CN106318979B

CN106318979B - Method for inducing transdifferentiation of mesenchymal stem cells into skin stem cells

Info

Publication number: CN106318979B
Application number: CN201510066904.9A
Authority: CN
Inventors: 黄兵; 殷勤伟
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-02-10
Filing date: 2015-02-10
Publication date: 2021-02-19
Anticipated expiration: 2035-02-10
Also published as: CN106318979A

Abstract

The invention discloses a method for inducing transdifferentiation of adipose-derived mesenchymal stem cells (AD-MSCs) into skin stem cells (AD-SSCs), which specifically comprises the following steps: s1, rapidly separating and purifying the homogenized adipose-derived mesenchymal stem cells; s2, selecting a plurality of small RNA molecules for epigenetic regulation of stem cells; s3, assembling and transfecting nucleic acid polypeptide nanoparticles; s4, preparing a culture medium for inducing the transdifferentiation of the mesenchymal stem cells into skin stem cells; s5, activating a variety of related genes that direct the self-renewal and transdifferentiation of skin stem cells. The invention uses human adipose-derived mesenchymal stem cells which are easy to obtain, have sufficient sources, have no ethical problems, are safe and effective as induction objects, and are transformed and differentiated into AD-SSCs rapidly and in high purity in a large scale under the action of a special serum-free culture medium by transfecting 2 small RNAs molecules. These induced skin stem cells have the same self-renewal capacity and the potential to differentiate into skin cells as skin stem cells naturally produced in the human body.

Description

Method for inducing transdifferentiation of mesenchymal stem cells into skin stem cells

Technical Field

The invention relates to the technical field of stem cell culture and transformation, in particular to a method for inducing mesenchymal stem cells to transdifferentiate into skin stem cells.

Background

White Adipose (WAT) which is a main source of Adipose-derived stem cells (AD-MSCs) is widely distributed in vivo, and abundant in reserves, and is an adult Mesenchymal Stem Cell (MSCs) which is widely distributed in different tissues, such as bone marrow, skeletal muscle, fat, cardiac muscle, liver and other different parts. The research finds that the MSCs have the following characteristics: (1) the number of adipose tissues is large, and 50ml of adipose tissue can produce about 5X 10⁵Mesenchymal stem cells, approximately stem cells obtained from bone marrow500 times the amount; (2) the materials are safe and simple to obtain, a large number of cells can be obtained under local anesthesia, and the in vitro amplification and self-renewal capacity is strong; (3) differentiation into various cell lines is possible: fat, bone, cartilage, skeletal muscle, smooth muscle, cardiac muscle, endothelium, liver cells, hematopoietic cells and neuronal cells, AD-MSCs are truly stem cells with multipotential differentiation potential; (4) can secrete a plurality of cytokines and participate in the repair, the renewal and the remodeling of skin tissues; (5) the number of AD-MSCs in adipose tissue did not decrease with increasing donor age. The AD-MSCs have strong in-vitro amplification and self-renewal capacity, and after primary culture for about 5-7 days, the cell fusion is over 90 percent, so that the cells can be stably passed for more than 20 generations. The AD-MSCs have specific surface markers of mesenchymal stem cells, and express a large amount of adhesion molecules: AD-MSCs persistently express CD9, integrins beta 1(CD29) and alpha 4(CD49d), intercellular adhesion molecule 1(ICAM-1, i.e., CD54), CD105, vascular cell adhesion molecule (VCAM, i.e., CD106), and activated lymphocyte adhesion molecule (ALCAM, i.e., CD166), expressing class I histocompatibility protein HLA-ABC. AD-MSCs do not express hematopoietic cell surface markers CD14, CD15, CD33, CD34 or CD45, class II histocompatibility antigen HLA-DR and endothelial cell marker CD31, and co-stimulatory factors B7-1(CD80), B7-2(CD86) and CD 40.

The adipose-derived mesenchymal stem cells (AD-MSCs) have the capacity of directionally differentiating into terminally differentiated cells required by tissue repair, and provide sufficient cell sources for tissue repair and tissue engineering skin construction. Researches show that AD-MSCs are treated by skin homogenate and cultured independently or co-cultured with heat shock damaged HeCat cells to induce the AD-MSCs to differentiate directionally to the epidermal cells to express CK10, CK14 and CK19, and the induced differentiation efficiency is higher than that of the AD-MSCs which are induced and differentiated to the epidermal cells by using the epidermal growth factor EGF alone. Some experimental results indicate that EGF, retinoic acid and other components are used for inducing AD-MSCs to differentiate, the cells show changes of paving stones after 10 days, the cells express CK19, differentiate towards epithelial cells, express an early surface marker of the epithelial cells, namely keratin CK18, and no longer express mesenchymal stem cell markers such as vimentin. In another group, AD-MSCs were cultured in a keratinocyte conditioned medium for 3 weeks, and were found to differentiate into keratinocytes and express CK5 and CK 14. However, in general, these methods induce less efficient transformation into skin cells, require a long period of time, and have poor cell uniformity. In particular, the key problem of how to induce efficient transdifferentiation into skin stem cells is not solved.

In addition to differentiating into various cells, AD-MSCs can secrete various cytokines such as VEGF, granulocyte/macrophage colony stimulating factor M-CSF, matrix derived factor SDF-1 alpha, HGF, IGF-1, KGF and bFGF. Animal wound model shows that TGF-beta 1[29] in ADSCs-CM promotes hyaluronic acid synthesis by increasing expression of hyaluronidase HAS-1 and HAS-2; the ADSCs-CM can also up-regulate mRNA levels of extracellular matrix I, III type collagen and fibronectin, down-regulate mRNA level of MMP-9, enhance secretion of type I collagen by HDF, stimulate collagen synthesis and fibroblast migration, and promote wound healing in vivo. The AD-MSCs are locally injected to the wound surface of a diabetic ischemia model, the VEGF content in blood plasma and tissues is increased, the area of the wound surface is obviously reduced, local new blood vessels are increased, the healing speed is accelerated, and the quality of the wound surface after healing is improved. In conclusion, AD-MSCs are capable of improving the microenvironment of skin tissue, promoting the metabolism and renewal of skin tissue cells, regulating extracellular matrix deposition and remodeling, repairing skin damage and preventing skin aging.

The AD-MSCs still maintain the characteristics of stem cells, proliferation and differentiation capacity and corresponding immunogenicity after long-term passage or frozen storage in a laboratory, have no obvious immunological characteristic change, but have the tendency of aging and deformation after multi-generation proliferation. A large number of AD-MSCs mice injected intravenously into immunodeficient mice survived without side effects and tumorigenesis. In human clinical trials, single injections of 1-4X 10⁹Cells, no side effect occurs after 36 months of follow-up visit, and tumor generation cannot be induced. In conclusion, the AD-MSCs have multidirectional differentiation potential, promote the formation of new blood vessels and accelerate the healing of wound surfaces. The AD-MSCs used for the treatment may be derived from the patient's own fat, and thus, it is expected to repair damaged tissues and renew and activate aged skin cells without causing any immune rejection. Because the AD-MSCs have sufficient sources, easy separation and safety without tumorigenicity, the AD-MSCs are used for autologous stem cell transplantationThe optimal cell source for preventing and treating skin diseases and aging is planted.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a method for inducing transdifferentiation of human adipose derived mesenchymal stem cells into skin stem cells, and the method solves the problems of low transformation rate, long required time, small number of transformed skin stem cells, low purity and the like in the existing induced stem cell transdifferentiation technology.

The purpose of the invention is realized by the following technical scheme: a method for inducing transdifferentiation of mesenchymal stem cells into skin stem cells, comprising the steps of:

s1, rapidly separating and purifying the homogenized adipose-derived mesenchymal stem cells;

s2, selecting a plurality of small RNA molecules for epigenetic regulation of stem cells;

s3, assembling and transfecting nucleic acid polypeptide nanoparticles;

s4, preparing a culture medium for inducing the transdifferentiation of the mesenchymal stem cells into skin stem cells;

s5, activating a variety of related genes that direct the self-renewal and transdifferentiation of skin stem cells.

As a preferred technical solution, the method for rapidly separating, purifying homogeneous and undifferentiated long-term expanded adipose-derived mesenchymal stem cells in step S1 further comprises the steps of:

s11, under the aseptic condition, the fine particle human adipose tissues extracted by the machine are rinsed for 3 times by PBS liquid to remove the residual blood and tissue fragments;

s12, mixing 30-60ml of crushed small adipose tissues in a ratio of 1: mixing the mixture with 1 proportion and 0.25 percent collagenase I solution, placing the mixture in a working tube of a ClinicMACS protegee tissue separating and purifying machine, adjusting the working temperature to 37 ℃, setting the rotating speed of the separating and purifying machine to be 60 revolutions per minute, and rotating positively and negatively for 15 minutes respectively;

s13, after the separation is finished, adding 10% FBS low-sugar DMEM to stop digestion, then carrying out centrifugal treatment at 1500 rpm for 10 minutes, taking precipitated cell particles, and washing the cell particles for 2 times by PBS;

s14, resuspended in low sugar DMEM/F12 containing 5-10% FBS, 5ng/ml FGF-b,5ng/ml PDGF-BB,5ng/ml IGF-1,3ng/ml HGF,3ng/ml EGF,3ng/ml VEGF,5ng/ml IL-8,0.5ng/ml TGF-beta,1ng/ml LIF,100uM Vc,100U penicillin and 100mg/L streptomycin;

s15, removing residual fibroblasts by differential adherence and removing residual mononuclear blood cells by dexamethasone.

As a preferred technical scheme, the plurality of small RNA molecules used for epigenetic regulation of the stem cells in the step S2 refer to sequences of antisense-24(anti-24) molecules and antisense-29(anti-29) molecules, target molecules of the small RNA molecules are human miR-24 and miR-29 respectively, and the target molecules can regulate a plurality of genes related to the skin stem cells and differentiated downstream functional cells thereof, particularly AKT, TCF3/4 and the like. And the weight ratio of the powder is 0.6: the mixture is prepared according to the proportion of 0.4.

The step S3 of assembling and transfecting nucleic acid polypeptide nanoparticles refers to a process of preparing different small RNA molecules and polypeptide transfection reagents according to a certain ratio, procedure and time (see the fourth embodiment), and achieves the optimal induced transformation efficiency by once-a-day transfection (first time is transfection after digestion, second time is non-digestion transfection) and at least 4 days of culture.

As a preferred technical solution, the step S4 includes the following steps: serum-free high-glucose DMEM/F12 medium was supplemented with 20ng/ml bFGF,20ng/ml EGF,10ng/ml IGF-1,5ng/ml KGF,50ng/ml WNT3a,10ug/ml Insulin, 0.5uM Retinoic Acid, 20ng/ml Delta1,0.5mM VPA, and 100uM Vc, as well as type IV collagen fibers and formed prebound-treated flasks and plates.

As a preferred technical scheme, the mesenchymal stem cells and the skin stem cells can be prepared into different culture kits according to the formulation of the medium for inducing and amplifying the mesenchymal stem cells and the skin stem cells, wherein the different culture kits comprise cell proliferation factors and cell differentiation inhibitors required by the proliferation of different mesenchymal stem cells (such as bone marrow mesenchymal stem cells, adipose mesenchymal stem cells and umbilical cord mesenchymal stem cells). The specific formulation ingredients are shown in table 1.

Preferably, the transdifferentiated skin stem cell culture is induced by the induction of skin stem cellsAmplification medium with 1-3X10⁵Cells were cultured at a density of/ml for at least 4 days.

As a preferred embodiment, the related genes in step S5 include, but are not limited to, Keratin 5/14/15/19, Notch-1, p63, α 6 β 4-and α 3 β 1-integrins, ABC transporters, c-kit/CD117, CD34, stat3, CD49f, sox9, EGFR, IGF-1/IGF-R1, WNT, Myc, TGF- β, Tcf/Lef, BMI-1, Lhx2, IKK- α, IRF6, and NFATc 1.

Compared with the prior art, the invention has the following beneficial effects:

1. can be used in large scale (1-3X 10)^8-9Cells) and rapidly (4-6 days) induces the transdifferentiation of mesenchymal stem cells into skin stem cells. Solves the technical bottle diameter problems of low conversion rate, long time, small quantity of skin stem cells after conversion and low purity in the prior induced stem cell transdifferentiation technology.

2. The transdifferentiated skin stem cells have the same self-renewal capacity and the potential of being differentiated into skin cells as the skin stem cells naturally generated in a human body, can be used for improving the skin characteristics of the human body and has the effect of delaying skin aging. The skin stem cells obtained by the method can be used for treating scalds and burns of the skin, beautifying and resisting aging of the skin and treating and repairing skin diseases.

The invention proves that the invention has better effect on inducing the transdifferentiation of the adipose-derived mesenchymal stem cells to the skin stem cells than the similar technology in the market at present through in vitro multiple experimental research and repeated detection demonstration.

Drawings

FIG. 1 is a flow chart of a method for inducing transdifferentiation of mesenchymal stem cells into skin stem cells according to the present invention;

FIG. 2 is an eosin staining pattern and FACS analysis pattern (C) of adipose-derived mesenchymal stem cells cultured in vitro at 5 th generation (A) and 15 th generation (B);

FIG. 3 is a graph showing differentiation of adipose-derived mesenchymal stem cells into adipocytes (a and b) and osteoblasts (c and d);

FIG. 4 is a sequence diagram (A) showing the molecular structure of the small RNAs (miR-24 and miR-29) and their target genes (TCF and AKT) and antisense nucleic acids (Anti-24 and Anti-29); and a luciferase relative expression level map (B) and a gene expression level map (C) under quantitative PCR analysis;

FIG. 5 is a graph showing the results of 2 days after transfection induction of adipose mesenchymal stem cells (AD-MSCs: A) (from spindle cells to round cells: B) to 4 days after transfection induction (pleomorphic skin stem cells: C) to 7 days after transfection induction (flattened skin cells: D);

FIG. 6 is a FACS analysis of transdifferentiation of adipose-derived mesenchymal stem cells into skin stem cells;

FIG. 7 is a graph showing immunostaining for a marker protein specific to skin stem cells (AD-SSCs) transdifferentiated from AD-MSCs;

FIG. 8 is a diagram of Anti-24 and Anti-29 induced transdifferentiation of adipose-derived mesenchymal stem cells (examples 1A-3A) into skin cells;

FIG. 9 is a quantitative PCR analysis chart of Anti-24 and Anti-29 induced transdifferentiation of adipose-derived mesenchymal stem cells (AD-MSCs) into skin stem cells (AD-SSCs).

Detailed Description

The following detailed description of embodiments of the invention refers to the accompanying drawings in which:

as shown in fig. 1, a method for inducing transdifferentiation of mesenchymal stem cells into skin stem cells, comprising the steps of:

s3, assembling and transfecting nucleic acid polypeptide nanoparticles;

As shown in fig. 2 to 8, the specific steps and operations are as follows:

firstly, separating, purifying and culturing human AD-MSC:

hassc isolation and culture: because the existing methods for separating and culturing the adipose-derived mesenchymal stem cells are time-consuming, labor-consuming, low in purity and easy to damage the cells, the invention develops a technology which is more time-saving, simple and convenient and has high separation and purification efficiency. The new technology is disclosed and described in detail as follows: 1) rinsing fine-particle human adipose tissues extracted by a machine for 3 times by using PBS (phosphate buffer solution) under an aseptic condition to remove residual blood and tissue fragments; 2) 30-60ml of minced adipose tissue was mixed with 1: 1, placing the solution in a working tube of a ClinicMACS Prot é (Miltenyi Biotec) tissue separating and purifying machine, adjusting the working temperature to 37 ℃, setting the rotating speed of the separating and purifying machine to 60 rpm, and rotating the separating and purifying machine forward and backward for 15 minutes respectively; 3) after separation, adding 10% FBS low-sugar DMEM to stop digestion, then centrifuging at 1500r/min for 10min, taking precipitated cell particles, and washing with PBS for 2 times; 4) resuspension in low sugar DMEM/F12 containing 5-10% FBS, 5ng/ml FGF-b,5ng/ml PDGF-BB,5ng/ml IGF-1,3ng/ml HGF,3ng/ml EGF,3ng/ml VEGF,5ng/ml IL-8,0.5ng/ml TGF-beta,1ng/ml LIF,100uM Vc,100U penicillin and 100mg/L streptomycin; 5) residual fibroblasts were removed by differential paring and residual mononuclear blood cells were removed with dexamethasone. Then, most of the cells are attached to the wall 48 hours after primary AD-MSC inoculation, and the cells except the attached cells are removed by changing the liquid; after 72h, the cell morphology changes, the cells grow polar, and the cells are vortex-shaped and river-shaped after fusion. After 2-3 generations, AD-MSCs with very uniform morphology were obtained (FIG. 2A). There was no significant change in the morphology of the cells within 20 passages (FIG. 2B). Usually we used 3 or 4 generations of AD-MSCs as the cellular material for other experiments.

Adipose mesenchymal stem cell phenotype flow cytometry (FACS) detection: after digesting the 3 rd generation AD-MSC with 0.25% trypsin, the cell concentration was adjusted to 10% with PBS containing 1% by volume of bovine serum albumin⁵The aunt/ml is digested by enzyme to prepare single cell suspension. The cells were aliquoted into 1.5ml EP tubes, 1ml per tube, to which were added the following murine anti-human monoclonal antibodies: CD31-FITC, CD34-FITC, CD45-FITC, CD33-FITC, CD44-FITC, CD73-FITC, CD90-FITC, CD29-FITC, CDl66-FITC, HLA-DR-FITC, IgG1-FITC/PE as isotype negative controls. Incubating at 4 ℃ in dark for 30min, and detecting by a flow cytometer. CD29 and CD90, CD44 and CDl66, CD73 all become positive expression, and the positive rate is more than 95%. Hematopoietic system, leukocyte, endothelial marker and tissue phaseThe capacitive complexes CD31 and CD34, CD45 and HLA-DR negative expression (see FIG. 2C).

II, identifying the induced differentiation potential of the human AD-MSC:

inducing AD-MSC to differentiate into fat cells in vitro: taking 3 rd generation AD-MSC, after the cells are fused to 90%, changing into fat induction culture medium, DMEM containing high sugar, FBS 8%, dexamethasone 1umol/L, IBMX 0.5mmol/L, indomethacin 200umol/L, and insulin 10 ug/ml. After 14 days of culture, oil red staining was performed to identify lipid droplets. After 2 weeks of adipogenic induction of AD-MSCs, lipid droplets were red when stained with oil-Red-O (FIGS. 3a and 3b), and the normal control group was negative. The oil red O dyeing method comprises the following steps: (1) washing the cell slide twice with PBS for 5min each time; (2) fixing with 4% paraformaldehyde for 15 min; (3) rinsing with PBS twice, each for 5 min; (4) rinse in 60% isopropanol. (5) Stain for 15 minutes in oil red O. Rinsing with 60% isopropanol, rinsing with double distilled water for 2 times, and observing under an inverted microscope.

Inducing AD-MSC to differentiate into osteoblasts in vitro: taking 3 rd generation AD-MSC, after the cells are fused to 90%, changing into osteoinduction group, DMEM containing high sugar, 8% FBS, 100nmol/L dexamethasone, 1mmol/L ascorbic acid and 10mmol beta sodium glycerophosphate. Alizarin red S staining was performed after 3 weeks of culture to identify calcium nodules. Alizarin red S staining method operation steps: (1) on induction day 21, the osteogenic conditioned medium was aspirated and washed 3 times with PBS; (2) alizarin is dyed for 2 min; (3) washing with distilled water for 5-10 s; (4) washing with differentiation solution for 15 s; (5) PBS was washed 3 times, then dehydrated, cleared and mounted, observed under a microscope and photographed. The calcium nodules were orange-red when alizarin red S was stained 21 days after osteogenic induction of AD-MSCs, i.e. alizarin red staining was positive (fig. 3c and 3d), and the normal control group was negative.

Sequence, structure and synthesis of small RNAs:

we screened and identified 2 miRNAs by bioinformatics techniques, small RNA chip analysis and associated prediction software (Target Scan) (see fig. 4A).

The first miRNA is miRNA-24 and Anti-sense nucleic acid Anti-24, and the sequences of the miRNA and the Anti-sense nucleic acid Anti-24 correspond to the small RNA sequences as follows: miR-24, 5-CUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCAUUUUACACACUGGCUCAGUUCAGCAGGAACAGGAG-3Anti-24, 5-GGCUGUUCCUGCUGAACUGAGCCAUU-3.

The second small RNA is miRNA-29, and the sequence of the small RNA is as follows:

5-AUGACUGAUUUCUUUUGGUGUUCAGAGUCAAUAUAAUUUUCUAGCACCAUCUGAAAUCGGUUAU-3。

Anti-29，5-GGUAACCGAUUUCAGAUGGUGCUAUU-3。

these miRNAs can be obtained by chemical synthesis, and the constituent monomers of these small RNAs can be chemically modified in whole or in part, such as methoxy or ethoxy modification, for enhanced stability. The preparation method of the active ingredients of the small RNAs comprises the following steps: the 2 different nucleotide monomers are synthesized into 2 different small RNA single strands according to specific designed sequences by an RNA/DNA synthesizer, and the sequences of the small RNA single strands are shown as above. The synthesized small RNA single strand is separated and purified to remove other components, and is chemically modified to form small RNAs with 2 characteristics. The 2 small RNA molecules are frozen and concentrated into dry powder to be used as the active ingredient of the small nucleic acid in the formula, and the dry powder is stored at low temperature for standby. In order to detect the activity inhibition effect of the Antisense-24 and the Antisense-29 on the miR-24 and miR-29 respectively, the Antisense-24 and the Antisense-29 are co-transfected with luciferase plasmids (pRL-TK) containing 3' UTR region sequences of TCF and AKT respectively, and the results of luciferase activity analysis after 36 hours of transfection show that the small RNA molecules (comprising the miR-24 and miR-29) can effectively down-regulate the respective target genes of TCF and AKT (see figure 4B). If co-transfected with their antisense nucleic acids, quantitative PCR analysis showed that these antisense nucleic acids ANTI-24 and ANTI-29 were effective in inhibiting their respective targets miR-24 and miR-29, resulting in up-regulation of TCF and AKT expression (see FIG. 4C). To enhance the inhibitory and inductive effect of small RNA, we used the binding strategy of these 2 small RNAs, and the final small nucleic acid active ingredient was 0.6: the mixture is mixed and prepared according to the requirement of 0.4 proportion.

And fourthly, transdifferentiation of the human AD-MSC into skin stem cells and skin cells:

in order to improve the transformation efficiency of adipose-derived mesenchymal stem cells into skin stem cells, the invention discloses a high-efficiency induced transformation technology, and the embodiment of the technology is as follows: 1) culturing AD-MSCs from generation 4 until the confluence degree is 90%, and treating with pancreatinDigesting to obtain discrete AD-MSCs; 2) preparing small nucleic acid polypeptide nanoparticles: 1 mixture of 60% of 1.2ug of Anti-24 and 40% of 0.8ug of Anti-29 (synthesized by Shanghai Jima Biotech Co., Ltd., www.genepham.com.) in DMEM, and 10-80 ug of transmembrane polypeptide (purchased from Beijing Gilio Biotechnology development Co., Ltd., www.geno-bio. com. cn) in DMEM, thoroughly mixed and incubated at 37 ℃ for 20 minutes; 3) mixing the small nucleic acid polypeptide nanoparticle solution with 1-3X10⁶Mixing AD-MSCs thoroughly at 1-3 × 10⁵The cell density of/ml is inoculated into a T75 culture flask pre-combined with IV type collagen; 4) added into a special induced amplification culture solution developed by the invention, and the specific formula is shown in table 1. The best culture solution comprises the following components: 10ng/ml bFGF,20ng/ml EGF,10ng/ml IGF-1,5ng/ml KGF,10uM Insulin, 0.5uM Retinoic Acid, 20ng/ml Delta1,0.5mM VPA,100ng/ml WNT3a in a serum-free high-sugar DMEM/F12 medium for in vitro induction culture; 5) transfection was performed once more on day 2, under the same dosage conditions as the first, but without digestion and exchange of fluid, in order to achieve optimal induction of transformation efficiency. The beginning of the day three, some cells changed from spindle to irregular shape of skin stem cells (FIGS. 5a-c), and 5-6 days later, antigens characteristic of skin stem cells were revealed by FACS and fluorescence immunoassay (FIGS. 6 and 7). Also in the above way, we can efficiently convert 1-3X10 in 4-6 days^8-9AD-MSCs (cultured in the corning cell factory HYPERSTACK-12) were transformed into skin stem cells. This large scale and rapid transdifferentiation is the first disclosure of the present invention.

Table 1. formulation of medium for induced expansion of skin stem cells:

cells gradually fused into a stone-like change beginning on day 5, with a large patch of flat, diverse skin cells visible after 7 days (fig. 5 and 8). FACS analysis showed positive CK-5, -15 and-19 expression, with positive rates of 50%, 86% and 60%, respectively (FIG. 6). Carrying out in-vitro induction culture on the AD-MSCs in an induction culture medium containing growth factors such as TGF beta 1 and bFGF, wherein after the AD-MSCs are induced for 7 days, the content of mRNA of a plurality of cutin proteins is 3 times that of the mRNA of a control group in the transcription level; the immunofluorescence results showed that the expression of group keratin was induced to increase compared to the negative control group (fig. 7). The conclusion is that the small RNA molecules can induce the human adipose-derived mesenchymal stem cells to transdifferentiate into epidermal cells under the condition of in vitro culture by combining with growth factors such as EGF, bFGF, IGF-1, KGF, WNT3a, Delta1 and the like.

Fifthly, detecting the surface markers of the transdifferentiated skin stem cells:

five days later, 0.25% trypsin digestion was performed on AD-SSCs transdifferentiated from AD-MSC, and the cell concentration was adjusted to 2X10 with PBS containing 1% by volume of bovine serum albumin⁵Perml, prepare single cell suspension. The cells were aliquoted into 1.5ml EP tubes, 1ml per tube, to which were added the following murine anti-human monoclonal antibodies: CK15-FITC, CD34-FITC, CD49f-FITC, CK19-FITC, beta 1-integrin-FITC, CD71-FITC, IgG1-FITC/PE as isotype negative controls. Incubated at 4 ℃ in the dark for 45 minutes and detected by a flow cytometer. CD71, CD90, CD34 and CD49f are all expressed in positive way, and the positive rate is above 95% (see figure 6). These results indicate that AD-MSCs do transdifferentiate into AD-SSCs that acquire the phenotype of skin stem cells and lose the phenotype of mesenchymal stem cells.

Sixthly, identifying the transformed AD-SSC cells by a cellular immunofluorescence method:

4% paraformaldehyde was fixed at room temperature for 20min, and 3% hydrogen peroxide in deionized water was incubated at room temperature for 10 min. 0.5% TritonX-100 was clear for 20 min. Blocking 2% BSA and 10% serum at room temperature for 30min, adding dropwise primary anti-mouse anti-human CK15, CK19, mouse anti-human 63, CD34, CD49f and beta 1-integrin protein clone antibody, respectively, washing with PBS for 3 times at 37 ℃ for 2 hours. And respectively dripping glycerol and a sealing sheet containing an anti-fluorescence quenching agent. The cell shape of the visible induction group under the light microscope is oval, the ratio of the nuclear plasma is high, the cells are closely arranged, the outline is clear, the refractivity is good, full clone can be formed in about 5-7 days, the cell shape is similar to the typical structure of skin stem cells, and the antibodies CK19, beta 1-integrin, CD34 and p63 are positive in staining (figure 7B). The cell morphology of the AD-MSC of the normal control group is long spindle, and is obviously different from that of the cell morphology of the induction group, wherein the antibody CK19 is stained negatively, and the antibody CD34 is stained negatively (FIG. 7A). The positive expression rate of both CD166 and CD73 is up to 98%.

Seventhly, real-time fluorescent quantitative PCR analysis shows the expression conditions of the cytokeratin 15/19(CK15/19), P63 and beta 1-integrin before and after induction:

detecting the expression of mRNA of related genes of skin stem cells (AD-SSCs) transformed from the AD-MSCs after induction by real-time fluorescent quantitative PCR: and extracting the total RNA of the cells of the same time group by using a Trizol method. And detecting the concentration and purity of the RNA by using an ultraviolet spectrophotometer. The cDNA synthesis is carried out strictly according to the reverse transcription kit instructions, and the reverse transcription reaction conditions are 30 ℃ for 10 minutes, 45 ℃ for 20 minutes, 99 ℃ for 5 minutes and 5 ℃ for 5 minutes. A PCR reaction system is prepared by the following components: l0 × RNA PCR buffer, 29; water, 18.5; taq enzyme, 0.5; the PCR upstream primer and the PCR downstream primer are both 1. Adding the reaction components into a PCR reaction tube after the reverse transcription is finished, slightly and uniformly mixing the reaction components with the total volume of 50ul per sample, and carrying out PCR reaction according to the following conditions; first, pre-denaturation at 95 ℃ for 2 minutes; then denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 90 seconds for 35 cycles; finally, the results were converted to 2 using B actin as the internal reference (the ACt target gene-Ct internal reference gene) at 72 ℃ for 7 minutes. RT-PCR assays showed that in the experimental groups AD-SSCs, transdifferentiated cells all expressed some key transcription factors and other marker genes such as sox9, stat3, Myc, TGF-. beta.Tcf/Lef, BMI-1, Lhx2, IKK-. alpha., IRF6, Keratin 5/14/15/19, Notch-1, p63,. alpha.6. beta.4-and. alpha.3. beta.1-integrins, ABC transporters, c-kit/CD117, CD34, CD49f, EGFR, IGF-1/IGF-R1, Wnt-R, and NFATc 1. While the control and the false small RNA groups were negative. These results indicate that AD-MSCs indeed transdifferentiated into AD-SSCs that acquired the genotype of skin stem cells (FIG. 9), losing the genotype of mesenchymal stem cells.

Further, the drawings are described in more detail as follows:

FIG. 2 eosin staining of adipose mesenchymal stem cells in vitro at passage 5 (A) and 15 (B). And C is FACS analysis, and shows that the cells can express surface markers of mesenchymal stem cells and do not express surface markers of hematopoietic stem cells and vascular endothelial cells.

FIG. 3 adipose-derived mesenchymal stem cells were able to differentiate into adipocytes (a and b) and osteoblasts (c and d) under specific culture conditions.

FIG. 4 (A) the molecular structural sequences of the small RNAs (miR-24 and miR-29) and their target genes (TCF and AKT) and antisense nucleic acids (Anti-24 and Anti-29). (B) Quantitative PCR analysis the expression levels of TCF and AKT of the target genes under different conditions. Different conditions are as follows: control (Control), pseudo-antisense (Anti24m) and (Anti29m), and true-antisense.

FIG. 5 adipose-derived mesenchymal stem cells (AD-MSCs: A) 2 days after transfection induction (shuttle cells to round cells: B) to 4 days after transfection induction (pleomorphic skin stem cells: C) to 7 days after transfection induction (flattened skin cells: D).

FIG. 6 shows that under the special culture medium and induction conditions developed by the present invention, the adipose-derived mesenchymal stem cells can be trans-differentiated into skin stem cells, and FACS analysis indicates that the cells can express surface markers of the skin stem cells, but not the vascular endothelial cells and the adipose-derived mesenchymal stem cells.

FIG. 7 immunostaining for specific marker proteins of skin stem cells transdifferentiated from AD-MSCs (AD-SSCs), which were found to express P63, CD34, CK19 and a6 integrin.

FIG. 8, under the specific culture conditions described above, Anti-24 and Anti-29 induced transdifferentiation of adipose-derived mesenchymal stem cells (examples 1A-3A) into skin cells (examples 1B-3B).

FIG. 9. quantitative PCR analysis indicates that under the specific culture conditions described above, Anti-24 and Anti-29 induced transdifferentiation of adipose-derived mesenchymal stem cells (AD-MSCs) into skin stem cells (AD-SSCs) and significant expression of transcription factors and key genes associated with skin stem cells, which could not occur in control of the pseudo-small RNA-transfected adipose-derived mesenchymal stem cells (AD-MSCm).

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A method for inducing the transdifferentiation of mesenchymal stem cells into skin stem cells is characterized by comprising the following steps:

s2, selecting a plurality of small RNA molecules for stem cell epigenetic regulation, wherein the plurality of small RNA molecules for stem cell epigenetic regulation refer to Anti-nucleic acids Anti-24 and Anti-29 of miR-24 and miR-29;

the sequence structure of the miR-24 is as follows:

5-CUCCGGUGCCUACUGAGCUGAUAUCAGUUCUCAUUUUACACACUGGCUCAGUUCAGCAGGAACAGGAG-3；

the sequence structure of Anti-24: 5-GGCUGUUCCUGCUGAACUGAGCCAUU-3;

the sequence structure of the miR-29 is as follows:

5-AUGACUGAUUUCUUUUGGUGUUCAGAGUCAAUAUAAUUUUCUAGCACCAUCUGAAAUCGGUUAU-3；

the sequence structure of Anti-29 is as follows: 5-GGUAACCGAUUUCAGAUGGUGCUAUU-3;

the antisense nucleic acids Anti-24 and Anti-29 of the miR-24 and miR-29 are prepared from the following components in a dry powder weight ratio of 0.6: 0.4 mixing and preparing;

s3, assembling and transfecting nucleic acid polypeptide nanoparticles;

s4, preparing a culture medium for inducing the mesenchymal stem cells to transdifferentiate into skin stem cells, and performing amplification culture on the transfected skin stem cells; the step S4 includes the following steps: serum-free high-glucose DMEM/F12 medium was supplemented with 10ng/ml bFGF,20ng/ml EGF,10ng/ml IGF-1,5ng/ml KGF,10uM Insulin, 0.5uM Retinoic Acid, 20ng/ml Delta1,0.5mM VPA, and 100ng/ml WNT3 a;

2. The method for inducing transdifferentiation of mesenchymal stem cells into skin stem cells according to claim 1, wherein the step S1 comprises the steps of:

s11, rinsing the fat particles with PBS to remove residual blood and tissue fragments;

s12, mixing the crushed small fat tissue in a ratio of 1: mixing the mixture with 1 proportion and 0.25 percent collagenase I solution, placing the mixture in a working tube of a ClinicMACS protegee tissue separating and purifying machine, adjusting the working temperature to 37 ℃, setting the rotating speed of the separating and purifying machine to be 60 revolutions per minute, and rotating positively and negatively for 15 minutes respectively;

s13, after separation, adding 10% FBS low-sugar DMEM to stop digestion, then carrying out centrifugal treatment at 1500 rpm for 10 minutes, taking precipitated cell particles, and washing with PBS;

3. The method for inducing transdifferentiation of mesenchymal stem cells into skin stem cells according to claim 1, wherein the step S3 of assembling and transfecting nucleic acid polypeptide nanoparticles comprises the steps of: 1) culturing the AD-MSCs from the 4 th generation until the confluence degree is 90%, and digesting the AD-MSCs by pancreatin to obtain discrete AD-MSCs; 2) preparing small nucleic acid polypeptidePeptide nanoparticles: mixing the mixture of 60% of 1.2ug Anti-24 and 40% of 0.8ug Anti-29 prepared by DMEM with 10-80 ug of transmembrane polypeptide prepared by DMEM, mixing completely, and placing in an incubator at 37 deg.C for 20min to obtain small nucleic acid polypeptide nanoparticles; 3) mixing the small nucleic acid polypeptide nanoparticle solution with (1-3) x10⁶Mixing AD-MSCs thoroughly at (1-3). times.10⁵The cells were seeded at a density of one ml in T75 flasks pre-treated with type IV collagen.

4. The method for inducing transdifferentiation of mesenchymal stem cells into skin stem cells according to claim 1, wherein the induction medium prepared in step S4 is manufactured into different culture kits comprising cell proliferation factors and cell differentiation inhibitors required for proliferation of skin stem cells.

5. The method of claim 1, wherein the related genes in step S5 include Keratin 5/14/15/19, Notch-1, p63, α 6 β 4-and α 3 β 1-integrins, ABC transporters, c-kit/CD117, CD34, stat3, CD49f, sox9, EGFR, IGF-1/IGF-R1, WNT, Myc, TGF- β, Tcf/Lef, BMI-1, Lhx2, IKK- α, IRF6, and NFATc 1.