A technology for controlling the Jak-Stat pathway to differentiate, dedifferentiate, and rejuvenate cells, and its use.

Abstract

A method for producing self-rejuvenating and safe cells, and a self-rejuvenating and safe repair cell are provided. [Solution] A technology and its use are provided for controlling the Jak-Stat pathway to differentiate, dedifferentiate, and rejuvenate cells. By quantitatively and / or periodically controlling gene or protein targets of the Jak-Stat signaling pathway in cells using combinations of small molecule compounds, cytokines, or recombinant proteins, gene editing technology, or genetic recombination technology, rejuvenated cell products and / or different types of cell products are obtained. This product can be applied to the reprogramming of cells, tissues, organs, and living organisms; the construction of tissue engineering materials; the repair of damaged and aging, degenerated tissues and organs in mammals; the delay or reversal of the progression of aging in cells, tissues, organs, and living organisms; and the immunomodulation of cells, tissues, organs, and living organisms.

Description

[Technical Field]

[0001] This invention belongs to the field of cell biology and relates to a technique for differentiating, dedifferentiating, and rejuvenating cells by controlling the Jak-Stat pathway, and its use, and more particularly to a method for regulating cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging, and apoptosis by controlling the JAK-STAT signaling pathway, thereby extending the lifespan of living organisms, as well as the manufacture and use of such cell products. [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 deterioration or degeneration of multiple organs, including bone, cartilage, heart, muscle, brain, skin, pancreas, liver, kidneys, and gastrointestinal tract. Abnormalities in immune system function are also related to aging. In fact, chronic inflammation of tissues and organs is a cause of their degeneration and aging. Senescent cells typically have problems such as DNA damage or mutation, telomere shortening, epigenetics, redox, abnormal energy metabolism, decreased proliferative capacity, and an increase in dead cells. Senescent stem cells lose the ability to differentiate into specific 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 older adults have a reduced ability to differentiate into neurons and an increased ability to differentiate into astrocytes. This is thought to be related to the decline in cognitive abilities among the elderly.

[0003] Stem cells, particularly mesenchymal stem cells, are promising candidates for treating or intervening in aging and related diseases due to their availability, scalability and versatility, growth factor release, and immunomodulatory capabilities. Clinically, mesenchymal stem cells are widely used to treat a variety of diseases, including graft-versus-host disease, multiple sclerosis, amyotrophic lateral sclerosis, spinal cord injury, lupus erythematosus, arthritis, and aging. Umbilical cord mesenchymal stem cells are widely considered for allogeneic use. However, long-term / repeated use of "non-self" cells always carries clinical risks. Autologous transplantation is considered safer, but unfortunately, mesenchymal stem cells also age with age. Aged mesenchymal stem cells lose many important functions, limiting their clinical application. Induced pluripotent stem cells (iPS cells), which are young cells obtainable from elderly individuals, were considered a suitable cell source for therapy. However, the induction efficiency of iPS cells is low, and the introduction of foreign genes often results in genetic mutations, making them unfavorable for clinical application. Recently, genetic modification has been used as a method of rejuvenation, but this method still carries the risks of off-target effects and tumor formation. Therefore, the development of methods for producing self-rejuvenating and safe cells, and obtaining self-rejuvenating and safe repair cells, is crucial for preventing, delaying, and reversing the progression of human aging, repairing tissue, organ structure or function, and improving health and quality of life. [Overview of the Initiative]

[0004] The present invention aims to provide a technology and its use for controlling the Jak-Stat pathway to differentiate, dedifferentiate, and rejuvenate cells, and in particular a method for controlling the JAK-STAT signaling pathway to regulate cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging, and apoptosis, thereby reversing aging and extending the lifespan of living organisms. The cell products obtained by this method (e.g., regenerative fibroblasts, rejuvenated mesenchymal stem cells) can be applied to the prevention, delay, and reversal of the progression of human aging, and the repair of tissue, organ structure, or function. The regenerative fibroblasts produced by the technology of the present invention possess the characteristics of both dermal fibroblasts and mesenchymal stem cells, and are therefore named regenerative dermal fibroblasts (rFib), and may also be named induced and rejuvenated mesenchymal stem cells (irMSC) or induced mesenchymal stem cells (iMSC), and will be referred to as rFib herein.

[0005] The technical means of the present invention are as follows: A technique and use thereof for differentiating, dedifferentiating, and rejuvenating cells by controlling the Jak-Stat pathway, which quantitatively and / or periodically activates or suppresses the Jak-Stat signaling pathway.

[0006] In the above method, the gene or protein target that is highly expressed or is highly expressed, or is lowly expressed or suppressed in the JAK-STAT signaling pathway is 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). ), SOCS3 (Genetic number / Accession: NM_003955.4), IL6ST (Genetic number / Accession: NM_001243835.1), STAT1 (Genetic number / Accession: NM_007315.3), STAT2 (Genetic number / Accession: NM_198332.1), STAT3 (Genetic number / Accession: NM_213662.1), STAT4 (Genetic number / Accession: NM_001243835.1), STAT6 (Genetic number / Accession: NM_0011 78081.1), STAT5A (Genetic number / Accession: NM_001288720.1), IRF9 (Genetic number / Accession: NM_006084.4), IL6 (Genetic number / Accession: XM_005249745.5), IL6R (Genetic number / Accession: NM_181359.2), IL2 (Genetic number / Accession: NM_000586.3) (e.g., IL2A and / or IL2B), PRKCD (Genetic number / Accession: NM_001354679.1), CXCL1 2 (Genetic code / Accession: NM_000609.6), CXCR4 (Genetic code / Accession: NM_003467.2), JAK2 (Genetic code / Accession: NM_004972.3), IL15RA (Genetic code / Accession: NM_001351095.1), IL20RB (Genetic code / Accession: XM_006713665.4), GHR (Genetic code / Accession: NM_001242406.2) and PRLR (Genetic code / Accession: NM_001204314).It includes at least one of the following:

[0007] The aforementioned 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, cardiomyocytes, smooth muscle cells, vascular endothelial cells, induced pluripotent stem cells, embryonic stem cells, osteoblasts, chondrocytes, adipocytes, and osteoclasts. The cells produced by the above method are defined herein as target cells and include various types of cells that originate from target cells and have different characteristics produced in the process of regulating JAK-STAT within the target cells, the characteristics of which include at least one of differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging, and apoptosis.

[0008] Quantitative activation or suppression of the JAK-STAT signaling pathway is defined as the upregulation or downregulation of at least one gene or protein target of the JAK-STAT signaling pathway in the target cell by a factor of 1 to 300 relative to the target cell.

[0009] The timing of activation or suppression of the JAK-STAT signaling pathway is controlled for 24 hours to 220 days so that at least one of the JAK-STAT signaling pathway gene or protein target is highly expressed, low, or not expressed in the target cells. The resulting target cells maintain high, low, or non-expression of at least one of the JAK-STAT signaling pathway gene or protein target for an extended period, or restore it to the same expression level as the target cells.

[0010] In the above 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 pathways, focal adhesion, cell cycle, citric acid cycle, TGF beta 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, adenylyl cyclase signaling pathway, HMT (histone methyltransferase), DNMT (DNA methyltransferase), and histone demethylase inhibitors.

[0011] Activating or inhibiting the cellular targets and cellular 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 plaque 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, and 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, 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 beta 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, 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 PKC gene or protein target is one or more species 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 adenylyl 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 aforementioned 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 above method employs at least one of the following: a combination of small molecule compounds, a combination of cytokines, or a combination of recombinant proteins, gene editing technology, and genetic recombination technology. The aforementioned combination of low molecular weight compounds is Histone deacetylase inhibitors: Sodium phenylbutyrate, butyrate, sodium butyrate, VPA, scriptide, apicidine, LBH-589 (panobinostat), MS-275, SAHA (vorinostat), trichostatin (TSA), psammaplin A, spritomycin, SRT1720, resveratrol, siltinol, APHA, CI-994, depdesin, FK-228, HC toxin, ITF-2357 (divinostat), thidamide, RGFP 966, PHOB, BG45, Nexturastat A, TMP269, CAY10603, MGCD-0103, Niltubacin, PXD-101 (Belinostat), Piroxamide, Tubacin, EX-527, BATCP, Canbinol, 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 bicindylmaleimide I (GF109203X) WNT / β-catenin agonists: MAY-262611, CHIR98014, CHIR99021, LiCl, Li2CO3, TD114-2, AZD2858, AZD1080, BIO, Kaempaulon, 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, Shiromilast, 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, thiazovibin, ripasdil (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, zebralin, and 5-azacitidine (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, Homoharingtonnin, Pyridone 6, Pyridone 6, Artesunate, Ruxolitinib, SH-4-54, Baricitinib, Ruxolitinib Sulfate, AG-490, Baricitinib Sulfate, SAR-20347, CYT387 Mesylate, AS1517499, Peficitinib, Ruxolitinib Sulfate, NSC 74859, Static, Tofacitinib citrate, Pimozide, Oclacitinib maleate, Ruxolitinib S enantiomer, SB1317, Niclosamide, Scutellarin, Sorucitinib, Mogrol, Nifloxazide, TG101348 (SAR302503), AG-1478 (Tilphostin AG-1478) (EGFR inhibitor) KX2-391 (Src inhibitor), PKI-402 (PI3Kα / β / γ / δ and mTOR inhibitor), NSC 74859 (S3I-201) (STAT3 inhibitor), Fludarabine (Fludara) (STAT-1 inhibitor), UO126-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-RafV600E and c-Raf-1Y340D / Y341D inhibitor) This includes one or more of the following: The aforementioned combination of cytokines or recombinant proteins 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-2 R alpha, TNF-alpha, TNF-beta, TPO, IGF2, IGFBP5, IGFBP6, IGFBP4, IGFBP7, IGFBP9, PDGF-BB, MMP3, GDF11, and TIMP2. The gene editing technology includes upregulating or knocking out genes or protein targets in the JAK-STAT signaling pathway, such as STAT5A, by CRISPR / Cas9 gene editing technology and TALEN gene editing technology. The gene recombination technology includes overexpressing or suppressing genes or protein targets in the JAK-STAT signaling pathway, such as STAT5A, by lentivirus or retrovirus.

[0013] The repaired fibroblasts produced by the method described above, wherein the JAK-STAT signaling pathway in the repaired fibroblasts is suppressed, and the genes or protein targets with low expression or suppressed expression in the JAK-STAT signaling pathway include 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] In the signal transduction pathway in the repaired fibroblasts, the following changes occur. The NOD-like receptor signal transduction pathway is suppressed, and / or the TGF-β receptor signal transduction pathway is suppressed, and / or the insulin signal transduction pathway is downregulated, and / or the Wnt signal transduction pathway is upregulated, and / or the Notch signal transduction pathway is downregulated, and / or the p53 signal transduction pathway is downregulated.

[0015] The method for producing the repaired fibroblasts, wherein the target cells of the repaired fibroblasts are normal fibroblasts, and the normal fibroblasts are derived from connective tissues (such as blood, skin, bone marrow, heart) of mammals (such as humans, monkeys, mice, pigs).

[0016] In the above manufacturing method, normal fibroblasts are treated with a combination of low-molecular-weight compounds to obtain repair-type fibroblasts. The combination of low-molecular-weight compounds includes at least one of the following: a Jak-Stat inhibitor, a WNT / β-catenin agonist, a histone deacetylase inhibitor, and a cAMP agonist.

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

[0018] In the method for producing the repair-type fibroblasts described above, the first-step compound and the second-step compound are used in a stepwise manner over time. 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. The second-step compound includes a histone deacetylase inhibitor, a TGF-β inhibitor, a WNT / β-catenin agonist, a cAMP agonist, a RAR agonist, an HMT inhibitor, ascorbate (ascorbic acid), a PKC inhibitor, a PKC inhibitor, and a ROCK inhibitor.

[0019] In the above manufacturing method, at least one of the following is used: 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-azacitidine, or 1-50 μM SP600125. Alternatively, ordinary fibroblasts are first treated with the first-step compound for 2-10 days. The first-step compound contains 0.05–10 mM VPA, 1–15 μM CHIR99021, 0.5–10 μM Repsox, and 3–50 μM forskolin. After the first-step treatment, the material is treated with the second-step compound for 4–20 days. The second-step compound contains 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 aforementioned repair-type fibroblasts is 1.5 to 12 times longer than that of early, normal fibroblasts, and is close to the level of allogeneic cells from juvenile individuals. Other types of cells produced from these repair-type fibroblasts (e.g., osteoblasts, chondrocytes) have longer telomeres and exhibit stronger functional activity compared to allogeneic cells derived from the same animal individual.

[0021] Cell products (e.g., cell secretions, cell lysates) produced from reparative fibroblasts manufactured by the method described above can be applied to the construction of tissue engineering materials, and to the delay or reversal of aging in cells, tissues, organs, and living organisms.

[0022] The use of the reparative fibroblasts in the construction of tissue engineering materials, and in delaying or reversing aging of cells, tissues, organs, and living organisms.

[0023] Super fibroblasts are produced by knocking out the STAT5 gene in ordinary fibroblasts.

[0024] In the method for producing the super fibroblasts described above, super fibroblasts are obtained that are rejuvenated and have elongated telomeres within 3 to 100 days after knocking out the STAT5 gene in ordinary fibroblasts.

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

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

[0027] Use of cell products (e.g., reparative fibroblasts) manufactured by the above method, or cell culture media / culture media of said cell products, lysates of said cell products, kits, drugs, health products, foods, cosmetics, or medical devices.

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

[0029] Use of cell products (e.g., reparative fibroblasts) produced by the aforementioned method in medical research or as immunomodulators.

[0030] Use of cell products (e.g., reparative fibroblasts) produced by the aforementioned methods in the prevention, delay, or reversal of the progression of aging in mammalian tissues, organs, and organisms in vitro / in vivo.

[0031] The use of cell products manufactured by the aforementioned method in the reprogramming of cells, tissues, organs, or living organisms, or in the rejuvenation of cells, tissues, organs, or living organisms.

[0032] The present invention aims to regulate cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging, and apoptosis, reverse aging, and extend the lifespan of living organisms by quantitatively and / or periodically controlling gene or protein targets in the Jak-Stat signaling pathway. Gene or protein targets in the Jak-Stat signaling pathway in cells are quantitatively and / or periodically controlled by combinations of small molecule compounds, cytokines, or recombinant proteins, gene editing technology, or genetic recombination technology. The gene or protein targets include 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 the following: Jak-Stat inhibitors, WNT / β-catenin agonists, histone deacetylase inhibitors and cAMP agonists, RAR agonists, DNMT inhibitors, HMT inhibitors, histone demethylase inhibitors, ascorbate (ascorbic acid), JNK inhibitors, PKC inhibitors, ROCK inhibitors, and TGF-β inhibitors. The aforementioned 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-2 R alpha, TNF-alpha, TNF-beta, TPO, IGF2, IGFBP5, IGFBP6, IGFBP4, IGFBP7, IGFBP9, PDGF-BB, MMP3, GDF11, and TIMP2.The gene editing technology upregulates or knocks out a gene or protein target in the JAK-STAT signaling pathway, such as STAT5A, using crispr / cas9 gene editing technology and TALEN gene editing technology. The gene recombination technology overexpresses or suppresses a gene or protein target in the JAK-STAT signaling pathway, such as STAT5A, using a lentivirus or retrovirus.

[0033] In this invention, reparative dermal fibroblasts are produced by repressing gene or protein targets (e.g., STAT5A, JAK1) of the Jak-Stat signaling pathway in fibroblasts using small molecule compounds. The Jak-Stat signaling pathway in the reparative dermal fibroblasts is repressed. The following changes occur in the signaling pathways in the reparative fibroblasts: the NOD-like receptor signaling pathway is repressed, and / or the TGF-β receptor signaling pathway is repressed, 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 reparative fibroblasts is 1.5 to 12 times longer than that of early normal fibroblasts and is close to the level of allogeneic cells from juvenile individuals. Other types of cells produced from the reparative fibroblasts (e.g., osteoblasts, chondrocytes) have elongated telomeres and exhibit stronger functional activity compared to allogeneic cells derived from the same animal individual. The aforementioned reparative fibroblasts and cell products produced from these cells (e.g., cell secretions, cell lysates) can be applied to the construction of tissue engineering materials, and to the delay or reversal of aging in cells, tissues, organs, and living organisms.

[0034] The technology provided in this invention, which quantitatively and / or timely controls gene or protein targets in the Jak-Stat signaling pathway to regulate cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging, and apoptosis, reverse aging, and extend the lifespan of living organisms, can promote transdifferentiation between different types of cells, can be applied to the production of different types of rejuvenated cells (e.g., reversing the aging of mesenchymal stem cells to produce super fibroblasts), and can be applied to promoting cellular aging and apoptosis. Cells and cell products produced by the above technology can be used in vitro / in vivo to prevent, delay, and reverse the progression of aging in mammalian tissues, organs, and living organisms, can be used for reprogramming cells, tissues, organs, and living organisms, and can be used to repair damaged and aged, degenerated mammalian tissues and organs as seed cells and scaffolds for tissue engineering materials.

[0035] Mechanism of the invention By controlling the expression of genes or protein targets in the Jak-Stat signaling pathway within cells, and quantitatively and / or periodically regulating changes in different metabolic pathways within the cells, the cellular state of target cells is modified, transforming them into different types of cells or cells with different cellular characteristics.

[0036] Compared to the prior art, the present invention has the following beneficial effects. The rejuvenating fibroblasts provided in this invention possess rejuvenating characteristics but do not exhibit tumorigenicity compared to fibroblasts derived from the same individual or from donors of the same age group. These rejuvenating characteristics include, for example, changes in epigenetics and / or changes in the expression levels of aging-related genes and / or telomere lengthening and / or increased cell proliferation rate and / or the ability of cells to be passed through the cell in a stable long-term manner. These rejuvenated cells and their cell products can reverse aging in mammalian organisms and extend lifespan. The technology provided in this invention, which modulates cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging, and apoptosis by quantitatively and / or timely control of gene or protein targets in the Jak-Stat signaling pathway, can systematically regulate cell differentiation, dedifferentiation, transdifferentiation, rejuvenation, aging, and apoptosis. Cells and cell products produced by this technology can be applied to the prevention, delay, and reversal of the progression of aging in mammalian tissues, organs, and organisms in vitro / in vivo, can be applied to the reprogramming of cells, tissues, organs, and organisms, and can be applied to the repair of mammalian tissues, organs, and aged, degenerated tissues and organs as seed cells for tissue engineering materials and as a source of scaffolds for tissue engineering materials. [Brief explanation of the drawing]

[0037] [Figure 1] This demonstrates the production of regenerative dermal fibroblasts (rFib). [Figure 2] This study demonstrates that rFib has rejuvenating properties compared to Fib and bMSC. [Figure 3] The ability of aged bMSCs to differentiate into bone and chondrogenic structures is worse than that of rFib, indicating that rFib does not exhibit tumorigenicity. [Figure 4] rFib demonstrates immunomodulatory function in vitro. [Figure 5] This demonstrates that rFib cells possess in vivo immunomodulatory functions. [Figure 6] rFib demonstrates the ability to repair bone defects without age restrictions. [Figure 7] This shows an in vivo cartilage repair experiment. [Figure 8] This study demonstrates that suppressing the STAT5 gene rejuvenates skin fibroblasts and enables them to acquire the ability to differentiate in multiple directions. [Figure 9] This shows the changes in STAT5 and H3K9me after knocking out STAT5. [Figure 10] This shows the detection of rejuvenation and differentiation ability after knocking out STAT5 in a 62-year-old (62Y) Fib strain from another strain. [Figure 11] This demonstrates the rejuvenation of MSC cells. Cells were treated with different compounds for 3 days, followed by β-galactosidase staining. 55Y = 55 years old, 82Y = 82 years old; Y = year indicates the age of the cell donor. [Figure 12] This study demonstrates that rFib cells can extend the lifespan and increase bone density in aged NOD / SCID mice. [Figure 13] This study demonstrates that rFib cells can be distributed to multiple organs in mice and differentiate into functional cells. [Figure 14] This study demonstrates that rFib cells can improve bone density in aged osteoporosis mice. [Figure 15] This study demonstrates that rFib culture media can significantly promote skin healing. The rFib culture media group achieved almost complete healing 12 days after model construction. [Figure 16] This study demonstrates that rFib can improve lower limb ischemic symptoms in mice. [Figure 17] Mix Y treatment suppresses the expression of the STAT5 and STAT3 genes in cells (AB), downregulates the CDKN1A gene, and lengthens telomeres (CD), indicating cell rejuvenation. [Figure 18] Mix Pn treatment downregulates cellular STAT5 expression (A) and suppresses the expression of ATF3, CDKN1A, GADD45B, and IL6 (BE), demonstrating cell rejuvenation. [Figure 19]This study shows that treatment with Mix Y-Mix Pn2 suppresses JAK1 expression in fibroblasts (A) and elongates their telomere length (B). [Figure 20] This shows the conversion of cutaneous fibroblasts to nerve cells. Figure 20A shows Tuj1 staining of nerve cells converted from cutaneous fibroblasts, and Figure 20B shows the measurement of Nestin expression levels. [Figure 21] This study demonstrates that small molecule compounds upregulate the expression of the STAT5 gene during the differentiation process from ES cells to nerve cells. [Figure 22] This shows modules that contain many KEGG routes. [Modes for carrying out the invention]

[0038] Example 1: Acquisition of reparative dermal fibroblasts and identification of their characteristics

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

[0040] 2. The cell culture medium was replaced with a Fib-inducible culture medium containing the low molecular weight cocktail combination Mix V, and the liquid was changed every two days.

[0041] 3. After culturing for 5 days in rFib-inducing culture medium containing low molecular weight cocktail combination Mix V, the culture medium was replaced with rFib-inducing culture medium containing low molecular weight cocktail combination Mix P, and the liquid was changed every 2 days.

[0042] 4. After culturing for 7 days in rFib-inducing culture medium containing the low molecular weight cocktail combination Mix P, 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, or HG-DMEM supplemented with 10% FBS, or cultured in rFib medium. After treating the cells for 3 days, they were subjected to long-term subculturing. After treating the cells for 3 days, identification was started.

[0043] 5. During long-term subculturing, rFib cells were cultured in MSC basal medium and subculturing was performed when cell confluence reached 90%.

[0044] Dermatophyte 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 with 10% FBS added (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, 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 were added to FibGro medium of brand HCell, product number FGS0040.

[0048] MSC basal medium: 10% FBS + LG-DMEM; Bone marrow mesenchymal stem cell complete medium, product number HUXMA-90011, purchased from Cyagen; 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] Figure A is a schematic diagram of the process of converting dermal fibroblasts (Fib) to rFib. After nearly aged Fib (P13 passage) was converted to rFib, it gained enormous proliferative potential (capable of proliferating for another 19 passages). A similar treatment process induced death in bone marrow mesenchymal stem cells.

[0052] B shows a comparison of growth curves when rFib and its homologous Fib are amplified over the long term. The growth rate of the rFib obtained after treatment was faster than that of homologous Fib.

[0053] C shows the histochemical analysis of the cell trisystem differentiation ability (differentiation into adipose tissue, bone, and cartilage) before and after conversion (parent skin cells were derived from P8 passaged cells of a single 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 adipose chondrocytes was detected. Osteoblasts (bone formation, identified by alizarin red staining), adipocytes (adipogenesis, identified by oil red staining), and chondrocytes (chondrogenesis, identified by Alcian blue staining). After multiple passages (P9 passage, P16 passage), rFib cells still maintained good trisystem differentiation ability (differentiation into adipose tissue, bone, and cartilage).

[0054] D shows the results of q-RT-PCR analysis of 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. As can be seen from the results, rFib cells, like young bMSCs, highly express genes related to the three differentiation pathways after induction. Compared to D0(Fib), *p<0.05, **p<0.01, ***p<0.001, and n≧3.

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

[0056] H shows the transcriptome analysis of rFib cells. Their aggregation is similar to that of Fib and bMSCs, and unlike iPSCs and ESCs, they possess characteristics of both dermal fibroblasts and mesenchymal stem cells. rFib cells are safe and non-tumor-forming.

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

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

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

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

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

[0062] H measured the relative telomere lengths of Fib, rFib, bMSCs and their differentiated osteoblasts (rFib-OB, bMSC-OB), and chondrocytes (rFib-CH, bMSC-CH) using q-RT-PCR and T / S values.

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

[0064] Figure 3: Aged bMSCs have worse osteogenesis and chondrogenesis differentiation capabilities than rFib, and rFib does not exhibit tumorigenicity.

[0065] A shows alizarin red staining of bMSCs and rFibs derived from individuals of different ages. As can be seen from the results, the bone-forming differentiation ability of bMSCs derived from aged individuals was significantly reduced, but rFibs from the same individuals retained good bone-forming differentiation ability.

[0066] B shows oil red O staining of bMSCs and rFibs from individuals of different ages for adipogenesis. As can be seen from the results, the adipogenesis ability of bMSCs from aged individuals was significantly improved, while rFibs from aged individuals retained adipogenesis ability consistent with that of juvenile individuals.

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

[0068] D shows COL2A1 and MMP13 immunohistochemical staining in bMSCs and rFibs from individuals of different ages during chondrogenic differentiation. After aged bMSCs underwent chondrogenic differentiation, COL2A1 was low and MMP13 was high, while rFibs from aged individuals showed similar COL2A1 / MMP13 expression to those from younger individuals.

[0069] FG shows the results of detecting the expression of COL2A1 and MMP13 in bMSCs and rFib cells derived from individuals of different ages after chondrogenesis by q-RT-PCR. The results were consistent with the staining results.

[0070] H indicates the detection of karyotypes in rFib passages P9 and P13, and its homologous Fib passage P6. As can be seen from the results, the karyotype of the rFib remained consistent with that of the Fib even after long passages.

[0071] Figure 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 teratoma formation (with clear trigerm layer structure), while transplantation into rFib mice did not result in tumor formation.

[0072] J shows an analysis of telomere length and telomerase expression in cells. rFib cells exhibited significantly longer telomeres than their homologous Fib cells, and these were stably maintained even after induction was complete. However, telomerase was transiently high during induction, and then restored to low levels. This differs from tumor cells (where telomerase is continuously high). This indicates that rFib cells rejuvenate without being tumorigenic.

[0073] 6.1 In vitro immunomodulatory studies

[0074] The cells were treated with mitomycin C for 2.5 hours, digested, and counted, and then 1 × 10⁶ cells were measured. 5 Cells were inoculated into 24-well plates at a rate of one cell per well. Lymphocytes were stained with carboxyfluorescein diacetate succinimimidyl ester (CFDA-SE) at 37°C for 30 minutes, and 2 × 10⁶ cells were collected. 5The cells were inoculated into 24-well plates at the specified density. Lymphocyte proliferation was stimulated with PHA at a final concentration of 2 μg / ml. Five groups were established: bMSC + lymphocyte + PHA co-culture group, rFib + lymphocyte + PHA co-culture group, cutaneous fibroblast + lymphocyte + PHA co-culture group, lymphocyte + PHA positive control group, and lymphocyte-only negative control group. After co-culture for 5 days, lymphocytes were collected from each well and rinsed three times with PBS. Lymphocyte proliferation was detected by flow cytometry using anti-CD3, CD4, and CD8 antibodies. All antibodies were purchased from BD Biosciences.

[0075] Figure 4: rFib has immunomodulatory function in vitro.

[0076] A shows that parental Fib, rFib, and bMSCs were cultured with T cells in a mixed lymphocyte reaction, respectively. PBMCs derived from healthy volunteers were labeled with CFSE. Compared to the "T+PHA" group, **p<0.01, ***p<0.001, n=3. As can be seen from the results, rFib has the ability to suppress T cell proliferation, while its homologous Fib cells do not have the ability to suppress T cell proliferation, indicating that rFib has immunomodulatory capabilities.

[0077] BC demonstrates that rFib has regulatory capabilities for CD4+ immune cell subtypes.

[0078] DE indicates that rFib has regulatory capabilities for CD8+ immune cell subtypes.

[0079] 6.2 In vivo immunomodulation experiments

[0080] 1 x 10 6 bMSCs, rFib, and dermal fibroblasts were cultured at a density in a 10 cm culture dish for 48 hours. The culture medium was then filtered through a 0.22 μm filtration membrane (Millipore) to remove cells or cell debris, and 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 concanavalin dissolved in PBS at a dose of 25 mg / kg body weight, while PBS alone was used as a control (Han et al., 2014). There were 6 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 samples were collected. Liver samples were stained with HE, and CD3+ T cells were detected by flow cytometry. ALT / AST levels were detected in the blood.

[0082] Serum ALT / AST quantification was performed using an ELISA kit (Shanghai Enzyme-linked Biotechnology Co., Ltd.) according to the instructions. Three independent repeat samples were prepared for each group, and the data are expressed as mean ± SD.

[0083] Figure 5: rFib cells possess in vivo immunomodulatory capabilities.

[0084] A shows the results of inducing acute liver injury in C57BL / 6 mice with concanavalin and treating them with concentrated rFib cell culture. As can be seen from the results, mice treated with rFib culture showed no obvious liver abnormalities (hemorrhage, necrosis, etc.).

[0085] B shows the absolute number of T lymphocytes measured in liver tissue 8.5 hours after injection of cell culture medium into the tail vein. As can be seen from the results, the rFib culture medium exhibited remarkable immunomodulatory capacity, similar to that of bMSCs.

[0086] CD indicates that mice treated with rFib culture medium have near-normal levels of both blood ALT and AST, and show no significant liver damage.

[0087] 6.3 PCR of ordinary genes

[0088] Total RNA extraction was performed according to the instructions for 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, and SYBR Premix EX Taq™II (Takara Bio) was used as the specific primer and SYBR Green. Circulation conditions were followed according to the manufacturer's (Takara) instructions. Relative expression levels were normalized using 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 ethics committee, a femoral defect model was constructed using 8-10 week old, 20-24g NOD / SCID mice, with 5 animals in each group. The model construction method involved incising the skin and subcutaneous tissue under pentobarbital sodium anesthesia, bluntly separating the rectus femoris and semitendinosus muscles, and exposing a sufficient portion of the mid-femur. Surgery was performed proximal to the center of the right femur. A continuous 4mm × 1mm bone defect was constructed surgically. Skin fibroblasts (Fib), bone marrow mesenchymal stem cells (bMSCs), and rFib were stained with Hoechst 33342 (Thermo, NucBlue live cell), then mixed with Matrigel, and 5 × 10⁶ samples were taken. 5 The cells were transplanted (administered) to the deficient site in mice.

[0091] Twenty-eight days after transplantation, mice were euthanized by injecting a lethal dose of pentobarbital sodium. The mouse thighs were bluntly isolated, fixed with 4% PFA, and detected using μCT imaging (SkyScan 1272, Bruker microCT). The collected data was then analyzed.

[0092] Figure 6: rFib has bone defect repair ability without age restrictions.

[0093] A is a schematic diagram of the surgical procedure for a mouse femoral midsection 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 labeled with Hoechst 33342 can self-fluoresce with blue fluorescence. Sections of the repair site show that rFib cells form new bone at the defect site, and their number and location correspond to newly formed osteocytes.

[0096] D shows micro-CT analysis of different experimental groups. rFib derived from aged individuals (62 years old) can repair bone defects, but its bone defect repair capacity is very weak.

[0097] E shows micro-CT analysis of different experimental groups. The repair capacity of rFib derived from young (39 years) and elderly (62 years) individuals was similar, indicating that the bone defect repair capacity of rFib 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 weighing 20-24g and 8-10 weeks old were used. The therapeutic effect of rFib was evaluated using an improved articular cartilage model (Cheng et al., 2014). An articular cartilage defect (1.5 mm × 1 mm) was constructed in the trochlear groove of the distal end of the femur using a biopsy punch. Cells (2.5 × 10) 5 A 35 μl Matrigel was labeled with Hoechst 33342 and implanted into the cell-defect area. A cell-free Matrigel was used as a control.

[0101] Figure 7: In vivo cartilage repair experiment

[0102] AB consisted of 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) were able to repair cartilage defects, and aged bMSC (62 years old) was able to generate new tissue but lacked neocartilage, while rFib (62 years old) derived from aged individuals was able to form cartilage tissue.

[0103] C represents the Pineda score for cartilage repair. As can be seen from the results, rFib derived from older individuals (62 years old) and rFib derived from younger individuals (39 years old) have similar cartilage repair capabilities.

[0104] As can be seen from the section in Figure D, rFib labeled with Hoechst 33342 formed new cartilage tissue. The newly generated cartilage tissue was similar in structure and morphology to normal cartilage, and there was no generation of abnormal tissue.

[0105] Examples 4-12: Repairing fibroblasts were obtained by treating cells for different durations using 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: Combinations of Examples and Processing Days [Table 1] JPEG2026062950000003.jpg117170

[0107] Example 13: Production of Super Fibroblasts

[0108] 1. Construction of a vector plasmid in which the stat5A gene of crispr / cas9 is knocked out: The following plasmid was 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 introduced according to the product instructions. On the first day after virus introduction, the culture medium containing the virus was removed and replaced with fresh complete medium. The culture was incubated in an incubator at 37°C and 5% CO2.

[0110] 3. From the second day after the introduction of the virus, the genes of the lentivirus began to be expressed, and the cells were continued to be cultured to further accumulate expression products or to alter the cellular phenotype.

[0111] 4. After amplifying the cells, the cells into which the virus had been introduced were purified with antibiotics, and the cells were subsequently cultured in HG-DMEM containing 10% FBS for 150 days.

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

[0113] A is a box plot of expression changes in two representative rFib modules containing many KEGG pathways selected from 12,036 genes identified by WGCNA, as well as two gene modules in several samples.

[0114] As can be seen from B, after knocking out the STAT5 gene and continuing to culture for 40 days, H4K20me3 (an indicator of aging, where higher levels are greater) was significantly reduced compared to the control group after knocking out STAT5 in Fib (Fib-STAT5-KO) by immunohistochemical staining.

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

[0116] As can be seen from D, Fib after STAT5 knockout shows bone formation (Alizarin Red S staining) and cartilage formation (Alcian Blue staining) capabilities.

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

[0118] H is a schematic diagram illustrating how it controls the Jak-Stat signaling pathway in fibroblasts, leading to cell and organismal rejuvenation.

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

[0120] Figure A shows that after knocking out STAT5 in Fib, STAT5A expression ceases.

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

[0122] Figure 10: Detection of rejuvenation and differentiation ability after knocking out STAT5 in a 62-year-old (62Y) Fib from a different strain.

[0123] A indicates the detection of aging-related genes.

[0124] B indicates the detection of osteogenesis and chondrogenesis / differentiation.

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

[0126] E indicates the detection of STAT5 expression status.

[0127] F indicates the detection of STAT5 in cells of the same strain 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-sugar DMEM containing 10% FBS.

[0130] 2. After treating cells with different compound combinations for 3 days, the cells were subsequently cultured for 3 days in a low-sugar DMEM culture medium containing 10% FBS, followed by β-galactosidase staining.

[0131] Table 2: Treatment by combination of different compounds [Table 2]

[0132] Figure 11: Rejuvenation of MSC cells Cells were treated with different compounds for 3 days, followed by β-galactosidase staining. 55Y = 55 years old, 82Y = 82 years old, Y = year: age of the cell donor.

[0133] Example 15: Intravenous injection of rFib can extend the lifespan of aging mice.

[0134] Fib cells from the same 39-year-old individual (P9 passage), rFib cells from the same 62-year-old individual (P13 passage), and rFib cells were labeled with Hoechst 33342 and dissolved in 200 μL of DMEM. 6 The solution was injected via tail vein into naturally aged NOD / SCID mice (43 weeks old; the average lifespan of these mice is 36-38 weeks old. 43 weeks old is equivalent to approximately 86 years in humans). In the solvent group, only 200 μL of DMEM was injected. After the mice died naturally, samples were taken and detected.

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

[0136] A shows the survival curve of aging mice. Mice injected with rFib cells effectively extended their lifespan, regardless of whether the cells were derived from young (39-year-old) or aged (62-year-old) individuals. Younger Fib cells did not have a lifespan-extending effect, and the survival curve was not significantly different from that of the solvent group.

[0137] B shows observations of the morphology of two groups of mice. The 43-week-old mice were clearly aged (their fur was unkempt, dull, and hunchbacked), and their condition did not clearly improve 4 weeks after injection of rFib cells. The mice injected with DMEM aged even further after 4 weeks.

[0138] The CE images are anatomical photographs of four mice aged 10 weeks, 25 weeks, 47 weeks (DMEM injected at 43 weeks, died at 47 weeks), and 49 weeks (rFib injected at 43 weeks, died at 49 weeks), respectively. The images show H&E-stained gastric mucosal sections and a micro-CT analysis of the third lumbar vertebra. As can be seen from the images, the gastrointestinal tract of mice injected with rFib cells had an appearance similar to that of young mice (10W, 25W), and the thickness and density of the gastric mucosa and the lumbar trabecular structure were similar to those of 25W mice. The aged mice in the DMEM-injected group had significant lesions in their gastrointestinal tract, with short and sparse gastric mucosa and severe defects and ruptures of the lumbar trabecular bones.

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

[0140] G is on page 16 Ink4a The expression analysis is shown. There was no significant difference between aged mice injected with rFib and 25-week-old and 10-week-old mice, and p16 in aged mice injected with DMEM. Ink4a The expression of [the substance] increased significantly.

[0141] H indicates staining analysis of osteoblasts (ALP) and osteoclasts (TRAP). Aged mice injected with rFib cells showed significantly increased osteoblast expression and decreased osteoclast expression compared to the DMEM group.

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

[0143] In sample K, as can be seen from immunohistochemical staining, the bones of mice injected with rFib showed positivity for human antibody (hCD29, green), 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 LM, the secretion levels of GDF11 (a protein with anti-aging function) and PDGFA (a protein favorable for bone formation) in rFib culture medium are significantly higher than those of homologous Fib, suggesting that the anti-aging and bone density-enhancing functions of rFib may be related to paracrine effects.

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

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

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

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

[0149] As can be seen from E, other paracrine substances of rFib (BFGF, HGF, VEGF) are the same as or have no significant difference from Fib, and they are not the main mechanisms for exerting anti-aging functions.

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

[0151] Figure 14 shows that rFib cells can improve the bone density of aged osteoporosis mice.

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

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

[0154] Example 17: The culture medium of rFib cells can promote the healing of animal skin wounds.

[0155] C57 mice were used, and a full-thickness skin defect with a width of 8 cm was created on their backs. In the control group, no treatment was given, and in the rFib medium group, the rFib culture medium was applied daily for treatment.

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

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

[0158] A lower limb ischemia model was constructed by ligating one femoral artery in NOD / SCID mice. After verifying the success of the model construction using laser Doppler postoperatively, 1 × 10¹⁶ ions were applied to the femoral artery ligation point and its distal and proximal ends. 6 Cells were injected only once. Blood flow was measured using laser Doppler on days 7 and 14 after the cell injection.

[0159] Figure 16 shows that rFib can improve lower limb ischemic symptoms in mice.

[0160] Figure A shows the results of detecting lower limb blood flow in mice using laser Doppler. As can be seen from the results, rFib and bMSC can significantly improve lower limb ischemia in mice.

[0161] Image B shows a photograph taken 7 days after ligation of the mouse hind limb. The control group shows the normal hind limb. Ischemic shows the ligated hind limb. As can be seen from the results, the necrosis of the ligated hind limb was significantly milder in the rFib group and the bMSC group.

[0162] Example 19: Production of rFib cells by combination of different compounds

[0163] 1. Dermatophytes were inoculated into 6-well plates and cultured in dermatophyte culture medium for 24 hours.

[0164] 2. The cell culture medium was replaced with an rFib-inducible culture medium containing the low molecular weight compound combination Mix Y, and the fluid was changed every two days, treating the cells for 10 days.

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

[0166] 5. During long-term subculturing, rFib cells were cultured in MSC basal medium and subculturing was performed when cell confluence reached 90%.

[0167] Dermatophyte 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, 5 μM Y-27632, 0.2 mM Vc, 5 μM EPZ004777, 10 μM forskolin, and 1 μM Repsox were added to FibGro medium of brand HCell, product number FGS0040.

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

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

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

[0173] 1. Dermatophytes were inoculated into 6-well plates and cultured in dermatophyte culture medium for 24 hours.

[0174] 2. The cell culture medium was replaced with an rFib-inducing culture medium containing the low molecular weight compound combination Mix Pn, and the fluid was changed every two days, treating the cells for seven days.

[0175] 3. After treatment with a combination of low molecular weight compounds (Mix Pn), the culture medium was replaced with HG-DMEM supplemented with 10% FBS, and the cells were cultured for 3 days before identification, or cultured in rFib medium.

[0176] 5. During long-term subculturing, rFib cells were cultured in MSC basal medium and subculturing was performed when 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 with 10% FBS added (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, 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 were added to FibGro medium of brand HCell, product number FGS0040.

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

[0181] Figure 18: Mix Pn treatment downregulates cellular STAT5 expression (A) and suppresses the expression of ATF3, CDKN1A, GADD45B, and IL6 (BE), demonstrating cell rejuvenation.

[0182] Example 21: Production of rFib cells by combination of different compounds

[0183] 1. Dermatophytes were inoculated into 6-well plates and cultured in dermatophyte culture medium for 24 hours.

[0184] 2. The cell culture medium was replaced with rFib-inducing culture medium containing the low molecular weight cocktail combination Mix Y, and the liquid was changed every two days.

[0185] 3. After culturing for 9 days in rFib-inducing culture medium containing the low molecular weight cocktail combination Mix Y, the culture medium was replaced with HG-DMEM supplemented with 10% FBS and treated for 3-7 days.

[0186] 4. After the treatment in step 3, rFib-inducible culture medium containing the low molecular weight compound combination Mix Pn2 was added, and the liquid was changed every two days.

[0187] 5. After culturing cells in an rFib-inducing culture medium containing the 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 the culture medium was replaced with HG-DMEM supplemented with 10% FBS and the cells were treated for 3 days, or the cells were cultured in rFib medium and identified after 3 days.

[0188] 6. During long-term subculturing, rFib cells were cultured in MSC basal medium and subculturing was performed when cell confluence reached 90%.

[0189] Dermatophyte 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, 5 μM Y-27632, 0.2 mM Vc, 5 μM EPZ004777, 10 μM forskolin, and 1 μM Repsox were added to FibGro medium of brand HCell, product number FGS0040.

[0192] Mix Pn: HG-DMEM with 10% FBS added (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, 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 were added to FibGro medium of brand HCell, product number FGS0040.

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

[0195] Figure 19 shows that Mix Y-Mix Pn2 treatment suppresses JAK1 expression in fibroblasts (A) and elongates their telomere length (B).

[0196] Example 22: Skin fibroblasts differentiate into nerve cells.

[0197] 1. Dermatophytes were inoculated into 6-well plates and cultured in dermatophyte culture medium for 24 hours.

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

[0199] 3. After culturing cells in a nerve induction culture medium containing the low molecular weight compound combination Mix Neu for 5 to 12 days, it was observed that the elongated spindle-shaped cell morphology changed to that of nerve cells. The culture medium was then replaced with nerve cell culture medium, and the cells were continuously subcultured.

[0200] 4. Differentiated nerve cells were identified by immunofluorescence and quantitative PCR.

[0201] Dermatophyte 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] Nerve cell culture medium: 5 mL of DMEM / F12, 5 mL of Neurobasal, 1 / 100 of N2, 1 / 50 of B27, 100 μM cAMP, 20 ng / mL of BDNF, 20 ng / mL of GDNF, 10% (v / v) of KOSR.

[0204] Figure 20: Shows the conversion of skin fibroblasts to nerve cells.

[0205] A shows Tuj1 staining of nerve cells converted from cutaneous fibroblasts.

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

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

[0208] 1. Embryonic stem cells cultured with adherent cells were digested and then suspended in nerve induction fluid.

[0209] 2. After culturing in nerve induction solution for 10-15 days, it was observed that the cells successively adhered to the wall. The suspended cells were then inoculated into a 6-well plate treated with Matrigel, and the cells were cultured to adhere to the wall and subsequently cultured in nerve induction solution for 5-7 days.

[0210] 3. After the cells adhered to the wall, the nerve induction fluid was replaced with nerve cell culture medium.

[0211] 4. Neuronal markers were identified from induced cells using immunofluorescence staining and quantitative PCR.

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

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

[0214] Figure 21 shows that the expression of the STAT5 gene is upregulated during the differentiation process from ES cells to nerve cells by acting on a small molecule compound.

[0215] Example 24: Characteristics of the signaling pathway in rFib cells produced in Example 1

[0216] In the manufacturing method shown in Example 1, transcriptome sequencing was performed on rFib cells derived from different individuals, and 12,036 genes in each cell sample were analyzed using WGCNA to obtain 12 clustering modules.

[0217] Figure 22 shows a module that contains many KEGG pathways.

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

Claims

1. A method for producing rejuvenated, repair-type fibroblasts, A method characterized by treating fibroblasts with a combination of low molecular weight compounds including VPA, CHIR99021, Repsox, forskolin, Go 6983, Y-27632, AM580, EPZ004777, Vc, TTNPB, and 5-Aza-2'-deoxycytidine.

2. The method according to claim 1, characterized in that the fibroblasts are derived from humans, mice, monkeys, or pigs.

3. The method according to claim 1, characterized in that the fibroblasts are derived from human skin.

4. Use of cells produced by the method of any one of claims 1 to 3 in the preparation of products for repairing mammalian tissue, organ damage, and aging, degenerated tissue and organs, as a source of seed cells for tissue engineering materials and scaffolds for tissue engineering materials.

5. Use of cells produced by the method according to any one of claims 1 to 3 in the preparation of an immunomodulator.

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