week old C57BL / 6 mice by tail vein injection of 25 mg / kg body weight of concanavalin dissolved in PBS, and PBS alone was injected as a control (Han et al., 2014). There were six mice in each group. After 30 minutes, different concentrated media or PBS were injected into the tail vein. 8.5 hours after tail vein injection of the media, the mice were euthanized. Blood and liver were collected. Livers were stained with HE and CD3+ T cells were detected by flow cytometer, and ALT / AST was detected in blood.

[0082] Serum ALT / AST was quantified using an ELISA kit (Shanghai Enzyme-linked Biotechnology Co., Ltd.) according to the manufacturer's instructions. Three independent replicate samples were set up for each group, and data are expressed as mean ± SD.

[0083] Figure 5: rFib cells have in vivo immunomodulatory capacity.

[0084] A shows the results of inducing acute liver injury in C57BL / 6 mice with concanavalin and treating them with concentrated rFib cell culture medium. As can be seen from the results, mice treated with rFib culture medium did not have obvious abnormalities (hemorrhage, necrosis, etc.) in the liver.

[0085] B shows the results of measuring the absolute number of T lymphocytes in liver tissue 8.5 hours after tail vein injection of the cell culture medium. As can be seen from the results, the culture medium of rFib had a significant immunomodulatory ability similar to that of bMSC.

[0086] CD shows that the blood ALT and AST levels of mice treated with rFib culture medium were both close to normal levels, and they showed no significant symptoms of liver injury.

[0087] 6.3 PCR of ordinary genes

[0088] Total RNA was extracted according to the instructions of the TRIzol kit (Takara Bio). RNA (1.0 μg) was reverse transcribed to cDNA using the Primescript RT kit (Takara Bio). In q-RT-PCR, cDNA was used as a template, SYBR Premix EX TaqTMII (Takara Bio) was used as a specific primer, and SYBR Green was used. The cycling conditions were as described by the manufacturer (Takara). Relative expression levels were normalized by an internal reference (ACTIN). In gPCR, genomic DNA was used as a template, and Premix Taq (Takara Bio) was used as the human-specific primer ACTIN.

[0089] Example 2: rFib cells have the ability to repair bone defects.

[0090] Under the approval of the ethical committee, femoral defect models were constructed using NOD / SCID mice aged 8-10 weeks and weighing 20-24 g, with five animals in each group. The model was constructed by incising the skin and subcutaneous tissue under sodium pentobarbital anesthesia, bluntly separating the rectus femoris and semitendinosus muscles, and exposing a sufficient amount of the midsection of the femur. Surgery was performed proximal to the center of the right femur. A continuous bone defect of 4 mm × 1 mm was constructed during surgery. Dermal fibroblasts (Fib), bone marrow mesenchymal stem cells (bMSC), and rFib were stained with Hoechst 33342 (Thermo, NucBlue live cell), mixed with Matrigel, and 5 × 10 5 Cells were transplanted (administered) at the defect site at 100 cells / mouse.

[0091] 28 days after transplantation, the mice were euthanized by a lethal injection of sodium pentobarbital. The mouse femurs were bluntly dissected, fixed in 4% PFA, and detected by μCT imaging (SkyScan 1272, Bruker microCT), and the collected data were analyzed.

[0092] Figure 6: rFib has the ability to repair bone defects without age restriction.

[0093] A is a schematic diagram of the surgical procedure for a mouse mid-femur defect model.

[0094] B shows mouse femur samples from different groups and H&E staining. Both young bMSCs (31 years old) and rFib (39 years old) have good bone defect repair ability.

[0095] C shows that rFib cells were labeled with Hoechst 33342 and were able to self-fluoresce blue. Sections of the repair site show that rFib cells formed new bone at the defect site and corresponded in number and location to newly formed bone cells.

[0096] D shows micro-CT analysis of different experimental groups. rFib derived from an old individual (62 years old) can repair bone defects, but has a very weak bone defect repair ability.

[0097] E shows micro-CT analysis of the different experimental groups. The repair capacity of rFibs from young (39 years old) and old (62 years old) individuals was similar, indicating that the ability of rFibs to repair bone defects is not limited by age.

[0098] Example 3: rFib cells have the ability to repair cartilage defects.

[0099] Articular cartilage defect model and cell transplantation

[0100] NOD / SCID mice aged 8–10 weeks and weighing 20–24 g were used. A modified articular cartilage model was used to evaluate the therapeutic effect of rFib (Cheng et al., 2014). Articular cartilage defects (1.5 mm × 1 mm) were constructed in the trochle groove of the distal femur using a biopsy punch. Cells (2.5 × 10 5 Cells were labeled with Hoechst 33342 and implanted into the defect site. Matrigel without cells was implanted as a control.

[0101] Figure 7: In vivo cartilage repair experiment

[0102] AB shows the cartilage tissue samples and 10 μm sections stained with Safranin Fast Green. In Safranin Fast Green staining, red indicates cartilage and green indicates bone tissue. Both young rFib (39 years old) and bMSC (31 years old) can repair cartilage defects, and old bMSC (62 years old) can generate new tissue but no neocartilage, while even rFib (62 years old) derived from an old individual can form cartilage tissue.

[0103] C shows the Pineda score of cartilage repair. As can be seen from the results, rFib derived from an old individual (62 years old) and rFib derived from a young individual (39 years old) have similar cartilage repair capabilities.

[0104] In D, Hoechst 33342 labeled rFib formed new cartilage tissue as seen in the section. The newly formed cartilage tissue resembled the structural morphology of normal cartilage, without the formation of abnormal tissue.

[0105] Examples 4 to 12: Repair-type fibroblasts were obtained by treatment for different periods of time with different combinations of low molecular weight compounds. The identification method was the same as in Example 1. The combinations are shown in the table below (Table 1).

[0106] [Table 1] JPEG0007689490000002.jpg134142

[0107] Example 13: Production of superfibroblasts

[0108] 1. Construction of the vector plasmid with the stat5A gene knocked out of crispr / cas9: The following plasmids were purchased from Cyagen: pLV[2gRNA]-EGFP:T2A:Puro-U6>hSTAT5A[gRNA#4]-U6>hSTAT5A[gRNA#10] pLV[Exp]-CBh>hCas9:T2A:Hygro

[0109] 2. The virus was transduced according to the manufacturer's instructions. One day after the virus transduction, the medium containing the virus was removed and replaced with fresh complete medium. Incubate at 37°C, 5% CO 2 The cells were incubated in an incubator at 4 °C for 1 h.

[0110] 3. From the second day after viral transfection, the gene carried by the lentivirus began to be expressed, and the cells were further cultured to further accumulate the expression product or to change the cell phenotype.

[0111] 4. After amplifying the cells, the virus-transduced cells were purified using antibiotics, and the cells were further expanded in HG-DMEM containing 10% FBS, and the cells were continuously cultured for 150 days.

[0112] Figure 8: By suppressing the STAT5 gene, dermal fibroblasts can be rejuvenated and acquire multidirectional differentiation capabilities.

[0113] A shows two representative modules enriched in KEGG pathways of rFib selected from 12,036 genes determined by WGCNA, and box-and-whisker plots of the expression changes of two gene modules in several samples.

[0114] As can be seen in B, after knocking out the STAT5 gene, the cells were subsequently cultured for 40 days, and immunohistochemical staining showed that after knocking out STAT5 in Fib (Fib-STAT5-KO), H4K20me3 (the more senescence markers there are, the more senescence there is) was significantly reduced compared to the control.

[0115] C. Identification of senescence marker genes (ATF3, GADD45B, IL6, CDKN1A, highly expressed in senescent cells). As can be seen from the results, the expression of senescence marker genes in STAT5-knockout Fib cells was significantly reduced.

[0116] As can be seen in D, after STAT5 knockout, Fib exhibits the ability to form bone (stained with Alizarin Red S) and cartilage (stained with Alcian Blue).

[0117] EG shows changes in cellular JAK1 and STAT5 expression (decreased after knockout), telomerase (TERT) expression (high expression after 43 days), and telomere length (significantly lengthened on day 54) after STAT5 gene knockout.

[0118] H is a schematic diagram showing the regulation of the Jak-Stat signaling pathway in fibroblasts to rejuvenate cells and the organism.

[0119] Figure 9: Changes in STAT5 and H3K9me after STAT5 knockout

[0120] A shows that STAT5A expression is abolished after STAT5 knockout against Fib.

[0121] B shows that knockout of STAT5 does not affect H3K9me.

[0122] FIG. 10: Detection of rejuvenation and differentiation capacity after STAT5 knockout for another line of 62-year-old (62Y) Fib.

[0123] A shows detection of senescence-related genes.

[0124] B shows detection of osteogenic and chondrogenic differentiation.

[0125] CD indicates detection of telomerase and telomere length.

[0126] E shows detection of STAT5 expression status.

[0127] F shows detection of STAT5 in cells of the same line treated with the Mix V+Mix P system.

[0128] Example 14: Rejuvenation of MSC cells

[0129] 1. Bone marrow mesenchymal stem cells derived from different individuals were cultured in low-glucose DMEM containing 10% FBS.

[0130] 2. After cells were treated with different compound combinations for 3 days, the cells were subsequently cultured in low-sugar DMEM medium containing 10% FBS for 3 days, and then stained for β-galactosidase.

[0131] [Table 2] Table 2: Treatment with different compound combinations

[0132] Figure 11: Rejuvenation of MSC cells After treating the cells with different compounds for 3 days, β-galactosidase staining was performed. 55Y=55 years, 82Y=82 years, Y=year: age of cell donor

[0133] Example 15: Intravenous injection of rFib can extend the life span of aged mice.

[0134] Fibs at passage P9, rFibs at passage P13, and rFibs at passage P13 from the same individual aged 39 years were labeled with Hoechst 33342 and dissolved in 200 μL DMEM. 6 The 1000 μL / animal) was injected via the tail vein into naturally aged NOD / SCID mice (43 weeks old; the average lifespan of this mouse is 36-38 weeks. 43 weeks of age corresponds to approximately 86 years in humans). In the vehicle group, only 200 μL DMEM was injected. After the mice died naturally, they were sampled and detected.

[0135] FIG. 12 shows that rFib cells can extend the lifespan and increase bone density in aged NOD / SCID mice.

[0136] A shows the survival curve of aged mice. Mice injected with rFib cells can effectively extend the life span of mice, whether the cells are derived from young (39 years old) or old (62 years old) individuals. Even young Fib cells do not have the effect of extending life span, and the survival curve is not significantly different from that of the vehicle group.

[0137] B shows the observation of mouse morphology in the two groups: 43-week-old mice were obviously aged (with messy, dull, and hunched fur), and 4 weeks after injection of rFib cells, the mice's condition was not obviously improved, while the mice injected with DMEM were even more aged after 4 weeks.

[0138] CE shows the autopsy photographs of 4 mice aged 10 weeks, 25 weeks, 47 weeks (injected with DMEM at 43 weeks, died at 47 weeks), and 49 weeks (injected with rFib at 43 weeks, died at 49 weeks), H&E stained gastric mucosa sections, and micro-CT analysis of lumbar vertebra 3. As can be seen from the figures, the appearance of the gastrointestinal tract of the mice injected with rFib cells was close to that of young mice (10W, 25W), the thickness and density of the gastric mucosa, and the lumbar trabecular bone structure were close to that of the 25W mice, and the gastrointestinal tract of the aged mice injected with DMEM had significant lesions, with short and sparse gastric mucosa and severe loss and fracture of the lumbar trabecular bone.

[0139] F shows Micro-CT data analysis. The bone mineral density (BMD), relative bone volume (BV / TV) of aged mice injected with rFib were close to those of 25-week-old mice, and the trabecular bone number (Tb.N) and separation of trabecular bone (Tb.Sp) were improved compared to those of mice injected with DMEM (5 animals in each group; *p<0.05, **p<0.01, ***p<0.001, n=5).

[0140] G, p16 Ink4a The expression analysis of p16 in aged mice injected with rFib was not significantly different from that in 25-week-old and 10-week-old mice, whereas the expression analysis of p16 in aged mice injected with DMEM was significantly different from that in aged mice injected with rFib. Ink4a The expression of was significantly improved.

[0141] H shows staining analysis of osteoblasts (ALP) and osteoclasts (TRAP). The expression of osteoblasts was significantly improved and the expression of osteoclasts was significantly reduced in the aged mice injected with rFib cells compared to the DMEM group.

[0142] IJ show quantitative analysis of osteoblasts (I) and osteoclasts (J). The expression of osteoblasts and osteoclasts in aged mice injected with rFib cells was close to that in 25-week-old mice.

[0143] In K, immunohistochemical staining showed that human antibody (hCD29, green) positivity appeared in the bones of rFib-injected mice, and osteocalcin, a bone formation marker, was expressed (red), indicating that rFib differentiated into osteoblasts in NOD / SCID mice.

[0144] As can be seen from the LM, the secretion levels of GDF11 (a protein with anti-aging function) and PDGFA (a protein favorable for bone formation) in rFib cultures were significantly higher than those of their homologous Fibs, and the anti-aging and bone mineral density-enhancing functions of rFib may be related to their paracrine action.

[0145] FIG. 13 shows that rFib cells are distributed to multiple organs in mice and can differentiate into functional cells.

[0146] AB shows that rFib cells are distributed in the stomach, spleen, lungs, and liver of mice (detected by fluorescence and PCR).

[0147] C shows that rFib cells are distributed in the bones of mice (detected by PCR method. 1#~10# indicate the mouse numbers, 1#~5# are mice injected with rFib cells, and 6#~10# are mice injected with DMEM).

[0148] In D, immunohistochemical staining shows that human antibody (hCD29, green) positivity appeared in the bones of rFib-injected mice, and osteocalcin, a bone formation marker, was expressed (red), indicating that rFib differentiated into osteoblasts in NOD / SCID mice.

[0149] As can be seen from E, other paracrine products of rFib (BFGF, HGF, VEGF) are the same as or have no significant difference from Fib, and are not the main mechanism by which it exerts its anti-aging function.

[0150] Example 16: Intravenous injection of rFib improves bone density in aged osteoporotic animals.

[0151] FIG. 14 shows that rFib cells can improve bone density in aged osteoporotic mice.

[0152] A shows the results of intervention of senile osteoporosis (28W NOD / SCID mice) using human cells. In the experimental group, 1*10 6 The rFib cells were dispersed in 200μL DMEM and injected via the tail vein. In the control group, only DMEM was injected, once a week for a total of 3 weeks. On the 28th day after the first injection, the mice were euthanized, sampled, and the density of the lumbar bone was measured. As can be seen from the Micro-CT results, the structure of the trabecular bone in the third lumbar vertebra of the experimental group mice was more compact.

[0153] B shows the Micro-CT data analysis results of the experimental mice. As can be seen from the results, the bone mineral density (BMD), relative bone volume (BV / TV) and trabecular bone number (Tb.N) were all improved.

[0154] Example 17: Culture medium of rFib cells can promote wound healing in animal skin.

[0155] C57 mice were used, and an 8 cm-wide full-thickness skin defect was created on the back of the mice. The control group was not treated, while the rFib culture medium group was treated with rFib culture medium applied daily.

[0156] Figure 15 shows that rFib culture medium can significantly promote skin healing. The rFib culture medium group was almost completely healed 12 days after model construction.

[0157] Example 18: rFib can improve limb ischemia in mice.

[0158] A unilateral femoral artery in a NOD / SCID mouse was ligated to create a lower limb ischemia model. After surgery, the success of the model construction was verified by laser Doppler, and 1 × 10 6 The cells were injected once. Blood flow was measured by laser Doppler on days 7 and 14 after cell injection.

[0159] FIG. 16 shows that rFib can ameliorate hindlimb ischemia symptoms in mice.

[0160] A shows the results of detecting the blood flow in the lower limbs of mice using a laser Doppler. As can be seen from the results, rFib and bMSCs can significantly improve the ischemic condition of the lower limbs of mice.

[0161] B is a photograph taken 7 days after the mouse lower limbs were ligated. The control group shows the normal lower limbs. Ischemic shows the lower limbs on the ligated side. As can be seen from the results, the necrotic state of the lower limbs on the ligated side in the rFib group and the bMSC group is significantly milder.

[0162] Example 19: Preparation of rFib cells with different compound combinations

[0163] 1. Dermal fibroblasts were seeded into 6-well plates and cultured in dermal fibroblast culture medium for 24 hours.

[0164] 2. The cell culture medium was replaced with rFib induction medium containing the small molecule compound combination Mix Y, and the liquid was replaced every 2 days, and the cells were treated for 10 days.

[0165] 3. After treatment with low molecular weight compound combination Mix Y, the culture medium was replaced with HG-DMEM supplemented with 10% FBS, and the cells were further cultured for 3 days and then identified or cultured in rFib medium.

[0166] 5. During long-term passage, rFibs were cultured in MSC basal medium and passaged when the cell confluence reached 90%.

[0167] Dermal fibroblast culture medium: 10% FBS + HG-DMEM; or Fibstar-CO medium, brand: HCell, product number: FMS003C.

[0168] Mix Y: High glucose (HG)-DMEM supplemented with 10% FBS (containing 5 μM Y-27632, 0.2 mM Vc, 5 μM EPZ004777, 10 μM forskolin, and 1 μM Repsox).

[0169] Alternatively, FibGro medium, brand HCell, product number FGS0040, was supplemented with 5 μM Y-27632, 0.2 mM Vc, 5 μM EPZ004777, 10 μM Forskolin, and 1 μM Repsox.

[0170] MSC basal medium: 10% FBS+LG-DMEM; Bone marrow mesenchymal stem cell complete medium purchased from Cyagen, product number HUXMA-90011; or rFib medium, brand HCell, product number CRM0016-01.

[0171] FIG. 17 shows that Mix Y treatment suppresses the expression of cellular STAT5 and STAT3 genes (AB), downregulates CDKN1A gene, lengthens telomeres (CD), and rejuvenates cells.

[0172] Example 20: Preparation of rFib cells by combination of different compounds

[0173] 1. Dermal fibroblasts were seeded into 6-well plates and cultured in dermal fibroblast culture medium for 24 hours.

[0174] 2. The cell culture medium was replaced with rFib induction medium containing the small molecule compound combination Mix Pn, and the liquid was replaced every 2 days, and the cells were treated for 7 days.

[0175] 3. After treatment with the small molecule compound combination Mix Pn, the culture medium was replaced with HG-DMEM supplemented with 10% FBS, and the cells were subsequently cultured for 3 days before identification or cultured in rFib medium.

[0176] 5. During long-term passage, rFibs were cultured in MSC basal medium and passaged when the cell confluence reached 90%.

[0177] Dermal fibroblast culture medium: 10% FBS + HG-DMEM; Hubland: HCell, product number: FMS003C; Fibstar-CO medium.

[0178] Mix Pn: HG-DMEM supplemented with 10% FBS (containing 0.5 mM VPA, 3 μM CHIR99021, 1 μM Repsox, 10 μM forskolin, 5 μM Go 6983, 5 μM Y-27632, 0.05 μM AM580, 5 μM EPZ004777, 0.2 mM Vc, 5 μM TTNPB, and 10 μM 5-Aza-2'-deoxycytidine).

[0179] Alternatively, FibGro medium (brand HCell, product number FGS0040) was supplemented with 0.5 mM VPA, 3 μM CHIR99021, 1 μM Repsox, 10 μM forskolin, 5 μM Go 6983, 5 μM Y-27632, 0.05 μM AM580, 5 μM EPZ004777, 0.2 mM Vc, 5 μM TTNPB, and 10 μM 5-Aza-2'-deoxycytidine.

[0180] MSC basal medium: 10% FBS+LG-DMEM; Bone marrow mesenchymal stem cell complete medium purchased from Cyagen, product number HUXMA-90011; or rFib medium, brand HCell, product number CRM0016-01.

[0181] FIG. 18: Mix Pn treatment down-regulates the expression of cellular STAT5 (A), suppresses the expression of ATF3, CDKN1A, GADD45B and IL6 (BE), and rejuvenates cells.

[0182] Example 21: Preparation of rFib cells with different compound combinations

[0183] 1. Dermal fibroblasts were seeded into 6-well plates and cultured in dermal fibroblast culture medium for 24 hours.

[0184] 2. The cell culture medium was replaced with rFib induction medium containing small molecule cocktail combination Mix Y, and the liquid was changed every 2 days.

[0185] 3. After culturing in rFib induction medium containing the small molecule cocktail Mix Y for 9 days, the medium was replaced with HG-DMEM supplemented with 10% FBS and treated for 3 to 7 days.

[0186] 4. After the treatment in step 3, rFib induction culture medium containing the small molecule compound combination Mix Pn2 was added, and the liquid was changed every 2 days.

[0187] 5. After culturing in rFib induction culture medium containing low molecular weight compound combination Mix Pn2 for 7 days, the culture medium was replaced with HG-DMEM supplemented with 10% FBS, 10 ng / ml bFGF, 100 ng / ml PDGF-AB and 10 ng / ml BMP4, and the cells were treated for 3 days, or replaced with HG-DMEM supplemented with 10% FBS, and the cells were treated for 3 days, or cultured in rFib medium and identified after 3 days.

[0188] 6. During long-term passage, rFibs were cultured in MSC basal medium and passaged when the cell confluence reached 90%.

[0189] Dermal fibroblast culture medium: 10% FBS + HG-DMEM; or Fibstar-CO medium, brand: HCell, product number: FMS003C.

[0190] Mix Y: High glucose (HG)-DMEM supplemented with 10% FBS (containing 5 μM Y-27632, 0.2 mM Vc, 5 μM EPZ004777, 10 μM forskolin, and 1 μM Repsox).

[0191] Alternatively, FibGro medium, brand HCell, product number FGS0040, was supplemented with 5 μM Y-27632, 0.2 mM Vc, 5 μM EPZ004777, 10 μM Forskolin, and 1 μM Repsox.

[0192] Mix Pn: HG-DMEM supplemented with 10% FBS (containing 0.5 mM VPA, 3 μM CHIR99021, 1 μM Repsox, 10 μM Forskolin, 5 μM Go 6983, 5 μM Y-27632, 0.05 μM AM580, 5 μM EPZ004777, 0.2 mM Vc, and 5 μM TTNPB)

[0193] Alternatively, FibGro medium (brand HCell, product number FGS0040) was supplemented with 0.5 mM VPA, 3 μM CHIR99021, 1 μM Repsox, 10 μM forskolin, 5 μM Go 6983, 5 μM Y-27632, 0.05 μM AM580, 5 μM EPZ004777, 0.2 mM Vc, and 5 μM TTNPB.

[0194] MSC basal medium: 10% FBS+LG-DMEM; Bone marrow mesenchymal stem cell complete medium purchased from Cyagen, product number HUXMA-90011; or rFib medium, brand HCell, product number CRM0016-01.

[0195] FIG. 19 shows that Mix Y-Mix Pn2 treatment suppresses fibroblast JAK1 expression (A) and increases their telomere length (B).

[0196] Example 22: Dermal fibroblasts transdifferentiate into neural cells.

[0197] 1. Dermal fibroblasts were seeded into 6-well plates and cultured in dermal fibroblast culture medium for 24 hours.

[0198] 2. The cell culture medium was replaced with neural induction culture medium containing the low molecular weight compound Mix Neu, and the medium was replaced every two days.

[0199] 3. After culturing the cells in neural induction medium containing the small molecule compound Mix Neu for 5 to 12 days, the long spindle-shaped cell morphology was observed to change to that of neural cells, and the medium was replaced with neural cell culture medium and the cells were continuously subcultured.

[0200] 4. Transdifferentiated neurons were identified by immunofluorescence and quantitative PCR.

[0201] Dermal fibroblast culture medium: 10% FBS + HG-DMEM; or Fibstar-CO medium, brand: HCell, product number: FMS003C.

[0202] Mix Neu: HG-DMEM supplemented with 10% FBS (containing 0.5 μM A8301, 10 ng / mL bFGF, 5 μM EPZ004777, 10 μM RG108, 2 μM parnate, 10 μM CHIR99021, 50 μM forskolin, 0.5 mM VPA, 0.05 μM AM580, and 1 μM BIX 01294).

[0203] Neuronal culture medium: 5mL DMEM / F12, 5mL Neurobasal, 1 / 100 N2, 1 / 50 B27, 100μM cAMP, 20ng / mL BDNF, 20ng / Ml GDNF, 10% (v / v) KOSR.

[0204] FIG. 20: Conversion of skin fibroblasts into neural cells.

[0205] A shows Tuj1 staining of neurons converted from skin fibroblasts.

[0206] B shows the measurement of Nestin expression level.

[0207] Example 23: Embryonic stem cells differentiate into neural cells.

[0208] 1. Adherent cultured embryonic stem cells were digested and then cultured in suspension in neural induction medium.

[0209] 2. After culturing in the neural induction solution for 10–15 days, the cells were observed to gradually adhere to the wall. The suspension-cultured cells were then seeded into a 6-well plate treated with Matrigel, and the cells were cultured for adhesion, followed by culturing in the neural induction solution for 5–7 days.

[0210] 3. After the cells attached to the wall, the neural induction medium was replaced with neural cell culture medium.

[0211] 4. The induced cells were subjected to immunofluorescence staining and quantitative PCR to identify neural markers.

[0212] Nerve induction solution: DMEM / F12 supplemented with 10% KOSR (containing 10 ng / mL bFGF, 5 μM Y-27632, 0.5 mM VPA, 5 μM EPZ004777, 10 μM forskolin, and 1 μM Repsox).

[0213] Neuronal culture medium: 5mL DMEM / F12, 5mL Neurobasal, 1 / 100 N2, 1 / 50 B27, 100μM cAMP, 20ng / mL BDNF, 20ng / Ml GDNF, 10% (v / v) KOSR.

[0214] FIG. 21 shows that the expression of STAT5 gene is upregulated during the differentiation process from ES to neurons by acting with a small molecule compound.

[0215] Example 24: Characterization of signal transduction pathways in rFib cells produced in Example 1

[0216] In the preparation method shown in Example 1, transcriptome sequencing was performed on rFib cells derived from different individuals, and 12036 genes in each cell sample were analyzed by WGCNA, and 12 clustering modules were obtained.

[0217] FIG. 22 shows modules that are enriched for KEGG pathways.

[0218] The bar graph shows the 12 modules that are enriched in KEGG pathways. Representative genes in each KEGG pathway are shown according to the order of gene members. The box plot shows the distribution of the average expression levels of genes in each module.

Claims

1. A method for producing rejuvenated repair fibroblasts, comprising treating normal fibroblasts with a first low molecular weight compound and a second low molecular weight compound in a time series, the first small molecule compound comprises VPA, CHIR99021, Repsox and forskolin, or comprises Y-27632, Vc, EPZ004777, forskolin and Repsox; The second small molecule compound comprises VPA, CHIR99021, Repsox, forskolin, SP600125, Go6983, Y-27632, Vc, TTNPB, AM580 and EPZ004777, or comprises VPA, CHIR99021, Repsox, forskolin, Go6983, Y-27632, Vc, TTNPB, AM580 and EPZ004777; A method for producing rejuvenated repair fibroblasts, comprising treating normal fibroblasts with the first low molecular weight compound for 2-10 days, and then treating the normal fibroblasts with the second low molecular weight compound for 4-20 days.

2. said normal fibroblasts being derived from mammalian connective tissue; The mammals include humans, monkeys, mice, and pigs; The method for producing rejuvenated reparative fibroblasts according to claim 1, characterized in that the connective tissues include blood, skin, bone marrow and heart.

3. The first small molecule compound comprises 0.05 to 10 mM VPA, 1 to 15 μM CHIR99021, 0.5 to 10 μM Repsox, and 3 to 50 μM forskolin, or 1 to 25 μM Y-27632, 0.2 mM Vc, 0.5 to 15 μM EPZ004777, 3 to 50 μM forskolin, and 0.5 to 10 μM Repsox; the second small molecule compound comprises 0.05-10 mM VPA, 1-15 μM CHIR99021, 0.5-10 μM Repsox, 3-50 μM forskolin, 1-50 μM SP600125, 1-20 μM Go6983, 1-25 μM Y-27632, 0.2 mM Vc, 0.2-20 μM TTNPB, 0.02-1 μM AM580, and 0.5-15 μM EPZ004777; The method for producing rejuvenated repair fibroblasts according to claim 1, characterized in that the method comprises: 0.05-10 mM VPA, 1-15 μM CHIR99021, 0.5-10 μM Repsox, 3-50 μM forskolin, 1-20 μM Go6983, 1-25 μM Y-27632, 0.2 mM Vc, 0.2-20 μM TTNPB, 0.02-1 μM AM580 and 0.5-15 μM EPZ004777.

4. The telomere length of the repaired fibroblasts is 1.5 to 12 times longer than that of the initial normal fibroblasts, approaching the level of allogeneic cells from minors; The different type of cells produced from the repair fibroblasts have longer telomeres and exhibit stronger functional activity than allogeneic cells derived from the same animal. The other types of cells include osteoblasts, chondrocytes, The method for producing rejuvenated reparative fibroblasts according to any one of claims 1 to 3, characterized in that the functional activity includes bone-forming activity and cartilage-forming activity.

5. Use of rejuvenated reparative fibroblasts produced by the method for producing rejuvenated reparative fibroblasts described in any one of claims 1 to 4 in producing a cellular product, characterized in that the cellular product includes cell secretions, cell lysates, and other types of cells into which the reparative fibroblasts have differentiated.

6. Use of rejuvenated repair fibroblasts produced by the method for producing rejuvenated repair fibroblasts according to any one of claims 1 to 4, The rejuvenated, reparative fibroblasts are used to prevent, slow or reverse the aging process in a mammalian tissue, organ or organism in vitro.

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