CN117625550A

CN117625550A - Kit for preparing anti-aging reprogrammed Cheng Gan cells and preparation method

Info

Publication number: CN117625550A
Application number: CN202311484327.6A
Authority: CN
Inventors: 约翰·泰勒·徐
Original assignee: Australian Health Industry Co Pty Ltd
Current assignee: Australian Health Industry Co Pty Ltd
Priority date: 2023-10-24
Filing date: 2023-11-09
Publication date: 2024-03-01
Also published as: AU2023254901B1

Abstract

The invention discloses a kit for preparing anti-aging reprogramming Cheng Gan cells and a preparation method thereof. The kit comprises the following reagent A and reagent B: reagent a is a reprogramming factor: comprises three transcription factors of Nr5a2, tbx3 and Sall 4; reagent B is a cell culture reagent: media and growth factors for maintaining and culturing stem cells. The kit for preparing the anti-aging reprogrammed Cheng Gan cells can reprogram adult cells derived from skin into stem cells, and the anti-skin aging activity test of the reprogrammed stem cells proves that the reprogrammed Cheng Gan cells prepared by the kit have the activity of inhibiting the aging of human dermal fibroblasts, and the reprogrammed cells can be used for preparing anti-skin aging medicines or cosmetics for resisting skin aging.

Description

Kit for preparing anti-aging reprogrammed Cheng Gan cells and preparation method

Technical Field

The invention relates to the field of biotechnology, in particular to a kit for preparing anti-aging reprogrammed Cheng Gan cells and a preparation method thereof.

Background

The stem cells have self-renewal and multidirectional differentiation potential, are seeds for realizing cell regeneration, and the organism can realize cell renewal through proliferation and differentiation of autologous stem cells; however, the stem cell number of each tissue organ gradually decreases with the increase of the age, and the proliferation and differentiation capacity also decreases, so that the damaged tissue organ cannot be repaired in time, and the aging or disease of the human body occurs. Stem cell therapy, one of cell therapies, offers promise for human life prolongation, and has been preliminarily confirmed in anti-aging experiments of adipose-derived stem cells, bone marrow mesenchymal stem cells.

These cells either produce new stem cells under specific conditions or differentiate to form new functional cells in a program that maintains the dynamic balance of tissue and organ growth and deterioration, manifesting as aging when the progression of deterioration is greater than the capacity to regenerate; if the cell regeneration capacity is stronger, the progress of tissue senescence will be delayed or even blocked. It can be seen that the anti-aging effect of stem cells is exerted depending on whether a sufficient number of desired stem cells can be mobilized.

The stem cells are anti-aging, except that the stem cells are multidirectional differentiated into tissue cells under the action of tissue microenvironment to replace cells which die after aging, the stem cells have strong secretion function, secrete some growth factors and cytokines, promote the capability of resisting free radicals of organisms, play the roles of promoting angiogenesis and cell proliferation and differentiation, inhibiting inflammatory reaction and chemotaxis, regulating cell adhesion and migration, stimulate regeneration and repair functions of the tissue cells, accelerate wound healing and tissue remodeling, and achieve the aim of resisting aging.

The exogenous adult stem cells which can be used for resisting aging mainly comprise adipose stem cells, bone marrow mesenchymal stem cells and mesenchymal stem cells derived from gestational tissues; highly undifferentiated embryonic stem cells also have a small number of applications, with multipotent stem cells of a wide range of sources and no ethical issues involved, becoming the most widely studied and used stem cell species internationally today.

Stem cells can secrete cytokines such as epidermal growth factor (promote synthesis of tissue fibers and collagen, strengthen fiber structure, improve cell metabolism, delay skin aging), vascular endothelial growth factor (promote angiogenesis, enrich vascular network, improve the nutritional status of tissue organs and skin), nerve cell growth factor (promote development, differentiation, growth, regeneration and expression of functional properties of central and peripheral neurons), fibroblast factor (greatly improve proliferation capacity of cells), etc.; the stem cells can secrete cell outer membrane vesicles, such as exosomes and the like, and the exosomes contain a large amount of protein factors and non-coding RNA, so that the stem cells can promote the repair of various tissues and organs of the organism and delay aging.

It was previously mentioned that stem cell depletion is the root cause of aging in the body, and stem cells do combat aging through a variety of functions, so scientists have proposed stem cell therapy to promote injury repair, tissue regeneration and combat aging by reinfusion of autologous-derived stem cells into the body.

Cells currently available for stem cell therapy include embryonic stem cells/induced pluripotent stem cells, adult stem cells such as adipose stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow mesenchymal stem cells, and the like.

Stem cell technology is always a social focus of attention in terms of anti-aging and extending health life, with great promise and potential. However, the existing stem cell anti-aging technology has the following defects:

(1) Safety problem: stem cell therapy involves the transplantation and implantation of stem cells, with potential safety issues. These problems include immune rejection after cell transplantation, risk of ectopic transplantation, cellular aberration or uncontrolled proliferation, etc. Before applying stem cells to anti-aging treatment, safety and reliability of the stem cells must be ensured.

(2) Operational complexity: the stem cell culturing, expanding and transplanting processes are complicated, and highly precise operations and conditions are required. The complexity of these procedures increases the difficulty and risk of treatment.

(3) Persistence of effects: the persistence of the current anti-aging therapeutic effect on stem cells is not clear. Stem cell therapy may require multiple injections or transplants to maintain its effect, while long-term efficacy still requires further investigation.

(4) Individual differences: the constitution and physiological state of each individual are different, and there may be individual differences in response to stem cell anti-aging treatment. Thus, there is a need for personalized treatment regimens, rather than a universal method of incisional utility.

Reprogramming a cell refers to the process of converting one cell type into another cell type by manually intervening in the gene expression and epigenetic status of the cell. During this process, the specific gene expression pattern and function of the cell is altered from a differentiated state back to a state similar to that of a stem cell, thereby having the ability to differentiate in multiple directions.

Disclosure of Invention

In view of this, the present invention proposes a kit for preparing anti-aging Cheng Gan cells and a preparation method thereof.

The invention provides a kit for preparing anti-aging reprogramming Cheng Gan cells, which comprises the following reagent A and reagent B:

reagent a is a reprogramming factor: comprises three transcription factors of Nr5a2, tbx3 and Sall 4;

reagent B is a cell culture reagent: comprising a medium and growth factors for maintaining and culturing stem cells.

Optionally, the kit further comprises the following reagent C: the reagent C is an anti-aging active ingredient: comprising antioxidants, cell cycle regulatory factors, DNA repair enzymes or apoptosis inhibitors.

Optionally, in the reagent B:

the culture medium for maintaining and culturing the stem cells is DMEM/F12 or DMEM basal medium;

the growth factor is one or more selected from basic fibroblast growth factor, epidermal growth factor, insulin growth factor-1, TGF-beta inhibitor and SIRT1 activator.

Optionally, in the reagent C:

the antioxidant is one or more selected from vitamin C, vitamin E, lipoic acid and polyphenol compounds;

the cell cycle regulatory factor is a p16.sup.INK4a inhibitor or a Telomerase activator;

the apoptosis inhibitor is a Bcl-2 family inhibitor or a Caspase inhibitor.

Optionally, the reagent a and reagent B are packaged separately.

Optionally, the reagent a, reagent B and reagent C are packaged separately.

The application of the kit for preparing anti-aging reprogramming Cheng Gan cells in preparing anti-aging products also belongs to the protection scope of the invention.

The invention also provides a preparation method of the anti-aging reprogrammed Cheng Gan cell, which comprises the following steps:

(1) Obtaining adult cells from skin tissue of an individual;

(2) Introducing a transcription factor into the adult cell obtained in the step (1) to obtain an intermediate adult cell; the transcription factors comprise three transcription factors of Nr5a2, tbx3 and Sall 4;

(3) Culturing the intermediate adult cells in a medium and growth factors for maintaining and culturing stem cells, causing cell reprogramming of the intermediate adult cells;

(4) Screening the stem cells subjected to reprogramming to obtain the reprogrammed Cheng Gan cells for resisting aging.

Optionally, the method further comprises the step of adding an anti-aging active ingredient after obtaining the reprogrammed Cheng Gan cells for anti-aging.

Optionally, in the step (1): the individual is a human or mouse; the adult cells are: skin fibroblasts, keratinocytes or adipocytes.

Optionally, in the step (3): the culture medium for maintaining and culturing the stem cells is DMEM/F12 or DMEM basal medium; the growth factor is selected from alkaline adultsOne or more of fibroblast growth factor, epidermal growth factor, insulin growth factor-1, TGF-beta inhibitor, SIRT1 activator; the culture conditions are as follows: in an in vitro incubator at 36-38 ℃, 4-6% CO ₂ Is cultured under an atmosphere of (2).

Optionally, the anti-aging active ingredient comprises an antioxidant, a cell cycle modulator, a DNA repair enzyme, or an inhibitor of apoptosis.

Further optionally, the antioxidant is selected from one or more of vitamin C, vitamin E, lipoic acid, a polyphenol compound; the cell cycle regulatory factor is a p16.sup.INK4a inhibitor or a Telomerase activator; the apoptosis inhibitor is a Bcl-2 family inhibitor or a Caspase inhibitor.

The obtained reprogrammed Cheng Gan cells also belong to the protection scope of the invention.

The invention also provides a composition for anti-aging comprising said reprogrammed Cheng Gan cells.

The kit for preparing anti-aging reprogrammed Cheng Gan cells provided by the invention can reprogram adult cells derived from skin into stem cells. The reprogramming factors Nr5a2, tbx3 and Sall4 in the kit have a plurality of common gene targets, which shows that the three transcription factors have synergistic effect and jointly mediate reprogramming. The reprogramming Cheng Gan cells prepared by the invention have the activity of inhibiting the aging of human dermal fibroblasts, and can be used for preparing anti-skin-aging medicines or cosmetics for resisting skin aging through the anti-skin-aging activity test of reprogramming stem cells.

Drawings

For purposes of illustration and not limitation, the invention will now be described in accordance with its preferred embodiments, particularly with reference to the accompanying drawings, in which:

FIG. 1 is the result of staining nuclei (400X) with DAPI nuclear stain.

FIG. 2 shows the results of observation of cells by a fluorescence microscope.

Detailed Description

General definition:

The singular terms "a" and "an" include plural referents unless the context clearly dictates otherwise. As an example, reference to "a cell" refers to one or more cells, and reference to "the method" includes reference to equivalent steps and methods disclosed herein and/or known to those of skill in the art, and the like. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.

As used herein, the term "comprising" is used in reference to compositions, methods, and their corresponding components that are essential to the methods or compositions, but is still open to inclusion of unspecified elements, whether or not necessary.

The term "consisting of … …" refers to compositions, methods and their corresponding components as described herein, excluding any elements not recited in the description of the embodiments.

The term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

The term "cell" as used herein refers to a single cell, a cell line, or a culture derived from such cells.

As used herein, the term "reprogramming" refers to a process of changing or reversing the differentiation state of a cell in culture or in vivo, as compared to the differentiation state that a cell would have under the same conditions without reprogramming.

1. Skin-derived adult cells

Skin-derived adult cells refer to cells in the mature, non-stem cell state isolated and cultured from human or animal skin tissue. Skin is one of the largest organs of the human body, and includes a plurality of cell types, so that a variety of adult cells can be isolated from the skin.

In some embodiments, the skin-derived somatic cells are skin fibroblasts.

In some embodiments, the skin-derived adult cells are keratinocytes.

In some embodiments, the skin-derived adult cells are adipocytes.

Skin-derived adult cells can be obtained by skin biopsy or surgical excision and can be cultured and expanded in the laboratory.

In some embodiments, the method of preparing a skin fibroblast is as follows:

(1) Skin biopsy: biopsies are taken from an individual's skin area, typically during a surgical or clinical diagnostic procedure. Biopsy samples should be collected as ethical rules and manipulated by a physician.

(2) Cell separation: fibroblasts were isolated from skin biopsy samples. This can be accomplished by cutting the skin tissue and then digesting the tissue with enzymes to release the cells. The released cells may be obtained by centrifugation.

(3) Cell culture: the isolated skin fibroblasts are transferred to a cell culture dish for growth and expansion of the cells in the culture medium. The formulation of the medium typically includes appropriate nutrients, growth factors and supplements.

(4) Culture conditions: the skin fibroblasts need to be cultured in a constant temperature incubator at 37℃under a moist environment. The culture dish is typically coated with a cell adhesive to aid in the attachment of cells to the surface of the dish.

(5) Cell expansion: skin fibroblasts will divide and expand under culture conditions to obtain a sufficient number of cells for subsequent experiments.

2. Reprogramming factors

Reprogramming factors are a class of transcription factors that are capable of reprogramming differentiated cells to Induce Pluripotent Stem Cells (iPSCs). These factors play a key role in iPSC technology, enabling the adult cells to return to a stem cell-like state, thus providing the potential for differentiation into a variety of different cell types.

In some embodiments, the reprogramming factor is an Nr5a2 transcription factor. Nr5a2 (Nuclear receptor subfamily 5group A member 2) is a member of the nuclear receptor family, also known as LRH-1 (liver receptor homolog-1). The role of Nr5a2 in the transcription factor network is closely related to maintaining pluripotency, which can promote the multipotency potential of cells.

In some embodiments, the reprogramming factor is a Tbx3 transcription factor. Tbx3 (T-box transcription factor 3) is a member of the T-box transcription factor family, and plays an important role in embryonic development and maintenance of stem cell status.

In some embodiments, the reprogramming factor is a Sall4 transcription factor. Sall4 (Sal-like protein 4) is a key embryonic stem cell marker belonging to the SALL family.

In some embodiments, the reprogramming factors are selected from at least two transcription factors in Nr5a2, tbx3, or Sall 4.

In some embodiments, the reprogramming factors comprise Nr5a2, tbx3, and Sall4 transcription factors.

In some embodiments, the reprogramming factors are a combination of three transcription factors Nr5a2, tbx3, and Sall 4.

In some embodiments, the transcription factor is prepared by genetic engineering methods, as follows:

(1) Cloning of the genes: first, the gene sequence of the target transcription factor needs to be cloned from the corresponding source. This can be achieved by PCR amplification or synthesis of gene fragments.

(2) Cloning into expression vector: the obtained gene sequence is cloned into an appropriate expression vector, typically a plasmid carrying a promoter, a selection marker (e.g., an antibiotic resistance gene), etc. This ensures that efficient expression of the target gene in the cell is achieved.

(3) Cell culture and transfection: the selection of target cells (e.g., cell lines, mouse embryonic fibroblasts, etc.) and the introduction of expression vectors carrying reprogramming factors into the cells is typically accomplished by transfection methods such as viral vector transfection, electroporation, etc.

(4) Screening and identification: following transfection, appropriate screening conditions (e.g., antibiotic selection) are used to identify successfully transfected cells. The expression of reprogramming factors can be detected by PCR, western blot, immunocytochemistry, and the like.

(4) Optimizing expression: for ensuring efficient expression, optimization of expression conditions, such as selection of appropriate promoters, cell culture conditions, etc., may be required.

3. Medium and growth factors for maintaining and culturing stem cells

In some embodiments, the medium used to maintain and culture the stem cells is DMEM/F12 or DMEM basal medium.

DMEM (Dulbecco's Modified Eagle Medium) is a commonly used cell culture medium that is widely used for culturing a variety of different types of mammalian cells. The specific composition of DMEM basal medium may vary depending on vendor and custom requirements, but typically includes the following components:

amino acid: providing amino acids such as glutamine, lysine, etc. required for cell growth.

Sugar: such as glucose, as a source of cellular energy.

Vitamins: including B vitamins and vitamin C, etc.

Salts: such as sodium, potassium, calcium, magnesium, etc., maintain the ionic balance of the cell.

Buffer solution: the pH of the medium was maintained stable.

Preservative: such as penicillin and streptomycin, to prevent bacterial contamination.

DMEM/F12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) is a cell culture medium commonly used to culture a variety of cell types including tumor cells, primary cells, stem cells, and the like. The DMEM/F12 culture medium is prepared by mixing two different culture media, namely, the DMEM and Ham's F-12 culture medium, and aims to provide a wide range of nutrients so as to meet the requirements of various cells. The specific composition of the DMEM/F12 medium may vary depending on vendor and custom requirements, but typically includes the following components:

DMEM (Dulbecco's Modified Eagle Medium): amino acids, sugars, salts, etc., required for cell growth are provided.

Ham's F-12 Medium: amino acids, sugars, salts, and the like, which are required for cell growth, are also provided.

L-Glutamine: providing amino acids required by the cell.

Salts: such as sodium, potassium, calcium, magnesium, etc.

Vitamins: including B vitamins and vitamin C.

Sugar: such as glucose.

Supplement: such as L-cystine, selenium, etc.

In some embodiments, the growth factor is selected from one or more of basic fibroblast growth factor, epidermal growth factor, insulin growth factor-1, TGF- β inhibitor, SIRT1 activator.

Basic fibroblast growth factor (bFGF, basic Fibroblast Growth Factor) bF is a multifunctional growth factor that has the effects of promoting proliferation, self-renewal and cell survival in a variety of cell types.

Epidermal growth factor (EGF, epidermal Growth Factor) is a growth factor that primarily affects the growth, differentiation and repair of epithelial cells. It has stimulating effect on proliferation and migration of epithelial cells, and can also be involved in wound healing and epidermal regeneration.

Insulin growth factor-1 (IGF-1, insulin-like Growth Factor 1) is a growth factor similar in structure to insulin and has a regulatory effect on cell growth, proliferation and survival. It is involved in cell signaling and metabolic regulation in a variety of cell types.

TGF-beta inhibitors (Transforming Growth Factor-beta Inhibitor) are a class of cytokines involved in regulating cell differentiation, proliferation and collagen synthesis. TGF-beta inhibitors are useful for blocking TGF-beta signaling pathways, thereby modulating cellular behavior and function.

SIRT1 activator (Sirtuin 1) is a protein deacetylase associated with growth, metabolism and aging, and has effects of regulating cellular metabolism and resisting oxidation. SIRT1 activators may promote SIRT1 activity, potentially affecting cell health and longevity.

In some embodiments, the growth factors are added in the following amounts: 2-5% of alkaline fibroblast growth factor, 5-10% of epidermal growth factor, 1-2% of insulin growth factor-1, 3-5% of TGF-beta inhibitor and 2-5% of SIRT1 activator.

4. Anti-aging active ingredient

In some embodiments, an anti-aging active ingredient: comprising antioxidants, cell cycle regulatory factors, DNA repair enzymes or apoptosis inhibitors.

In some embodiments, the antioxidant is selected from one or more of vitamin C, vitamin E, lipoic acid, a polyphenol compound.

Vitamin C (ascorbic acid) is a water-soluble vitamin, is a powerful antioxidant, and can help neutralize free radicals and reduce damage to cells caused by oxidative stress.

Vitamin E: is a fat-soluble vitamin and an antioxidant, and can protect cell membrane and lipid components from oxidative damage. It helps to maintain the stability of the cell membrane and protect the cell from free radicals and oxidative stress.

Lipoic acid is a natural organic sulfur compound with antioxidant and anti-inflammatory properties. It can help cells cope with oxidative stress, promote anti-inflammatory responses, and participate in the regulation of various cell signaling pathways.

Polyphenols are a natural product, including anthocyanin, catechin, flavone, etc. They are potent antioxidants, which neutralize free radicals and reduce oxidative stress and cell damage.

In some embodiments, the cell cycle regulatory factor is a p16.sup.INK4a inhibitor or a Telomerase activator.

In some embodiments, the apoptosis inhibitor is a Bcl-2 family inhibitor or a Caspase inhibitor.

In some embodiments, the anti-aging active ingredients are added in the following amounts: 1 to 2 percent of vitamin C, 1 to 2 percent of vitamin E, 2 to 5 percent of lipoic acid, 3 to 6 percent of flavone, 0.25 to 0.75mM of p16INK4a inhibitor and 3 to 6mM of Caspase inhibitor.

5. Kit for preparing anti-aging reprogrammed Cheng Gan cells

The kit for preparing anti-aging reprogrammed Cheng Gan cells comprises the following reagents A and B:

reagent B is a cell culture reagent: media and growth factors for maintaining and culturing stem cells.

In some embodiments, the kit further comprises reagent C; the reagent C is an anti-aging active ingredient: comprising antioxidants, cell cycle regulatory factors, DNA repair enzymes or apoptosis inhibitors.

In some embodiments, the agent a and agent B are packaged separately.

In some embodiments, the agent a, agent B, and agent C are packaged separately.

6. Preparation method of anti-aging reprogrammed Cheng Gan cells

The preparation method of the anti-aging reprogrammed Cheng Gan cell provided by the invention comprises the following steps:

(1) Obtaining adult cells from skin tissue of an individual;

In some embodiments, the method further comprises the step of adding an anti-aging active ingredient after obtaining the reprogrammed Cheng Gan cell for anti-aging.

In some embodiments, the individual is a human or mouse; the adult cells are: skin fibroblasts, keratinocytes or adipocytes.

In some embodiments, the medium used to maintain and culture stem cells is DMEM/F12 or DMEM basal medium.

In some embodiments, the culturing conditions are: in an in vitro incubator at 36-38 ℃, 4-6% CO ₂ Is cultured under an atmosphere of (2).

In some embodiments, the anti-aging active ingredient comprises an antioxidant, a cell cycle modulator, a DNA repair enzyme, or an inhibitor of apoptosis.

In some embodiments, culturing the somatic cells in the presence of a transcription factor causes at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the somatic cells to be reprogrammed to stem cells.

Transcription factors are molecules that, upon contact with a cell (e.g., expressed by the cell, transformed into the cell for expression, provided exogenously to the cell, etc.), can cause reprogramming, alone or in combination with other molecules. Transcription factors may be provided from exogenous sources, e.g., by addition to a culture medium, and may be introduced into cells by methods known in the art, such as by coupling to a cell entry peptide, protein or nucleic acid transfection agents, lipofection, electroporation, biolistics particle delivery system (biolistic particledelivery system) (gene gun), microinjection, and the like. In certain embodiments, the transcription factor is added to the culture medium without coupling to any other components.

In certain embodiments, the adult cells are cultured in the presence of the transcription factor for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 days, 1.5 months, 2 months, or 3 months. In certain embodiments, the adult cells are cultured in the presence of the transcription factor for no more than 3 months, 2 months, 1.5 months, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day. In certain embodiments, the adult cells are cultured in the presence of the transcription factor for 1 day to 3 months, 1 day to 2 months, 1 day to 1 month, 1 day to 25 days, 1 day to 20 days, 1 day to 19 days, 1 day to 18 days, 1 day to 17 days, 1 day to 16 days, 5 days to 16 days, 7 days to 16 days, 8 days to 16 days, 9 days to 16 days, 10 days to 16 days, 11 days to 16 days, 12 days to 16 days, 1 day to 15 days, 1 day to 14 days, 1 day to 13 days, 1 day to 12 days, 2 days to 12 days, 3 days to 12 days, 4 days to 12 days, 5 days to 10 days, 6 days to 10 days, 7 days to 10 days.

7. Reprogramming stem cells

In one aspect, the invention discloses a reprogrammed Cheng Gan cell prepared according to the method provided herein.

In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% are reprogrammed Cheng Gan cells.

In one aspect, the invention provides a composition comprising a reprogrammed Cheng Gan cell prepared according to the method provided herein. The composition may include one or more pharmaceutically acceptable carriers and diluents.

The term "pharmaceutically acceptable" means that the indicated carrier, vehicle, diluent, excipient and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with its recipient. Pharmaceutically acceptable carriers for the disclosed pharmaceutical compositions can include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agents, chelating/dispersing agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

In some embodiments, the compositions described herein may further comprise components that facilitate implantation. The compositions described herein may be pyrogen-free or substantially pyrogen-free and pathogen-free, wherein the pathogen comprises bacterial contaminants, mycoplasma contaminants, and viruses.

In some embodiments, compositions comprising recombinant stem cells described herein may further comprise an immunosuppressant or an tolerizing agent.

EXAMPLE 1 preparation of anti-aging reprogrammed Stem cells

1. Preparation of fibroblast derived from mouse skin

Materials:

c57BL/6 mice (healthy, age-appropriate) (from Australian BioResources (ABR));

centrifuge tubes and culture dishes;

DMEM (Dulbecco's Modified Eagle's Medium);

fetal bovine serum (Fetal Bovine Serum, FBS);

Penicillin/Streptomycin (Penicillin/Streptomycin);

0.25% trypsin-EDTA solution;

PBS (phosphate buffered saline).

The steps are as follows:

mice were euthanized: mice were euthanized according to ethical and legal regulations and ensured to minimize pain and discomfort.

Alcohol sterilization: the mice were placed under sterile conditions, the coat of the mice was smeared with alcohol, and then the mice were placed on a clean bench.

Scissors and forceps sterilization: the skin of the mice was cut open using sterilized scissors and forceps, exposing the internal tissues.

Tissue sampling: subcutaneous fat and muscle tissue of mice were carefully removed and placed in a petri dish containing PBS.

Tissue separation: the tissue was cut into small pieces (about 1-2 mm) using scissors and forceps, and the pieces were placed into centrifuge tubes containing 0.25% trypsin-EDTA solution.

Tissue digestion: the tubes containing the tissue pieces are placed in a 37℃incubator and digested, typically for 15-30 minutes. Periodically, the tube was gently shaken to ensure adequate dissociation of the tissue mass.

Stopping digestion: an equal volume of DMEM medium containing 10% FBS was added to neutralize the effect of trypsin.

And (3) centrifuging: the cell suspension was subjected to low-speed centrifugation at about 1000rpm for 5 minutes to pellet the cells.

Cell culture: the supernatant was decanted, the cell pellet resuspended and the cell suspension transferred to a petri dish containing complete DMEM medium and 10% FBS, 1% penicillin/streptomycin; placing the culture dish in a 37 deg.C incubator, providing proper humidity and CO ₂ Concentration, medium was changed periodically.

Cell separation: once the cells reach the proper density, cell separation can be performed using a 0.25% trypsin-EDTA solution and the cells are periodically passaged to maintain their growth.

2. Introduction of transcription factor into fibroblast derived from mouse skin

1. Preparation of transcription factor Nr5a2

1) DNA was extracted by conventional phenol/chloroform method, and the quality of DNA was measured by electrophoresis and OD260/280 measurement.

2) A pair of specific primers was designed using Primer 5 (sequence P1:5'-ATGGACCTCCGGCCTGCAG-3' (SEQ ID NO. 1), P2:5'-TTAAAGGAACGCAGTGGGCG-3' (SEQ ID NO. 2)), the mouse Nr5a2 gene sequence was PCR amplified.

20. Mu.L of the total PCR reaction system, 15-100ng of template DNA, 0.2. Mu.L of Taq polymerase (5U/. Mu.L), 0.5. Mu.L of dNTP (10 mM), 0.5. Mu.L of each primer, and MgCl ₂ (25 mM) 1.4. Mu.L, 10 Xbuffer 2. Mu.L, sterilized double distilled water was added to 20. Mu.L. The PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 35s,35 cycles; extending at 72℃for 7min. The PCR products were subjected to 1% agarose gel electrophoresis, and the amplification results were detected by Ethidium Bromide (EB) staining.

2. Preparation of transcription factor Tbx3

2) A pair of specific primers was designed using Primer 5 (sequence P3:5'-ATGGAGAGTGGGGACGGAAGC-3' (SEQ ID NO. 3)), P4:5'-TCAGCGCTGTTTTTGGGAAC-3' (SEQ ID NO. 4)), the mouse Tbx3 gene sequence was PCR amplified.

3. Preparation of transcription factor Sall4

2) A pair of specific primers was designed using Primer 5 (sequence P5:5'-ATGGAGAGTGGGGACGGAAGC-3' (SEQ ID NO. 5)), P6:5'-TCAGCGCTGTTTTTGGGAAC-3' (SEQ ID NO. 6)), the Sall4 gene sequence of the mouse was amplified by PCR.

20. Mu.L of the total PCR reaction system, 15-100ng of template DNA, 0.2. Mu.L of Taq polymerase (5U/. Mu.L), 0.5. Mu.L of dNTP (10 mM), 0.5. Mu.L of each primer, and MgCl ₂ (25 mM) 1.4. Mu.L, 10 Xbuffer 2. Mu.LL, sterilized double distilled water was added to 20. Mu.L. The PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 35s,35 cycles; extending at 72℃for 7min. The PCR products were subjected to 1% agarose gel electrophoresis, and the amplification results were detected by Ethidium Bromide (EB) staining.

4. Preparation of lentiviral vectors

The lentiviral vector used for constructing transcription factor expression is pLenti-CMV (purchased from Biotium), C57BL/6 mouse cDNA is used as a template, the primers P1-P6 are used for amplification, and amplified PCR products are subjected to electrophoresis and then cut into gel to recover products. And (3) carrying out enzyme digestion on the PCR recovery product, and simultaneously carrying out enzyme digestion on the pLenti-CMV vector, wherein the enzyme digestion reaction condition is 37 ℃ for 1 hour. And (3) carrying out electrophoresis after enzyme cutting of the PCR product and the carrier, cutting glue, recovering the enzyme-cut product, and carrying out a connection reaction. The PCR cleavage product and the vector cleavage product were subjected to ligation reaction using T4 ligase at 18℃for 10 hours. The ligation product was transformed with Salmonella typhimurium (Salmonella enterica) (from Bio-Rad) as competent strain, the transformation procedure being:

1) Melting the competent cells on ice, adding all the connection products into competent cells, mixing the walls of the flick tube uniformly, and standing on ice for 25 minutes.

2) Heat shock is carried out for 1min at 42 ℃, and the mixture is rapidly placed on ice for incubation for 3min.

3) 500 μl of LB medium without antibiotics was added and shaken for 25 min at 37 ℃.

4) After centrifugation, 300. Mu.l of the supernatant was discarded, and the remaining suspension was then spread evenly over kanamycin-positive agar plates and incubated overnight at 37 ℃. After 12h, observing the growth condition of the bacteria, selecting single large and full bacteria to shake for 10h in LB kanamycin positive culture medium, freezing and storing one part of the bacteria liquid, sequencing the other part of the bacteria liquid, and marking the identified correct bacteria as pLenti-CMV-Nr5a2, pLenti-CMV-Tbx3 and pLenti-CMV-Sall4.

5. Lentivirus package

Material and reagent preparation:

packaging cell line: the lentiviral packaging cell line was HEK293T (purchased from millipore sigma) with good viral packaging capacity.

Plasmid DNA: including lentiviral vectors pLenti-CMV-Nr5a2, pLenti-CMV-Tbx3 and pLenti-CMV-Sall4, helper vectors (e.g., pMD2.G and psPAX 2).

Virus packaging reagent: polyvinyl alcohol (PEI).

The steps are as follows:

1) Cell culture: HEK293T cells were cultured in petri dishes to a density of 80% -90%.

2) Transfection: plasmid vectors (lentiviral vectors pLenti-CMV-Nr5a2, pLenti-CMV-Tbx3 and pLenti-CMV-Sall4, pMD2.G, psPAX 2) were mixed with a transfection reagent (PEI) to generate a DNA-transfection reagent complex.

3) The DNA-transfection reagent complex was added dropwise to the above-prepared fibroblast cells derived from mouse skin.

4) Culturing: cells were continued to be cultured in DMEM medium after transfection, allowing them to produce viral particles.

5) Collecting virus particles: after 24-48 hours, cells in DMEM medium were lysed and cell lysates were collected.

6) The cell lysate is centrifuged to remove cell debris and debris.

7) Filtration and concentration: the cell lysate is filtered through a filter membrane to remove cell debris. The virus particles were concentrated using a centrifugal concentration method.

8) Virus titer assay: the titre of the viral particles was determined using the TCID50 method.

6. Lentivirus infected mouse fibroblasts

1) Cell preparation: culturing and amplifying the prepared fibroblast from the mouse skin until the cell density reaches 70-80%.

2) Virus preparation: the lentiviral particles prepared above (containing the desired gene expression sequence) were used.

3) Viral titers were determined to determine the appropriate infectious dose.

4) Infection: the virus particles were added to DMEM medium and co-cultured with the cells. The dish was gently shaken to ensure even distribution of the virus particles.

5) Incubation: incubating the cells at the infectious dose at 37℃CO ₂ In an incubator to allow the virus to interact with the cells.

6) Medium replacement: after 24 hours following infection, the medium was changed to remove unadsorbed virus particles.

7) Cell detection: using protein mass spectrometry techniques, it was identified that the transcription factors Nr5a2, tbx3 and Sall4 had been introduced into fibroblasts derived from the skin of mice to obtain intermediate adult cells.

3. Reprogramming intermediate adult cells into stem cells

Cell culture: the intermediate adult cells obtained above were transferred into DMEM/F12 medium (1:1 mixture) and cultured for 8 to 12 hours, and growth factors (Table 1) were initially added to the medium, and in an in vitro incubator at 37℃at 5% CO ₂ Is cultured for 3 to 5 days under the atmosphere of the culture medium.

TABLE 1 growth factor composition and concentration

Culturing and maintaining: cells were further cultured in DMEM/F12 medium in the presence of the above growth factors. The morphology and growth state of the cells were observed periodically.

Adding anti-aging active ingredients: after 10 to 15 cultures, anti-aging active ingredients were added to the DMEM/F12 medium (Table 2).

TABLE 2 anti-aging active ingredient addition

Composition of the components	Concentration (volume percent%)
		Vitamin C	1％
Vitamin E	1％
		Lipoic acid	3％
Flavone	5％
		p16INK4a inhibitors	0.5mM
Caspase inhibitors	3mM

Transfer to serum-free medium: when cells begin to form stem cell-like clones, they can be transferred to serum-free medium, such as KSR (Knockout Serum Replacement) medium, to further facilitate maintenance of stem cell state.

Cell identification and validation: immunocytochemistry was used to verify whether the cells were successfully reprogrammed to stem cell state.

Immunocytochemistry (ICC) methods can detect the presence and distribution of specific proteins at the cell level, thereby identifying stem cells. The following is a method for identifying stem cells using ICC:

materials and reagents:

cell culture medium and cell culture dish

Cell fixatives (e.g., 4% Paraformaldehde)

Penetrant (e.g. 0.1% Triton X-100)

Blocking agents (e.g. 5% BSA or 5% keto acid)

Suitable primary antibodies: protein for detecting target (OCT 4)

Suitable fluorescent-labeled secondary antibodies

Fluorescent dyes (e.g. DAPI for nuclear staining)

Microscope and fluorescent microscope lens

The steps are as follows:

cell fixation: the cultured stem cells were transferred to a medium containing 4% Paraformaldyde, and the cells were fixed. The fixing time is usually 15 to 30 minutes.

Penetration: cells were permeabilized with 0.1% Triton X-100 to allow antibodies to enter the interior of the cells.

Preventing: non-specific binding was prevented by adding 5% BSA or 5% keto acid to the cells.

Primary antibody incubation: primary antibodies are added at appropriate concentrations to bind to the stem cell marker proteins to be detected. Cells were incubated at 4℃overnight or at room temperature for 1-2 hours.

Washing: cells were washed with PBS to remove unbound antibodies and byproducts.

Secondary antibody incubation: a suitable fluorescent-labelled secondary antibody is added which will bind to the primary antibody. Incubate at room temperature for 1-2 hours.

Washing: the cells were washed again with PBS to remove unbound secondary antibodies.

Nuclear staining: nuclei were stained using DAPI nuclear stain to observe the nuclei of cells.

Sealing piece: an anti-fade agent may be added to the slide, and the cells placed in the coverslip, followed by covering the slide cover.

And (3) observing by a microscope: cells were observed using a fluorescence microscope and fluorescent signals were observed under appropriate channels.

The nuclear staining (400×) results are shown in fig. 1, and cells can be seen to have blue nuclear staining (using DAPI staining). The results of fluorescence microscopy of the cells are shown in FIG. 2, where a green fluorescent label (using a fluorescently labeled secondary antibody) is visible, indicating the presence of OCT4 protein. These green signals are located around the nucleus, indicating the expression of OCT4 protein in these cells. The results demonstrate that intermediate adult cells have been reprogrammed to stem cells.

Example 2 anti-skin aging Activity test of reprogrammed Stem cells

A DMEM-HG culture medium (glucose concentration is 30 mmol/L) is used for culturing and preparing an HDFs (Human Dermal Fibroblasts, human skin fibroblasts) aging model, and the aging of the HDFs is accelerated according to the principle of high sugar environment. The cell senescence beta-galactosidase staining experiment steps are as follows:

HDFs (available from PromoCell) with good growth status were prepared at a concentration of 2×10 in DMEM-LG medium (glucose concentration 5.5 mmol/L) containing 10% fbs and 1% diab (available from CellGro) ⁵ Cell suspension/mL, inoculated in 24-well plate at 1mL per well under culture conditions of 37℃and 5% CO ₂ Saturated humidity. After 24h, the medium was aspirated, the control group was replaced with fresh DMEM-LG medium containing 10% FBS, 1% diabody, the aging model group was replaced with DMEM-HG medium (glucose concentration 30 mmol/L) containing 10% FBS, 1% diabody (purchased from CellGro), the reprogramming Cheng Gan cell group was replaced with DMEM-HG medium containing 10% FBS, 1% diabody, 100ng/mL of reprogramming Cheng Gan cells, and the culture was continued, with 3 multiple wells each. Culture was continued for 8d, during which time the medium was replaced every 2d with the corresponding medium. Staining is carried out by referring to the instruction book of the aging beta-galactosidase detection kit, and the cell staining condition is observed under an inverted microscope. The non-aged cells were not stained, but the nuclei of the aged cells were blue-green, the percentage of stained cells per field was counted for each well, 3 different fields were taken for each well, and the positive rate of beta-galactosidase staining for each group was calculated.

Data were statistically processed using GraphPad Prism 5.0 software, expressed as x±s, and comparisons between the two groups were tested using t-test, with P < 0.05 indicating significant differences.

The dyeing positive rate of the beta-galactosidase of each group is shown in table 3, and compared with the control group, the dyeing positive rate of the beta-galactosidase of the aging model group is obviously increased (P is less than 0.05), which indicates that the aging model modeling is successful; compared with the aging model group, the beta-galactosidase staining positive rate of the reprogramming stem cell group is obviously reduced (P is less than 0.05). The results indicate that the prepared reprogrammed Cheng Gan cells of the present invention can inhibit the aging activity of HDFs.

TABLE 3 positive rate of beta-galactosidase staining for each group

Group of	Positive rate of beta-galactosidase staining
		Control group	(6.38±1.95)％
Aging model group	(17.92±2.12)％
		Reprogramming stem cell groups	(6.59±2.63)％

The above-mentioned research results show that the prepared reprogrammed Cheng Gan cell group has the activity of inhibiting the aging of human dermal fibroblast, and the reprogrammed cell can be used for preparing anti-skin-aging medicines or cosmetics for resisting skin aging.

The above embodiments do not limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications, combinations, sub-combinations and alternatives can occur depending upon design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of the present invention.

Claims

1. A kit for preparing anti-aging reprogrammed Cheng Gan cells, characterized by: comprising the following reagents A and B:

2. The kit for preparing anti-aging, reprogrammed Cheng Gan cells of claim 1, wherein: the kit also comprises the following reagent C:

The reagent C is an anti-aging active ingredient: comprising antioxidants, cell cycle regulatory factors, DNA repair enzymes or apoptosis inhibitors.

3. The kit for preparing anti-aging, reprogrammed Cheng Gan cells of claim 1, wherein: in the reagent B:

4. The kit for preparing anti-aging, reprogrammed Cheng Gan cells of claim 2, wherein: among the reagents C:

the apoptosis inhibitor is a Bcl-2 family inhibitor or a Caspase inhibitor.

5. A kit for preparing anti-aging, reprogrammed Cheng Gan cells according to any of claims 1 to 3, wherein: the reagent A and the reagent B are packaged separately.

6. The kit for preparing anti-aging reprogrammed Cheng Gan cells of claim 2 or 4, wherein: the reagent A, the reagent B and the reagent C are packaged separately.

7. Use of the kit of any one of claims 1 to 6 for preparing anti-aging reprogrammed Cheng Gan cells in the preparation of a product for anti-aging.

8. A method of preparing a reprogrammed Cheng Gan cell for anti-aging comprising the steps of:

(1) Obtaining adult cells from skin tissue of an individual;

9. The method of preparing a reprogrammed Cheng Gan cell for anti-aging of claim 8, wherein: the method further comprises the step of adding an anti-aging active ingredient after obtaining the reprogrammed Cheng Gan cells for anti-aging.

10. The method of preparing a reprogrammed Cheng Gan cell for anti-aging of claim 8, wherein: in the step (1):

the individual is a human or mouse;

The adult cells are: skin fibroblasts, keratinocytes or adipocytes.

11. The method of preparing a reprogrammed Cheng Gan cell for anti-aging of claim 8, wherein: in the step (3):

the growth factors are selected from one or more of basic fibroblast growth factors, epidermal growth factors, insulin growth factor-1, TGF-beta inhibitors and SIRT1 activators;

the culture conditions are as follows: in an in vitro incubator at 36-38 ℃ with 4-6% CO ₂ Is cultured under an atmosphere of (2).

12. The method of preparing a reprogrammed Cheng Gan cell for anti-aging of claim 9, wherein: the anti-aging active ingredient comprises an antioxidant, a cell cycle regulatory factor, a DNA repair enzyme or an apoptosis inhibitor.

13. The method of preparing a reprogrammed Cheng Gan cell for anti-aging of claim 12, wherein:

The apoptosis inhibitor is a Bcl-2 family inhibitor or a Caspase inhibitor.

14. A reprogrammed Cheng Gan cell prepared by the method of any one of claims 8 to 13.

15. A composition for anti-aging comprising the reprogrammed Cheng Gan cell of claim 14.