Composition and method capable of reversing cellular senescence, and use thereof

Abstract

The present invention belongs to the technical field of cellular anti-aging, and specifically relates to a small-molecule compound composition capable of reversing the senescence of an adult stem cell and restoring the regeneration activity thereof, and a method for reversing cellular senescence by means of using a chemical small-molecule composition and the use thereof. The composition capable of reversing cellular senescence comprises a WNT / β-catenin agonist, a TGF-β receptor inhibitor, an RAR agonist, a Smoothened receptor agonist, a multi-target inhibitor of the VEGFR and PDGFR families, a histone methyltransferase inhibitor, and a JAK1 / 2 inhibitor. The composition effectively solves the problem of the weak proliferation and regeneration potential of senescent adult stem cells, effectively reverses the senescence of the adult stem cells and maintains the intrinsic biological characteristics of the cells, and can thus improve and enhance the efficacy of clinical autologous cell transplantation therapy and cell derivative therapy.

Description

A composition, method and application for reversing cellular senescence

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202411047142.3, filed on July 31, 2024, entitled "A composition, method and application of reversible cellular senescence", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of cell anti-aging technology, specifically relating to a small molecule compound composition that can reverse the aging of adult stem cells and restore their regenerative vitality, as well as a method and application of using a chemical small molecule composition to reverse cell aging. Background Technology

[0004] Adult stem cells, also known as progenitor cells or precursor cells, are a type of pluripotent stem cells found in differentiated tissues. Adult stem cells have the ability to self-renew and differentiate into mature cells with specific phenotypes and functions within the tissue, thereby achieving the effects of repairing damaged tissues and maintaining the stability of bodily functions.

[0005] Currently, cell therapy, primarily based on adult stem cells, has been widely used in the treatment of diseases that seriously impair human health, including respiratory diseases, demonstrating good therapeutic effects and broad clinical application prospects. However, clinical observation data has revealed a significant negative correlation between the therapeutic effect of stem cells and the patient's age. A clinical study on autologous reinfusion of lung progenitor cells for chronic obstructive pulmonary disease (COPD) showed that for patients with older donor cells (usually over 70 years old), the lung progenitor cells exhibited poor clonal morphology and significantly reduced cell proliferation capacity during in vitro expansion, displaying morphological and molecular characteristics related to cellular aging. Consequently, the expected efficacy of autologous cell reinfusion for COPD was poor, failing to achieve the anticipated therapeutic effect. Comparative studies of mesenchymal stem cells (MSCs) have revealed significant age-related differences in MSC proliferation. Compared to cells isolated from younger donors, older or senescent MSCs showed a significant decline in cell quality, differentiation capacity, and migration ability. Clinical studies have also indicated that the clinical therapeutic effect of older MSCs is unsatisfactory. This phenomenon suggests that the aging of adult stem cells is the main reason for their reduced regenerative and repair capabilities and poorer treatment effects.

[0006] Research indicates that cellular senescence can be caused by telomere shortening or DNA damage, or by abnormal epigenetic regulation. Current studies show that abnormal epigenetic regulation, particularly the level and rate of DNA methylation, increases with cell age. Partial somatic cell reprogramming can eliminate age-related imprints in cells, essentially resetting the epigenetic map of somatic cells through epigenetic reprogramming. This effectively reverses cell age while maintaining the somatic cell's identity, restoring somatic cells to a more youthful state, achieving cellular rejuvenation, and restoring cell vitality and function.

[0007] Currently, the most widely used method for partial reprogramming is to use retroviruses as vectors to integrate reprogramming transcription factors into the cellular genomic DNA, inducing partial reprogramming through transient induction of transcription factor expression. However, this method of inserting transcription factors into genomic DNA carries risks such as insertional mutations and reactivation of endogenous reprogramming factors, which significantly increase oncogenicity. Furthermore, this method is difficult to implement and lacks control, and retrovirus vectors cannot be used in clinical research. This severely limits the application of transcription factor-induced controlled reprogramming in clinical translational research for reversing aging, necessitating the development of new, safer, and more reliable reprogramming methods to reverse cellular senescence.

[0008] Building upon this foundation, the method of using small-molecule chemical drugs to target and regulate the epigenetic induction of partial reprogramming of somatic cells has greater potential for clinical translational applications. On the one hand, small-molecule drugs can induce changes in the expression of age-related epigenetic genes in somatic cells, thereby rejuvenating aging adult stem cells. On the other hand, because small-molecule drugs do not need to integrate with genomic DNA, they avoid the ethical and genetic safety issues associated with other traditional programming methods. This allows for the effective reversal of cellular aging while maintaining the cellular identity and biological characteristics of adult stem cells, offering advantages such as high controllability and broad applicability. This provides a flexible and safe optimization method for reversing aging with clinical-grade stem cells and improving the efficacy of autologous cell infusion therapy, offering new clues for the treatment of age-related diseases.

[0009] Summary of the Invention

[0010] Therefore, the technical problem to be solved by this application is to provide a small molecule compound composition that can reverse cell senescence, the composition of which can reverse the senescence of adult stem cells and restore their regenerative vitality, effectively solving the problems of weak proliferation and regeneration potential of senescent adult stem cells;

[0011] The second technical problem to be solved by this application is to provide a method for reversing cell senescence using the above-mentioned chemical small molecule composition, which can effectively reverse the senescence of adult stem cells and maintain the original biological characteristics of cells, thereby improving the therapeutic effect of cell transplantation.

[0012] The third technical problem to be solved by this application is to provide the application of the above-mentioned chemical small molecule composition and the method for reversing cell senescence in the field of tissue and organ regeneration.

[0013] To address the aforementioned technical problems, this application provides a composition for reversing cellular senescence, which contains components that promote cell proliferation and inhibit cell methylation, including at least one of the following components in molar amounts: 1-10 molar amounts of WNT / β-catenin agonist, 1-10 molar amounts of TGF-β receptor inhibitor, 0.2-2 molar amounts of RAR agonist, 0.05-0.5 molar amounts of Smoothened receptor agonist, 0.1-1 molar amounts of VEGFR and PDGFR family multi-target inhibitors, 0.002-0.02 molar amounts of histone methyltransferase inhibitor, and 0.1-1 molar amounts of JAK1 / 2 inhibitor.

[0014] It should be noted that the term "senescence" used in this application has the same meaning as "aging," and rejuvenated cells refer to cells before aging. However, "senescence" as used in this application does not include cells that have permanently stopped dividing.

[0015] Specifically, the composition that reverses cellular senescence:

[0016] The WNT / β-catenin agonist includes at least one of MAY-262611, CHIR98014, CHIR99021, LiCl, Li2CO3, TD114-2, AZD2858, AZD1080, BIO, Kenpaullone, TWS119, LY2090314, CBM1078, SB216763, SKL2001, or ARA014418; and / or,

[0017] The TGF-β receptor inhibitor includes at least one of LY2109761, Pirfenidone, Repsox (E-616452), SB431542, A77-01, Tranilast, Galunisertib (LY2157299), A8301, GW788388, ITD-1, SD208, SB525334, LY364947, ASP3029, D4476, or SB505124; and / or,

[0018] The RAR agonist comprises at least one of TTNPB, Bexarotene, Ch55, Tamibarotene, Retinol, AM580, ATRA, 13-cisRA, BMS493, Adapalene, Vitamin A, or a Vitamin A derivative; and / or,

[0019] The smoothed receptor agonist includes at least one of SAG, SAG-d3, SAG hydrochloride, SAG dihydrochloride, or (Rac)-SAG; and / or,

[0020] The VEGFR and PDGFR family multi-target inhibitors include at least one of ABT-869, PP121, Vorolanib, Ki20227, SU11652, Sorafenib, Regorafenib, Sunitinib, Nintedanib, Ponatinib, Axitinib, Pazopanib, or SU 5402; and / or,

[0021] The histone methyltransferase inhibitors include at least one of EPZ004777, EPZ5676, GSK503, BIX01294, DZNep, DZNep·HCl, SGC0946, or Chaetocin; and / or,

[0022] The JAK1 / 2 inhibitors include at least one of Ruxolitinib, iJak-381, Tuspetinib, JAK1 / 2-IN-1, JAK-IN-1, Cerdulatinib, or JAK / HDAC-IN-2.

[0023] Optionally, the Wnt / β-catenin agonist is CHIR99021; the TGF-β receptor inhibitor is E-616452; the RAR agonist is TTNPB; the smoothed receptor agonist is SAG, which can activate the Hedgehog signaling pathway; the VEGFR and PDGFR family multi-target inhibitor is ABT-869; the histone methyltransferase inhibitor is DZNep; and the JAK1 / 2 inhibitor is Ruxolitini.

[0024] Specifically, the composition for reversing cellular senescence comprises the following components in molar amounts: 1-10 molar amounts of the WNT / β-catenin agonist CHIR99021, 1-10 molar amounts of the TGF-β receptor inhibitor E-616452, 0.2-2 molar amounts of the RAR agonist TTNPB, 0.05-0.5 molar amounts of the smoothed receptor agonist SAG, 0.1-1 molar amounts of the VEGFR and PDGFR family multi-target inhibitor ABT-869, 0.002-0.02 molar amounts of the histone methyltransferase inhibitor DZNep, and 0.1-1 molar amounts of the JAK1 / 2 inhibitor Ruxolitinib.

[0025] This application also discloses a storage solution for reversing cellular senescence, comprising the composition for reversing cellular senescence, and a pharmaceutically acceptable solvent or diluent.

[0026] For example, in the storage solution for reversible cell senescence, the "pharmaceutically acceptable solvent or diluent" includes conventional reagents such as water, PBS, and DMSO, or mixtures thereof. Optionally, the solvent or diluent is DMSO.

[0027] Specifically, the storage solution for reversing cellular senescence comprises the following components at the following concentrations: WNT / β-catenin agonist CHIR99021 1-10mM, TGF-β receptor inhibitor E-616452 1-10mM, RAR agonist TTNPB 0.2-2mM, Smoothened receptor agonist SAG 0.05-0.5mM, VEGFR and PDGFR family multi-target inhibitor ABT-869 0.1-1mM, histone methyltransferase inhibitor DZNep 0.002-0.02mM, and JAK1 / 2 inhibitor Ruxolitinib 0.1-1mM.

[0028] It should be noted that the effective concentration range of the specific small molecule compounds in the above composition is only for reference and can be adapted accordingly. If other small molecules are used to replace the proposed small molecules, the concentration can also be adjusted accordingly.

[0029] This application also discloses a reversible cell senescence induction culture medium, including a basal culture medium, and the composition of the reversible cell senescence and / or the storage solution of the reversible cell senescence.

[0030] Specifically, in the reversible cell senescence induction culture medium, the volume ratio of the storage solution to the basal culture medium is 1:100 to 1:100000.

[0031] Specifically, the reversible cellular senescence induction medium includes a basal medium comprising DMEM / F12 basal medium, with selective addition of 5-15 vol% fetal bovine serum, 0.5-2 mM L-glutamine, 3-8 ng / mL insulin, 0.05-0.2 ng / mL epidermal growth factor, 3-8 μg / mL adenine, and / or 3-8 μg / mL hydrocortisone.

[0032] This application also discloses a method for reversing cell senescence, including the step of inducing culture of senescent adult stem cells using the reversible cell senescence-inducing medium.

[0033] Specifically, the method for reversing cellular senescence described in this application is a method for reversing adult stem cell senescence using a small chemical molecule composition with reprogramming potential, comprising the following steps:

[0034] S1: Provides aging adult stem cells;

[0035] S2: Provides an induction culture medium containing small chemical molecule compositions;

[0036] S3: Use the aforementioned induction culture medium to induce the culture of senescent adult stem cells to obtain rejuvenated adult stem cells.

[0037] Optionally, the cell rejuvenation described in S3 is characterized by a decrease in cell diameter, an increase in roundness, a more uniform morphology, fewer elongated cell morphologies, a more compact clonal morphology with smooth edges, and no significant cell differentiation.

[0038] Specifically, the induction culture step in the method for reversing cell senescence includes the following conditions: culture temperature of 35-38℃, culture at 5-10% CO2 for 4-10 days.

[0039] Specifically, the method for reversing cellular senescence:

[0040] The induction culture step begins 0-24 hours after the adult stem cell inoculation; and / or,

[0041] The induction culture step also includes changing the induction medium every 2-3 days.

[0042] Specifically, in the method for reversing cellular senescence, the adult stem cells include lung progenitor cells, kidney progenitor cells, and / or mesenchymal stem cells;

[0043] Specifically, the senescent adult stem cells include, but are not limited to, lung progenitor cells, kidney progenitor cells, or mesenchymal stem cells isolated from elderly donors.

[0044] Optionally, the lung progenitor cells are derived from bronchial basal layer cells of healthy adults or patients, and can be obtained through non-invasive or minimally invasive methods such as fiberoptic bronchoscopy. Under certain conditions, the cells can be isolated and expanded to achieve the quantity and quality required for clinical treatment; and / or,

[0045] Optionally, the renal progenitor cells are derived from the urine of healthy or diseased patients, and are isolated and expanded under certain conditions to achieve the quantity and quality required for clinical treatment; and / or,

[0046] Optionally, the mesenchymal stem cells are derived from the umbilical cord of newborns delivered by cesarean section, and the cells are isolated and expanded under certain conditions to achieve the quantity and quality required for clinical treatment.

[0047] This application also discloses the use of the composition for reversible cell senescence, the storage solution for reversible cell senescence and / or the induction medium for reversible cell senescence in preparing a product having at least one of the following (1)-(5);

[0048] (1) Inhibit the expression of P16 or P21 or a combination thereof in cells;

[0049] (2) Inhibits the activity of cellular methyltransferases;

[0050] (3) Increase the expression of the early reprogramming factor OCT4;

[0051] (4) Inhibits inflammatory infiltration and enhances regenerative potential;

[0052] (5) Slow down the aging process and treat age-related diseases.

[0053] This application also discloses the application of the composition for reversing cell senescence, the storage solution for reversing cell senescence, the induction culture medium for reversing cell senescence, and / or the method for reversing cell senescence in the field of cell and organ transplantation therapy.

[0054] The composition for reversing cellular senescence described in this application specifically relates to a small molecule chemical composition with reprogramming potential, specifically including a WNT / β-catenin agonist, a TGF-β receptor inhibitor, a RAR agonist, a smoothed receptor agonist, a multi-target inhibitor of the VEGFR and PDGFR families, a histone methyltransferase inhibitor, and a JAK1 / 2 inhibitor. This composition for reversing cellular senescence can delay / reverse aging by inhibiting the expression of P16 or P21 or combinations thereof, inhibiting methyltransferase activity, effectively increasing the expression of the early reprogramming factor OCT4, inhibiting inflammatory infiltration, and improving cell differentiation capacity. This composition for reversing cellular senescence effectively addresses the problem of weak proliferation and regeneration potential of aging adult stem cells, effectively reversing the senescence of adult stem cells while maintaining their original biological characteristics, and can improve and enhance the clinical efficacy of autologous cell transplantation therapy.

[0055] The composition for reversing cellular senescence described in this application, based on the synergistic effect of selected components containing ingredients that promote cell proliferation and inhibit cell methylation, regulates aging or senescent cells through epigenetic regulation, or by activating early development-related transcription factors, such as activating the Hedgehog and RAR signaling pathways, inhibiting the Wnt / β-catenin and TGF-β signaling pathways, and suppressing methylation levels, thereby regulating cell proliferation capacity, influencing cell differentiation fate, and eliminating age-related epigenetic imprints, thus achieving the prevention, delay, or reversal of cellular senescence. The small molecule compound combination described in this application can be used to stimulate the upregulation of early reprogrammed stem gene OCT4 and proliferation-related gene Ki67 without affecting the expression of late reprogrammed stem genes such as Nanog, and inhibits the expression of one or more senescence-related genes P16 and P21, without altering the adult stem cell identity, and has the advantages of safety and efficacy.

[0056] The composition for reversing cellular senescence described in this application exhibits excellent performance in reversing lung progenitor cell senescence: adult stem cells treated with the combination of small molecule drugs show significantly improved cell morphology and uniformity, increased expression of cell proliferation-related gene Ki67 and stemness-related gene OCT4, reversed expression of senescence-related markers, and restoration of the original biological characteristics of adult stem cells.

[0057] The composition for reversing cellular senescence described in this application enhances the proliferative capacity and multi-lineage differentiation potential of adult stem cells, thereby improving the efficacy of age-related respiratory diseases such as COPD through autologous transplantation of lung progenitor cells, and promoting the clinical application of adult stem cells. Mice in the cell therapy group treated with small molecule drugs showed better results in terms of improvement in pathological damage, indicating that the rejuvenated adult stem cells obtained using the method described in this application have good tissue regeneration potential.

[0058] The method for reversing cellular senescence described in this application utilizes the small molecule composition as an effective component of the induction culture medium. This method can reverse the senescent state of adult stem cells, enhance their anti-aging capabilities, and achieve the rejuvenation and regeneration of senescent cells. Compared with other methods such as nuclear transplantation and transcription factor expression, it avoids ethical issues and risks such as gene insertion mutations. Furthermore, the method allows for control over both drug treatment time and concentration, offering advantages such as ease of operation and low cost. The resulting cells are of more uniform quality, enabling large-scale processing of lung progenitor cells and other adult stem cells. This optimizes the adult stem cell preparation process and provides strong technical support and theoretical basis for further clinical translational research. The method for reversing cellular senescence described in this application can be used in mammalian cells or tissues to prevent, delay, or reverse the aging process, obtaining rejuvenated cells, organs, and cell derivatives. These can be further applied in cell and organ transplantation therapy or used as cell derivatives in the treatment of aging-related diseases.

[0059] The method for reversing cellular senescence described in this application can target adult stem cells, including lung progenitor cells, kidney progenitor cells, and / or mesenchymal stem cells, for anti-aging reversal. The resulting rejuvenated cells and cell derivatives have the characteristics of inhibiting inflammatory infiltration and improving regenerative potential. The resulting rejuvenated lung progenitor cells can be used in drugs for treating respiratory diseases, or the rejuvenated cells and cell derivatives can be used through autologous or allogeneic transplantation. Attached Figure Description

[0060] To make the content of this application easier to understand, the following detailed description is provided based on specific embodiments and accompanying drawings.

[0061] Figure 1 shows the results of culturing adult stem cells from different sources on cells with and without a trophoblast layer in Example 1;

[0062] Figure 2 is a microscopic observation of lung progenitor cell clones obtained after induction and culture for 5 days using the chemical small molecule composition of this application in Example 2 within the range of 0.5-10× drug concentration;

[0063] Figure 3 is a microscopic observation of cell clones obtained after 5 days of induction culture of human lung progenitor cells aged 30-75 years using the small molecule composition of this application in Example 2;

[0064] Figure 4 shows a comparison of cell roundness and cell diameter between the five groups of human lung progenitor cells in Example 2 and the control group.

[0065] Figure 5 shows the immunofluorescence detection of Ki76 and P63 expression in the human lung progenitor cell experimental group and control group in Example 2;

[0066] Figure 6 shows the qRT-PCR detection results of OCT4 gene expression in the experimental group and control group of human lung progenitor cells in Example 2;

[0067] Figure 7 shows the bioinformatics analysis results of the human lung progenitor cell experimental group and the control group in Example 2;

[0068] Figure 8 shows the immunofluorescence staining of frozen sections of lung tissue from the drug-treated experimental group and the untreated control group in Example 3, which were transplanted into an immunodeficient mouse lung injury model for lung regeneration and repair.

[0069] Figure 9 shows the HE staining images of paraffin sections of tumor tissue formed by transplanting human lung progenitor cells into the subcutaneous tissue of immunodeficient mice in the drug-treated experimental group and the untreated control group in Example 4, as well as the results of inflammatory cell infiltration analysis.

[0070] Figure 10 shows the immunofluorescence staining images of paraffin sections of tumor tissue formed by transplanting human lung progenitor cells into the subcutaneous tissue of immunodeficient mice in the drug-treated experimental group and the untreated control group in Example 4, along with the cell differentiation analysis results.

[0071] Figure 11 is a microscopic observation of renal progenitor cells obtained after induction and culture for 4 days using the chemical small molecule composition of this application in Example 5 within the range of 0.5 to 5× drug concentration.

[0072] Figure 12 shows the qRT-PCR detection and analysis of related gene expression after treatment of renal progenitor cells with the small molecule composition in Example 5;

[0073] Figure 13 is a microscopic observation of umbilical cord mesenchymal stem cells obtained after induction and culture for 4 days using the chemical small molecule composition of this application in Example 6 within the range of 0.5-50× drug concentration.

[0074] Figure 14 shows the qRT-PCR detection and analysis of related gene expression after treatment of umbilical cord mesenchymal stem cells with the small molecule composition in Example 6. Detailed Implementation

[0075] The present application will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0076] The following embodiments are provided to facilitate a better understanding and use of this application by those skilled in the art, and are not limited to the preferred embodiments described herein, nor do they constitute a limitation on the content and scope of protection of this application. Those skilled in the art will readily be able to make various modifications to these embodiments and apply the general principles described herein to other embodiments without inventive effort. Therefore, any product identical or similar to this application, derived by any person based on the teachings of this application or by combining features of this application with other prior art, falls within the protection scope of this application.

[0077] Unless otherwise specified, all raw materials used in the following examples are commercially available products that are routinely available to those skilled in the art.

[0078] Example 1: Adult Stem Cell Acquisition

[0079] In this embodiment, the selected human lung progenitor cells specifically refer to adult lung stem cells derived from the basal layer of the human bronchi. These cells are easy to isolate and obtain; detailed isolation, extraction, and primary culture steps can be found in Chinese Patent CN111944737A. After obtaining the above cells, they were cultured and passaged once before being used in experiments.

[0080] Continue to seed the isolated fresh lung progenitor cells or cryopreserved and thawed cells onto a substrate lined with 1.8–2.2 × 10⁻⁶ cells. 4 cells / cm 2 The trophoblast cells were cultured in cell culture plates at 37°C in a 7.5% CO2 incubator using lung progenitor cell culture medium, with an inoculation density of 0.5-2 × 10⁻⁶ cells / mL. 4 cells / cm 2 The fluid should be changed every 2-3 days.

[0081] As culture time increased, senescent lung progenitor cells exhibited senescence-related morphological characteristics, such as poor clonal morphology, loose cell clones, indistinct edges, elongated filamentous cells, increased cell volume, and increased nucleocytoplasmic ratio and length-to-width ratio. Five days after inoculation, when cells continued to grow to a confluence of 70%-90%, the original culture medium was discarded, and the cells were washed once with PBS. 0.05% trypsin was added, and the cells were incubated at 37°C for 1 minute until microscopic observation showed complete detachment of the feeder layer cells. The liquid was discarded, and 0.25% trypsin was added, followed by incubation at 37°C for another 4-8 minutes. Microscopic observation showed complete detachment of the lung progenitor cells. Digestion was terminated by adding twice the volume of trypsin in lung progenitor cell culture medium. The cell suspension was collected in 15 mL centrifuge tubes, centrifuged at 1100 rpm for 5 minutes, and the supernatant was discarded to obtain the lung progenitor cell pellet for subsequent experiments.

[0082] In this embodiment, the selected adult mesenchymal stem cells refer to umbilical cord mesenchymal stem cells, which can be obtained from the umbilical cord of normal full-term cesarean section newborns, and used for subsequent experiments after expansion culture and bank construction.

[0083] Umbilical cord mesenchymal stem cells or uterine epithelial mesenchymal stem cells were used at a dose of 0.5-1×10⁻⁶. 4 cells / cm 2 Cells were seeded and cultured in cell culture plates using Icosé serum-free mesenchymal stem cell culture medium. Microscopic observation revealed an increase in the number of atypical cells with each culture passage, exhibiting characteristics of senescence. Cells were cultured for 3-5 days, with medium changes performed every 2-3 days. When cells reached 80%-90% confluence, the original culture medium was discarded, and the cells were washed once with PBS. 0.25% trypsin was added, and the cells were incubated at 37°C for 1-4 minutes until complete detachment of mesenchymal stem cells was observed under a microscope. Digestion was then stopped by adding twice the volume of trypsin-free culture medium, and the cell suspension was collected in 15 mL centrifuge tubes. After centrifugation at 1100 rpm for 5 minutes, the supernatant was discarded, yielding the mesenchymal stem cell pellet, which was used for subsequent experiments.

[0084] In this embodiment, the selected renal progenitor cells refer to the renal progenitor cell precipitate obtained from the urine of healthy or chronic kidney disease patients through multiple centrifugation and washing. After being expanded and cultured in renal stem cell culture medium, it is used for subsequent experiments. For details of the extraction and separation methods, please refer to Chinese Patent CN112430567A.

[0085] The obtained renal progenitor cells were seeded onto culture plates with or without a feeder layer and cultured at 37°C and 5% CO2 for 3-7 days in renal progenitor cell culture medium, with medium changes performed every 2-3 days. When the cell confluence reached 80%-90%, the original culture medium was discarded, the cells were washed once with PBS, and 0.25% trypsin was added. The cells were then incubated at 37°C for 1-4 minutes until all mesenchymal stem cells were observed to have detached under a microscope. Digestion was then stopped by adding twice the volume of trypsin in stop culture medium, and the cell suspension was collected into 15 mL centrifuge tubes. After centrifugation at 1100 rpm for 5 minutes, the supernatant was discarded to obtain the mesenchymal stem cell pellet, which was used for subsequent experiments.

[0086] In this embodiment, the culture results of the adult stem cells from the different sources mentioned above are shown in Figure 1.

[0087] Example 2

[0088] This embodiment uses a selected small molecule composition to reverse the aging of lung progenitor cells.

[0089] In this embodiment, the prepared storage solution is composed as follows: selected small molecule compounds are dissolved in DMSO or sterile water and mixed according to the concentrations of CHIR99021 (5mM), E-616452 (1mM), TTNPB (1mM), SAG (0.05mM), ABT-869 (0.2mM), DZNep (0.02mM), and Ruxolitinib (0.5mM) to form the desired storage solution. All the above small molecule compounds are well-known names in the art, i.e., conventional commercially available products. The substances selected in this embodiment were all purchased from MCE Corporation; products from other companies have essentially the same effect.

[0090] In this embodiment, the induction medium for lung progenitor cells was prepared by diluting the basal medium and the storage solution at a ratio of 5000-fold. The basal medium was formulated as follows: DMEM / F12 basal medium, 10 vol% fetal bovine serum, 1 mM L-glutamine, 5 ng / mL insulin, 0.1 ng / mL epidermal growth factor, 5 μg / mL adenine, and 5 μg / mL hydrocortisone.

[0091] In the storage solution described in this application, the basal culture medium can be any conventional culture medium in the art that can achieve similar functions. In this embodiment, as an exemplary culture medium, the DMEM / F12 basal culture medium described in this embodiment is the commercially available Gibco DMEM / F12 culture medium.

[0092] Example 3

[0093] This embodiment uses the induction culture medium described in Example 2 to conduct an experiment to reverse senescent cells.

[0094] The lung progenitor cells obtained in Example 1 were resuspended in 2 mL of lung progenitor cell culture medium, thoroughly mixed by pipetting, and counted using a cell counting chamber. Based on the counting results, the cells were divided into 1-2 × 10⁻⁶ cells. 4 pcs / cm 2 The inoculation density was 2×10⁶ ppm in the bedding. 4 pcs / cm 2 Induction culture of feeder cells was performed in six-well plates, specifically by replacing the lung progenitor cell culture medium with a small molecule induction medium within 24 hours of cell seeding. The cells were then cultured at 37°C and 7.5% CO2 for 48-72 hours, after which the induction medium was replaced with fresh medium. After 5-10 days of drug treatment and culture, the lung progenitor cells were recovered following the same procedures as cell passage.

[0095] In this embodiment, cell viability is assessed by detecting markers related to aging, proliferation, cell identity, and early reprogramming in drug-treated lung progenitor cells.

[0096] (1) Statistical analysis of cell morphology was performed using ImageJ software.

[0097] Cells with high stemness have larger nuclei, are nearly round, and have an aspect ratio close to 1. ImageJ software was used to analyze cell roundness and diameter (or aspect ratio), and statistical analysis was performed on photographs of drug-treated experimental cells and untreated control cells under an optical microscope.

[0098] (2) Detection of the expression of proliferation-related gene Ki67 and cell identity marker P63 using immunofluorescence staining.

[0099] Cellular immunofluorescence staining was used to label cell proliferation marker Ki67 and cell identity marker P63. The specific steps are as follows: lung progenitor cell clones in good growth condition were selected, and after washing, fixing, permeabilization, and blocking, they were sequentially labeled with primary antibody, secondary antibody and nuclear dye DAPI. The staining results were observed and photographed under a fluorescence microscope.

[0100] (3) Detection of early reprogramming factor-related gene expression using qRT-PCR

[0101] Total RNA was extracted from cells in the experimental group treated with the small molecule composition and from the untreated control group using the Trizol method (see reagent instructions). The RNA from both groups was reverse transcribed into cDNA using a reverse transcription kit (Novizan Biosciences) (see kit instructions). The cDNA was then used as a template for RT-PCR amplification. The expression of the early reprogramming factor OCT4 gene was detected using the GAPDH gene as an internal reference.

[0102] (4) Analyze changes in the cell transcriptome using RNA sequencing

[0103] The transcriptomes of drug-treated and untreated lung progenitor cells were sequenced using Bulk RNA sequencing, and the differences in gene expression were compared.

[0104] Figure 2 shows a microscopic observation of lung progenitor cell clones obtained after 5 days of induction culture with the small molecule composition at drug concentrations ranging from 0.5 to 10× (1× after dilution of the storage solution at 1:5000). It is evident that the small molecule composition screened in this application can effectively improve clonal morphology within a certain drug concentration range. The lung progenitor cells in the experimental group treated with the small molecule composition for 5 days exhibited consistent morphology, smaller size, larger nuclei, decreased aspect ratio, increased roundness, and clearer clonal edges.

[0105] Figure 3 shows a microscopic observation of cell clones obtained after 5 days of induction culture of lung progenitor cells from patients aged 30-75 years using a small molecule composition. When the drug concentration of the small molecule composition was 1×, the cell and clonal morphology of lung progenitor cells from patients of different ages treated with the small molecule composition for 5 days were significantly and effectively improved. The cells were uniform in morphology, smaller in size, with rounder and clearer clonal boundaries, and the senescence state of the cells was reduced.

[0106] Figure 4 shows the comparison of cell roundness and cell diameter between the experimental group and the control group of five human lung progenitor cells. Under the same culture time, the experimental group cells had higher roundness, smaller diameter (width), and lower length-to-width ratio, indicating that the lung progenitor cells treated with the small molecule composition had a younger cell morphology.

[0107] Figure 5 shows the immunofluorescence detection of Ki76 and P63 expression in the experimental and control groups of human lung progenitor cells. The results showed that the number of Ki67-positive cells in the control group (without the small molecule composition treatment) was lower, while the number of Ki67-positive cells in the experimental group (treated with the small molecule composition treatment) was significantly increased. Furthermore, the expression level of P63 was consistent in both groups. This indicates that treatment with the small molecule composition enhanced the proliferative capacity of lung progenitor cells without affecting the expression of the cell identity marker P63. When cell proliferation capacity is maintained well, damaged or aging cells can be effectively replaced, helping to maintain the normal function of tissues and organs. Therefore, enhanced cell proliferation capacity is beneficial in combating the aging process, thereby delaying aging.

[0108] Figure 6 shows the qRT-PCR results of OCT4 gene expression in the experimental and control groups of human lung progenitor cells. Quantitative analysis results indicate that the OCT4 gene expression level in the experimental group was significantly increased compared with that in the control group, suggesting that the cells in the experimental group underwent reprogramming. Maintaining or increasing OCT4 expression during aging helps restore or maintain a youthful cellular state, thereby supporting tissue repair and regeneration and counteracting the effects of aging.

[0109] Figure 7(a) shows the bioinformatics analysis results of the human lung progenitor cell experimental group and the control group. PCA analysis revealed significant differences between cells treated with the small molecule composition and untreated cells. Differential gene enrichment analysis showed that the expression of genes related to aging and inflammation was significantly downregulated in cells treated with the small molecule composition, while the expression of genes related to endothelial cell fate, metabolism, and stress regulation was upregulated. This indicates that drug treatment partially inhibited / reversed cellular senescence, increased cell viability, and enhanced differentiation potential.

[0110] In this embodiment, bulk RNA-seq was performed on 16 lung progenitor cells from donors of different ages, and a cell age prediction model was constructed, as detailed in Figure 7(b). Analysis using this model showed that the lung progenitor cells treated with the small molecule composition all achieved senescence reversal, generally appearing several decades younger, demonstrating the significant effect of this small molecule composition in reversing cellular senescence.

[0111] In summary, the method of reversing cellular senescence using the small molecule composition specified in this application can significantly downregulate / inhibit the senescence characteristics of lung progenitor cells and enhance their anti-aging ability.

[0112] Example 4

[0113] This embodiment uses the small molecule composition from Example 2 to enhance the in vivo anti-aging and regenerative repair capabilities of lung progenitor cells. A mouse lung injury model is constructed, and cell transplantation therapy and therapeutic effects are measured.

[0114] The mouse model was established as follows: 6-8 week old immunodeficient mice were anesthetized with isoflurane, and then bleomycin was administered intratracheally at a dose of 3 U / kg, with a perfusion volume of 40 μL per mouse. The model was established on day 7, at which point cell transplantation therapy could be performed.

[0115] Experimental group: 5×10 6 Lung progenitor cells treated with the small molecule composition were resuspended in 200 μL of PBS and administered at a dose of 1 × 10⁶ cells per mouse. 6 Five injured mice were treated with a dose of 40 μL via intratracheal infusion. The administration was performed once, and the therapeutic effect was evaluated after 25 days of treatment.

[0116] Control group: 5×10 6 Untreated lung progenitor cells were resuspended in 200 μL of PBS and administered at a dose of 1 × 10⁶ cells per mouse. 6 Five injured mice were treated with a dose of 40 μL via intratracheal infusion. The administration was performed once, and the therapeutic effect was evaluated after 25 days of treatment.

[0117] Twenty-five days after cell transplantation therapy, mice were anesthetized and sacrificed to obtain lungs. The lungs were then fixed, embedded, frozen sectioned, and immunofluorescently stained, and observed and photographed under a microscope.

[0118] Figure 8 shows immunofluorescence staining of frozen sections of lung tissue from the experimental group treated with the small molecule composition and the untreated control group, transplanted into an immunodeficient mouse lung injury model for lung regeneration and repair. The staining results indicate that the cells treated with the small molecule composition, after transplantation into mice, can differentiate and express RAGE (type I alveolar epithelial marker). This demonstrates that the transplanted lung progenitor cells, after in vivo differentiation, can replace damaged or aging alveolar epithelial cells, maintain normal lung function, and thus counteract tissue damage and aging processes, playing a significant role in regeneration and repair.

[0119] In summary, using lung progenitor cells treated with the small molecule composition specified in this application to reverse aging directly for tissue repair can promote tissue rejuvenation and regeneration and repair of damaged tissues.

[0120] Example 5

[0121] This embodiment verifies that lung progenitor cells treated with the small molecule composition resist aging by inhibiting inflammation and enhancing cell differentiation capacity.

[0122] Following the aforementioned culture method, lung progenitor cells cultured for 5 days were collected and counted, and 1×10⁶ cells were added to the culture. 6 Cells were resuspended in 100 μL of 50% matrix gel and subcutaneously injected into the axillary region of immunodeficient mice. After 2-4 weeks, when a mass with a diameter of about 0.5-1 cm formed at the injection site, the mass was collected, washed and fixed with PBS, embedded in paraffin, and sectioned. The infiltration of inflammatory cells in the mass was evaluated by HE staining, and the cell differentiation in the mass was evaluated by immunofluorescence staining (using Lamin A / C to indicate human cells and FOXJ1 to indicate differentiated ciliated cells). The percentage of FOXJ1+ cells was also counted. The results are shown in Figures 9-10.

[0123] Figure 9 shows the HE staining images (a) and inflammatory cell infiltration analysis results (b) of paraffin sections of subcutaneous tumor tissue formed by transplantation of human lung progenitor cells treated with the small molecule composition into immunodeficient mice. As can be seen from the figure, the subcutaneous tumor tissue derived from cells untreated with the small molecule composition in the control group had a greater number of cells with deeply stained nuclei, while the tumor tissue derived from cells treated with the small molecule composition in the experimental group had fewer cells with deeply stained nuclei. This indicates that the experimental group had less inflammatory cell infiltration, suggesting that lung progenitor cells treated with the small molecule composition can effectively inhibit cellular inflammation. With age, the human body tends to experience a state called "chronic low-grade inflammation," also known as "inflammatory aging." Therefore, controlling inflammation may provide an effective way to delay aging and prevent age-related diseases.

[0124] Figure 10 shows the immunofluorescence staining (a) and cell differentiation analysis results (b) of paraffin sections of tumor tissue formed by transplanting human lung progenitor cells from the experimental group treated with the small molecule composition and the untreated control group into the subcutaneous tissue of immunodeficient mice. As can be seen from the figure, compared with the control group, the proportion of FOXJ1+ cells in the tumor tissue derived from cells treated with the small molecule composition in the experimental group was higher, indicating that the cell differentiation capacity was enhanced.

[0125] Therefore, lung progenitor cells treated with the small molecule composition specified in this application to reverse aging may delay the overall aging of the body by inhibiting inflammation and improving cell differentiation capacity after in vivo transplantation.

[0126] Example 6

[0127] This embodiment uses a small molecule composition to reverse the aging of renal progenitor cells.

[0128] In this embodiment, the induction culture medium for the renal progenitor cells is obtained by diluting the basal culture medium and the storage solution at a ratio of 5000 times. The composition of the storage solution and the basal culture medium is the same as in Example 2.

[0129] Resuspend the cultured renal progenitor cells in 2 mL of renal progenitor cell culture medium, mix thoroughly by pipetting, and count using a cell counting chamber. Based on the counting results, divide the cells at a rate of 1-2 × 10⁻⁶. 4 pcs / cm 2 The cells were inoculated at the appropriate seeding density into six-well plates for induction culture, and cultured at 37°C and 5% CO2 for 48-72 hours, after which the induction medium was replaced with fresh medium. After drug treatment and culture for 4-10 days, the renal progenitor cells were recovered for further analysis. The specific recovery steps were the same as those described in the cell passage procedure.

[0130] Total RNA was extracted from renal progenitor cells of both the experimental group treated with the aforementioned induction medium and the untreated control group using the Trizol method (see reagent instructions). The RNA from both groups was reverse transcribed into cDNA using a reverse transcription kit (Novizan Biosciences), following the kit's instructions. The cDNA was then used as a template for RT-PCR amplification. The expression of renal progenitor cell markers SOX9, early reprogramming factor OCT4, proliferation-related factor Ki67, and aging-related genes P16 and P21 was detected using the GAPDH gene as an internal control. The results are shown in Figures 11-12.

[0131] Figure 11 shows a microscopic observation of renal progenitor cells obtained after induction and culture for 4 days in the range of 0.5-5× drug concentration using the small molecule composition. The results show that the renal progenitor cells treated with the 0.5-5 times diluted small molecule composition exhibited highly consistent morphology, smaller cell volume, more rounded shape, and a reduced number of cells elongated into filaments, indicating a trend towards younger cell morphology.

[0132] Figure 12 shows the qRT-PCR analysis of related gene expression in renal progenitor cells after treatment with the small molecule composition. The results indicated that, compared with the control group renal progenitor cells not treated with the small molecule composition, the expression levels of SOX9, OCT4, and Ki67 genes were significantly increased in the experimental group, while the expression of P16 and P21 genes was significantly downregulated. Although P16 and P21 genes act on different cell cycle checkpoints, their combined effect strengthened cell cycle arrest and promoted the appearance of senescence phenotypes. Therefore, the increased expression of SOX9, OCT4, and Ki67 genes and the decreased expression of P16 and P21 genes indicate that the senescence state of renal progenitor cells treated with the small molecule composition was reduced, and cell viability was increased.

[0133] Therefore, the method of reversing aging using the small molecule composition specified in this application can promote the morphological rejuvenation of renal progenitor cells, stimulate the cells' self-renewal and differentiation capabilities, restore their youthful gene expression patterns, thereby reversing aging and maintaining the youthful state of cells.

[0134] Example 7

[0135] This embodiment uses the small molecule composition to reverse the aging of umbilical cord mesenchymal stem cells, and the specific induction culture medium is the same as in Example 2.

[0136] After resuspending the cultured umbilical cord mesenchymal stem cells in culture medium, mixing them thoroughly by pipetting, and counting them, they were then subjected to a 0.5-1×10⁻⁶ ppm precipitate. 4 pcs / cm 2 Cells were inoculated into culture dishes at the specified seeding density for induction culture at 37°C and 5% CO2 for 48-72 hours, after which the induction culture medium was replaced with fresh medium. After culturing for 4-7 days within the concentration range used, cells were recovered for further analysis, following the same recovery procedure as cell passage.

[0137] Total RNA was extracted from umbilical cord mesenchymal cells in both the experimental group treated with the small molecule composition and the untreated control group using the Trizol method (see reagent instructions). The RNA from both groups was reverse transcribed into cDNA using a reverse transcription kit (Novizan Biosciences), following the kit's instructions. The cDNA was then used as a template for RT-PCR amplification. The expression of early reprogramming factor OCT4, proliferation-related factor Ki67, and aging-related genes P16 and P21 in umbilical cord mesenchymal cells was detected using the GAPDH gene as an internal control. The results are shown in Figures 13-14.

[0138] Figure 13 shows a microscopic observation of umbilical cord mesenchymal stem cells obtained after induction and culture for 4 days using a small molecule composition at drug concentrations ranging from 0.5 to 50×. Compared with the control group, there was no significant difference in cell morphology among the umbilical cord mesenchymal stem cells treated with the small molecule composition.

[0139] Figure 14 shows the qRT-PCR analysis of gene expression in umbilical cord mesenchymal stem cells after treatment with the small molecule composition. The quantitative analysis results indicate that, compared with the control group of umbilical cord mesenchymal stem cells without treatment with the small molecule composition, the expression levels of OCT4 and Ki67 genes in the experimental group were significantly increased, while the expression of P16 was significantly downregulated. This suggests that the proliferation capacity of umbilical cord mesenchymal stem cells treated with the small molecule composition was enhanced, the senescence process was inhibited, and the senescent state was reversed.

[0140] Therefore, the method of reversing aging using the small molecule composition specified in this application can delay / inhibit the aging process of umbilical cord mesenchymal stem cells and weaken / reverse the aging state of cells.

[0141] Example 8

[0142] In the reversible cell senescence storage solution described in this embodiment, the concentration ranges of each small molecule are as follows: CHIR99021 1mM, E-616452 10mM, TTNPB 0.2mM, SAG 0.05mM, ABT-869 1mM, DZNep 0.02mM, and Ruxolitinib 0.1mM.

[0143] The reversible cell senescence storage solution described in this embodiment, after being diluted with a suitable basal culture medium for cell culture, can be used for the reverse culture of senescent cells.

[0144] Example 9

[0145] In the storage solution for reversible cell senescence described in this embodiment, the concentration ranges of each small molecule are as follows: CHIR99021 10mM, E-616452 1mM, TTNPB 2mM, SAG 0.5mM, ABT-869 0.1mM, DZNep 0.002mM, and Ruxolitinib 1mM.

[0146] The reversible cell senescence storage solution described in this embodiment, after being diluted with a suitable basal culture medium for cell culture, can be used for the reverse culture of senescent cells.

[0147] Example 10

[0148] In the reversible cell senescence storage solution described in this application, the WNT / β-catenin agonist, TGF-β receptor inhibitor, RAR agonist, Smoothened receptor agonist, VEGFR and PDGFR family multi-target inhibitors, histone methyltransferase inhibitors, and JAK1 / 2 inhibitors are functional components, and the above functional components can also play a cell reversal role on their own.

[0149] Based on the composition and ratio of the composition in Example 2 above, this embodiment verified the cell reversal effect of seven compositions without the addition of the corresponding CHIR99021 (1mM), E-616452 (1mM), TTNPB (0.2mM), SAG (0.05mM), ABT-869 (0.1mM), DZNep (0.002mM) or Ruxolitinib (0.1mM). After 5 days of induction culture of lung progenitor cells, improved cell morphology was observed.

[0150] Similarly, this embodiment, based on the composition and ratio of the composition in the aforementioned embodiment 2, verified the cell reversal effect of five compositions without the addition of TTNPB+ABT869, TTNPB+CHIR99021, TTNPB+Ruxolitinib, TTNPB+SAG, and DZNep+Ruxolitinib. After 5 days of induction culture of lung progenitor cells, improved cell morphology was observed.

[0151] Example 11

[0152] Based on the composition ratio of the composition in Example 2 above, this embodiment verifies the effects of the following combinations of different components: WNT / β-catenin agonist, TGF-β receptor inhibitor, RAR agonist, smoothed receptor agonist, VEGFR and PDGFR family multi-target inhibitors, histone methyltransferase inhibitors, and JAK1 / 2 inhibitors.

[0153] The WNT / β-catenin agonist includes at least one of MAY-262611, CHIR98014, CHIR99021, LiCl, Li2CO3, TD114-2, AZD2858, AZD1080, BIO, Kenpaullone, TWS119, LY2090314, CBM1078, SB216763, SKL2001, or AR-A014418; and / or,

[0154] The TGF-β receptor inhibitors include at least one of LY2109761, Pirfenidone, Repsox (E-616452), SB431542, A77-01, Tranilast, Galunisertib (LY2157299), A8301, GW788388, ITD-1, SD208, SB525334, LY364947, ASP3029, D4476, or SB505124;

[0155] The RAR agonist includes at least one of TTNPB, Bexarotene, Ch55, Tamibarotene, Retinol, AM580, ATRA, 13-cisRA, BMS493, Adapalene, Vitamin A, or a Vitamin A derivative.

[0156] The smoothed receptor agonist includes at least one of SAG, SAG-d3, SAG hydrochloride, SAG dihydrochloride, or (Rac)-SAG.

[0157] The VEGFR and PDGFR family of multi-target inhibitors include at least one of ABT-869, PP121, Vorolanib, Ki20227, SU11652, Sorafenib, Regorafenib, Sunitinib, Nintedanib, Ponatinib, Axitinib, Pazopanib, or SU 5402.

[0158] The histone methyltransferase inhibitors include at least one of EPZ004777, EPZ5676, GSK503, BIX01294, DZNep, DZNep·HCL, SGC0946, or Chaetocin.

[0159] The JAK1 / 2 inhibitors include at least one of Ruxolitinib, iJak-381, Tuspetinib, JAK1 / 2-IN-1, JAK-IN-1, Cerdulatinib, or JAK / HDAC-IN-2.

[0160] The above-mentioned optional functional ingredients can all contribute to the reversal and improvement of aging cells.

[0161] It is evident that the composition described in this application has a significant effect on reversing senescent cells.

[0162] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A composition for reversing cellular senescence, characterized by, comprises at least one of the following components in the following molar amounts: WNT / p-catenin agonist 1-10 molar parts, TGF-p receptor inhibitor 1-10 molar parts, RAR agonist 0.2-2 molar parts, Smoothened receptor agonist 0.05-0.5 molar parts, VEGFR and PDGFR family multi-target inhibitor 0.1-1 molar parts, histone methyltransferase inhibitor 0.002-0.02 molar parts, JAK1 / 2 inhibitor 0.1-1 molar parts.

2. The composition for reversing cellular senescence according to claim 1, wherein: the WNT / p-catenin agonist comprises at least one of MAY-262611, CHIR98014, CHIR99021, LiCl, Li2CO3, TD114-2, AZD2858, AZD1080, BIO, Kenpaullone, TWS119, LY2090314, CBM1078, SB216763, SKL2001, or ARA014418; and / or, the TGF-p receptor inhibitor comprises at least one of LY2109761, Pirfenidone, Repsox (E-616452), SB431542, A77-01, Tranilast, Galunisertib (LY2157299), A8301, GW788388, ITD-1, SD208, SB525334, LY364947, ASP3029, D4476, or SB505124; and / or, the RAR agonist comprises at least one of TTNPB, Bexarotene, Ch55, Tamibarotene, Retinol, AM580, ATRA, 13-cisRA, BMS493, Adapalene, Vitamin A, or Vitamin A derivative; and / or, the Smoothened receptor agonist comprises at least one of SAG, SAG-d3, SAG hydrochloride, SAG dihydrochloride, or (Rac)-SAG; and / or, the VEGFR and PDGFR family multi-target inhibitor comprises at least one of ABT-869, PP121, Vorolanib, Ki20227, SU11652, Sorafenib, Regorafenib, Sunitinib, Nintedanib, Ponatinib, Axitinib, Pazopanib, or SU 5402; and / or, the histone methyltransferase inhibitor comprises at least one of DZNep, BIX01294, EPZ004777, GSK343, BIX01294, DZNeP, EPZ-6438, GSK343, or UNC0223; and / or, the JAK1 / 2 inhibitor comprises at least one of TG101348, Baricitinib, Filgotinib, Cibisatinib, Pacritinib, Momelotinib, Oclacitinib, Ruxolitinib, Fedratinib, or PF-06651600. The histone methyltransferase inhibitor comprises at least one of EPZ004777, EPZ5676, GSK503, BIX01294, DZNep, DZNep·HCL, SGC0946 or Chaetocin; and / or, The JAK1 / 2 inhibitor comprises at least one of Ruxolitinib, iJak-381, Tuspetinib, JAK1 / 2-IN-1, JAK-IN-1, Cerdulatinib or JAK / HDAC-IN-2.

3. The composition for reversing cellular senescence according to claim 1 or 2, wherein The components include WNT / β-catenin agonist CHIR99021 1-10 molar parts, TGF-β receptor inhibitor E-616452 1-10 molar parts, RAR agonist TTNPB 0.2-2 molar parts, Smoothened receptor agonist SAG 0.05-0.5 molar parts, VEGFR and PDGFR family multi-target inhibitor ABT-869 0.1-1 molar parts, histone methyltransferase inhibitor DZNep 0.002-0.02 molar parts, JAK1 / 2 inhibitor Ruxolitinib 0.1-1 molar parts.

4. A storage solution that reverses cellular senescence, characterized in that, The composition for reversing cellular senescence comprises the composition for reversing cellular senescence according to any one of claims 1-3, and a pharmaceutically acceptable solvent or diluent.

5. The storage solution for reversing cellular senescence of claim 4, wherein, The storage solution comprises the components in the following concentrations: WNT / β-catenin agonist CHIR99021 1-10 mM, TGF-β receptor inhibitor E-616452 1-10 mM, RAR agonist TTNPB 0.2-2 mM, Smoothened receptor agonist SAG 0.05-0.5 mM, VEGFR and PDGFR family multi-target inhibitor ABT-869 0.1-1 mM, histone methyltransferase inhibitor DZNep 0.002-0.02 mM, JAK1 / 2 inhibitor Ruxolitinib 0.1-1 mM.

6. An induction medium that reverses cellular senescence, characterized in that, The base medium comprises DMEM / F12 base medium, and 5-15 vol% fetal bovine serum, 0.5-2 mM L-glutamine, 3-8 ng / mL insulin, 0.05-0.2 ng / mL epidermal growth factor, 3-8 μg / mL adenine and / or 3-8 μg / mL hydrocortisone are selectively added.

7. The reversible cell senescence induction culture medium according to claim 6, characterized in that, The volume ratio of the storage solution to the base medium is 1:100-1:100000.

8. The senescence induction medium according to claim 6 or 7, wherein the medium is a medium for inducing reverse senescence. The base medium comprises DMEM / F12 base medium, and 5-15 vol% fetal bovine serum, 0.5-2 mM L-glutamine, 3-8 ng / mL insulin, 0.05-0.2 ng / mL epidermal growth factor, 3-8 μg / mL adenine and / or 3-8 μg / mL hydrocortisone are selectively added.

9. A method of reversing cellular senescence, comprising, The step of inducing culture of the senescent somatic stem cells with the induction medium for reversing cellular senescence according to any one of claims 6-8 is included.

10. The method of claim 9, wherein the cells are human cells. The conditions of the step of inducing culture include culturing at a temperature of 35-38°C in a 5-10% CO2condition for 4-10 days.

11. The method of claim 9 or 10, wherein: the inducing culturing step is performed at 0-24 hours after the seeding of the somatic stem cells; and / or, the inducing culturing step further comprises a step of replacing the inducing medium every 2-3 days. the cells are somatic stem cells, including lung precursor cells, kidney precursor cells and / or mesenchymal stem cells, of human and / or animal origin; optionally, the lung precursor cells are derived from bronchial basal layer cells of healthy adults or patients; and / or, 12. The method of reversing cellular senescence according to any one of claims 9-11, wherein, optionally, the kidney precursor cells are derived from urine of healthy or diseased patients; and / or, optionally, the mesenchymal stem cells are derived from mesenchymal stem cells of the umbilical cord of caesarean section neonates.

13. Use of the composition of any one of claims 1-3, the storage solution of any one of claims 4-5, and / or the inducing medium of any one of claims 6-8 for the preparation of a product having at least one of the following effects (1)-(5): (1) inhibiting the expression of P16 or P21 or a combination thereof in cells; (2) inhibiting the activity of methyltransferase in cells; (3) increasing the expression of early reprogramming factor OCT4; (4) inhibiting inflammatory infiltration and improving regenerative potential; (5) delaying the aging process and treating aging-related diseases.

14. Use of the composition of any one of claims 1-3, the storage solution of any one of claims 4-6, the inducing medium of any one of claims 7-9, and / or the method of any one of claims 10-13 in the field of cell, organ transplantation therapy and cell derivative therapy. ​ ​

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