CN101760447B - Method for inducing and acclimating epidermal stem cells into nerve cells - Google Patents

Abstract

The invention discloses a method for inducing and domesticating epidermal stem cells into nerve cells. The method is to mix the eplife medium and the induction medium in different proportions, so that the epidermal stem cells have sufficient time to adapt to the eplife medium and continuously reduce the induction medium Gradually increase, and it is beneficial to reduce the differentiation to the epidermis and increase the probability of transformation to the target cell. The method of the present invention is simple and very effective for directional induction and domestication of epidermal stem cells into nerve cells. And overcome the prejudice of those skilled in the art, for the first time subvert the conclusion that epidermal stem cells are unipotent stem cells, and prove that they have multi-directional differentiation potential.

Description

Method for Inducing and Domesticating Epidermal Stem Cells into Nerve Cells

technical field

The invention relates to a method for inducing cells, in particular to a method for directional induction and domestication of epidermal stem cells into nerve cells.

Background technique

Humans have been studying epidermal stem cells for decades, but due to their difficulty in inducing differentiation, they have always been considered as unipotent stem cells, that is, they can only differentiate into related components of the epidermis. Regenerative medicine is hot in the field of medicine, and the research on stem cells has occupied an important position in the field of regenerative medicine. Stem cells, due to their multi-differentiation potential, can differentiate into various tissues and organs under specific conditions have attracted the attention of scientists. . Embryonic stem cells are totipotent stem cells that can differentiate into all tissues and organs except the placenta. However, because research on embryonic stem cells involves many ethical issues, those skilled in the art have been working on finding another kind of totipotent stem cells to replace embryonic stem cells.

People are gradually targeting adult stem cells. At present, those skilled in the art have successfully induced MSC (mesenchymal stem cells), ADSC (adipogenic stem cells) and other adult stem cells to differentiate into other types of cells, such as chondrocytes, bone cells, cardiomyocytes, etc. Originally found in bone marrow, MSCs have attracted increasing attention because of their multi-lineage differentiation potential, hematopoietic support and promotion of stem cell implantation, immune regulation and self-replication. For example, mesenchymal stem cells can be differentiated into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and other tissue cells under specific induction conditions in vivo or in vitro, after continuous subculture and cryopreservation It still has multi-directional differentiation potential and can be used as an ideal seed cell for the repair of tissue and organ damage caused by aging and disease. Therefore, MSC is currently used as a seed cell and scaffold material for bone tissue engineering to repair damaged tissues in clinical practice. Universal application. Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells belonging to the mesoderm. They mainly exist in connective tissue and organ interstitium, and are most abundant in bone marrow tissue. Since bone marrow is its main source, they are collectively called bone marrow mesenchymal cells. stem cells. However, the disadvantages of using MSCs to induce differentiation of nerve cells are: the source of MSCs is limited, and the materials are very troublesome, and the materials are relatively limited; Lots of obstacles.

Epidermal stem cells, also known as specific stem cells or unipotent cells, refer to cells that can only produce one type of cell, but have self-renewal properties. Those skilled in the art have always classified epidermal stem cells as unipotent cells. The scope of application of epidermal stem cells is also limited by the influence of unipotent cells.

Contents of the invention

The purpose of the present invention is to make up for the deficiencies of the prior art, and to provide a method for directional differentiation of pluripotent adult stem cells into nerve cells through domestication induction. It provides a potential seed cell for the application of clinical tissue engineering in the future, which can be widely used in wound repair, gene therapy, beauty treatment, etc., and provides convenience for human beings.

In order to achieve the above object, the idea of the present invention is: in the in-depth study of epidermal stem cells, we have learned that there are stem cells with multi-directional differentiation potential in the appendage hair follicles and sweat glands of the epidermis, and there are also such stem cells in the dermis adjacent to the epidermis. Could it be that the most abundant stem cells in the body are unipotent stem cells that can only differentiate epidermal cells? With this question in mind, we began a lengthy study on the pluripotency of epidermal stem cells.

In the process of research, we have fully understood the relevant characteristics of epidermal stem cells, and also learned that epidermal stem cells were a kind of stem cells that were very difficult to cultivate before the advent of the eplife culture system. It must be cultured in a culture system with low calcium ion concentration or no calcium ion In order to maintain its dryness and reduce differentiation to the epidermis. The Eplife culture system not only provides such an environment, but also adds many growth factors that are more conducive to the maintenance of the stemness of epidermal stem cells, and during our experiments, we also found that the stemness of epidermal stem cells can increase after primary culture. However, this growth characteristic has brought difficulties to the induction of differentiation. The target cells for induction of differentiation require the presence of a certain concentration of calcium ions, which is necessary for their development.

Therefore, how to keep the stemness of epidermal stem cells relatively stable and actively guide them to differentiate into target cells has become the primary problem to be solved in the present invention. Since the eplife culture system has a definite significance for the maintenance of the stemness of epidermal stem cells (it has been confirmed by experiments), we chose to mix the eplife medium and the induction medium in different proportions in the same way as making a cocktail, so that the epidermal stem cells have Sufficient time to adapt to the continuous reduction of the eplife medium and the gradual increase of the induction medium is beneficial to reduce the probability of the transformation from epidermal differentiation to target cells. We call this training process domestication.

The concrete technical scheme that the present invention adopts is:

A method for inducing and acclimating epidermal stem cells into nerve cells, characterized in that the specific steps of the method are:

(1) After the epidermal stem cells are cultured, take the epidermal stem cells with a density greater than 80%-95%, wash the cells twice with PBS, replace with fresh Eplife medium, and after 20-30h, in 1%-5% neural induction medium and Cultivate in the mixed medium of 95%-99% dry maintenance medium for 30-40 hours;

(2) Replace the culture medium with 15%-25% neural induction medium 2 and 75%-85% dryness maintenance medium and continue culturing for 3-5 days;

(3) The medium is replaced every 3-5 days thereafter; the second component of the neural induction medium is increased by 15% to 25% each time, until the second component of the neural induction medium is 100% without dry maintenance medium.

In the above-mentioned method for inducing and domesticating epidermal stem cells into nerve cells, the composition and dosage of the nerve induction medium are as follows:

DMEM/F122% FBS; 40-45ng/ml basic fibroblast growth factor b-FGF; 15-25ng/ml epidermal growth factor EGF; 0.3-0.5mM retinoic acid (retinoic acid); 0.3-0.5mM valproic acid (valproic acid); 1500～2000U/ml leukemia inhibitory factor (leukemia inhibitory factor); 80～100mM sodium pyruvate (sodium pyruvate); 0.5mM β-mercaptoethanol (β-mercaptoethanol);

In the above-mentioned method for inducing and domesticating epidermal stem cells into nerve cells, the two components and dosages of the nerve induction medium are:

DMEM/F12 2% FBS; 15～20ng/ml basic fibroblast growth factor b-FGF; 15～20ng/ml epidermal growth factor EGF; 0.5uM retinoic acid (retinoic acid); 1×B27; 100ug/ml D Butylated hydroxyanisole.

The above-mentioned method for inducing and acclimating epidermal stem cells into nerve cells, the composition and dosage of the stem maintenance medium are:

94-98% Epilife, 1% PS double antibody and 1-5% HKGS additive.

The above-mentioned method for inducing and acclimating epidermal stem cells into nerve cells, the preparation steps of the epidermal stem cells used in the step (1) are as follows:

(1) Take the human epidermal tissue that has been isolated for 1 to 4 hours and stored at 4°C, soak it in dilute povidone iodine solution for 1 minute, wash it with PBS/normal saline for 3 times, remove the fat tissue, and trim it into ^1cm2 tissue pieces;

(2) Place the tissue block in a neutral protease-containing solution for 14-16 hours at 4°C or directly in a 37°C incubator for more than 3 hours;

(3) Rinse the specimen 3 times with PBS at 4°C; separate the epidermis;

(4) After chopping, add an equal volume of 0.25% Typsin-EDTA 3-8ml, treat at 37°C for 30-90min, stop digestion with 3-8ml MEFs medium, and then layer by vigorous shaking;

(5) Transfer the cell suspension in the middle layer to another centrifuge tube, and centrifuge at 600-1000rpm for 3-8min;

(6) Discard the supernatant and resuspend with human epidermal stem cell culture medium;

(7) Transfer to a culture bottle, let stand for 30-90 minutes, discard the culture supernatant and rinse with PBS 2-3 times, replace with fresh epidermal stem cell medium; place in a 37°C, 5% CO ² incubator to continue culturing.

In the above method for inducing and domesticating epidermal stem cells into neurons, the culture medium for human epidermal stem cells consists of 94-98% Epilife, 1% PS double antibody, and 1% HKGS additive. The advantages and benefits of the present invention are: the inventors of the present invention have confirmed through many experiments that the method of the present invention is simple and very effective in directional induction and domestication of epidermal stem cells into nerve cells, and overcomes the prejudice of those skilled in the art, subverting the epidermis for the first time. The assertion that stem cells are unipotent stem cells demonstrates their multilineage differentiation potential.

Description of drawings

Figure 1 is the characteristic markers of CD90, CD24 and CD29 detected by immunofluorescence, wherein the positive rate of CD29 is greater than 15%, the positive rate of CD24 is greater than 80%, and the positive rate of CD29 is greater than 90%;

Figure 2 is flow cytometry detection of undifferentiated keratinocytes derived from the basal layer of human foreskin. CD90, CD24, and β1 integrin are surface markers of differentiated keratinocytes. The left picture of each group is the control group, and the right picture is the detected epidermal stem cell group. The results showed that the stemness of the obtained keratinocytes was strong;

Figure 3 is RT-PCR identification of stemness genes OCT4, SOX2, Klf4, Nanog, c-myc, CRIPTO, REX1, KRT14, differentiation markers KRT1, KRT5, KRT10, KRT19, and other cell markers in epidermal stem cells at each time point of culture Expression of MTTF, TYR, c-kit;

Figure 4 is the initial state of epidermal cells and the changes after two days of induction;

Figure 5 shows the generation of neural cell lines from domesticated induced keratinocytes. A represents keratinocytes before induction and cells two days after induction; B arrows indicate Nestin-positive cells derived from keratinization two weeks after induction; C arrows indicate induction three Immunofluorescence results of nerve-specific marker β3-tublin after one week; D arrows indicate GFAP-positive cells, and the right picture shows keratinocyte-induced GFAP-positive typical glial cell differentiation morphology; E is RT three weeks after nerve induction -PCR analysis of gene expression, M, marker; K, differentiated keratinocytes; S, sample;

Figure 6 Nerve (nestin)-specific staining was positive after directional induction.

Detailed ways

Example 1 Preparation of Epidermal Stem Cells Before Induction and Domestication

1. Preparation of epidermal stem cells:

Direct source and original source: With the consent of the patient, it was collected from healthy circumcision patients in the Surgical Outpatient Department of 301 Hospital.

(1) Take fresh adult foreskin (in vitro time is about 1-4 hours, stored at 4°C);

(2) soak the foreskin in dilute iodophor solution (containing iodophor 20%) for 1 min;

(3) Rinse with PBS/normal saline for 3 times to remove blood clots (to prevent affecting the digestion of enzymes);

(4) Trim the foreskin, remove adipose tissue, rinse several times with PBS/normal saline, and finally cut into tissue squares of about ^1cm2 ;

(5) Place the tissue block in a 6cm dish containing neutral protease (3mg/ml; D-hanks as solvent) solution and treat it at 4°C for 14-16h. If the treatment is not enough, it can also be placed in a 37°C incubator (or directly Place in a 37°C incubator for more than 3 hours—observe the treatment effect at any time);

(6) Rinse the specimen 3 times with PBS at 4°C;

(7) Separate the epidermis (tear it with sterile tweezers);

Cut it into pieces in the vial and add an equal volume of 0.25% Typsin-EDTA (the concentration of trypsin is 0.25%, the concentration of EDTA is 0.05%, the above-mentioned concentration of trypsin and EDTA are mixed by 1:1) a total of 4ml, at 37 Treat at ℃ for 30-90 minutes (the younger the sample age, the longer the digestion time, generally <30 years old, digest at 37°C for 60 minutes to 90 minutes, generally >30 years old, digest at 37°C for 45 minutes), stop with 4ml of serum-containing medium (MEFs medium) Digestion, and then stratified by vigorous shaking: stratum corneum (upper layer), cell layer (middle layer), tissue (lower layer);

(8) Carefully transfer the cell suspension in the middle layer to another centrifuge tube, and centrifuge at 1000rpm for 5min;

(9) Discard the supernatant and resuspend with human epidermal stem cell culture medium;

(10) Transfer to a culture bottle (pretreated with 100 μg/ml type IV collagen), let it stand for 60 minutes (stem cells are inert, adhere to the wall first), discard the culture supernatant and wash it twice with PBS, and replace with fresh epidermal stem cell culture medium ; Place in a 37°C, 5% CO ² incubator to continue culturing.

Note: Human epidermal stem cell culture medium composition

98% Epilife (Cascade Biologics, cat. no. M-EPI-500-CA)

1% PS (double antibody Invitrogen, cat.no.15070)

1%, additive (HKGS, Cascade Biologics, cat.no.S-001-5)

2. Growth state of epidermal stem cells before induction and domestication:

1. After 1 day of dry culture of fresh epidermis, replace the medium (the purpose is to remove dead cells).

2. After 3 days of culture, replace the human epidermal stem cell culture medium again. At this time, most of the cells are attached to the wall and grow in the shape of a cobblestone road. The cells are spindle-shaped and oval (the cell wall boundary is clear), and a large number of small round bright cells are seen, which grow on the upper layer of the adherent cells and show clonal growth. .

3. Identification of epidermal stem cells:

Whether the epidermal stem cells obtained by the above method are the epidermal stem cells we need, combined with the characteristic molecular markers of stem cells, and the characteristics of epidermal stem cells, we use RT-PCR (Figure 3), immunofluorescence (Figure 1), flow cytometry ( Figure 3) Detection of markers of epidermal stem cells.

1. Identification method:

In order to confirm that the above-adhered cells are the epidermal stem cells we want, we use RT-PCR and its immunofluorescence method to detect its stemness molecules and specific molecular markers.

1.1 RT-PCR

1.1.1 Extraction of RNA

(1) Discard the prepared medium in IPS cells, epidermal stem cells, and MEF cells, and wash the cells twice with PBS.

(2) Add 1ml of 4°C pre-cooled Tribule Lysis Solution to each six-well plate, and see that the cells are all lysed, about 5min.

(3) Use a nuclease-free gun tip to pipette the cell lysate into a 1.5ml centrifuge tube.

(4) Add 200ul of chloroform, shake vigorously up and down four to five times to make it fully mixed.

(5) 12000rpm, 5min 4°C. The liquid is divided into three layers, and the upper transparent liquid layer is sucked into a new 1.5ml centrifuge tube.

(6) Add an equal volume of isopropanol, mix upside down four or five times, and let stand at room temperature for 10 minutes.

(7) 12000rpm, 5min, 4°C, RNA precipitation can be seen at this time, discard the supernatant, and be careful not to suck out the precipitate.

(8) Add 700ul of pre-cooled 70% ethanol, 12000, 2min, 4°C, discard the supernatant, and repeat the operation once.

(9) Dry the remaining liquid in the RNA, and add nuclease-free water to dissolve the RNA depending on the volume of the precipitate (note that the RNA is too dry and difficult to dissolve)

1.1.2 Reverse transcription of RNA to cDNA

(10) Configure the reverse transcription system according to the reverse transcription kit, see Table 1

Table 1

*The amount of RNA added should depend on the amount of precipitation, and the amount of nuclease-free water should be supplemented to 10ul

After adding according to the above table, 37°C, 15min, 85°C, 5s. -20 Save for later use.

1.1.3PCR amplification

(11) The PCR reaction system is as follows

PCR mixture 12.5ul

Primer 1 1ul

Primer 2 1ul

cDNA 1ul

dd H2O 9.5ul

(11) PCR reaction conditions, see Table 2

Table 2

Gene name and alias upstream primer downstream primer Annealing temperature (℃) number of cycles Oct4(POU5F1)-new CGTGAAGCTGGAGAAGGAGAAGCTG GAACATGTGTAAGCTGCGGCCCTTG 62 40 Sox2 GCTGCACATGAAGGAGCACCC CGGACTTGACCACCGAACCCA 58 33 Klf4 CAAGTCCCGCCGCTCCATTAC TGGCTGGGCTCCTTCCCTCAT 58 33 c-Myc ACTCTGAGGAGGAACAAGAA TGGAGACGTGGCACCTCTT 58 33 Nanog CCCAAAGGCAAACAACCCACT ATTGCTATTCTTCGGCCAGTT 58 33 CRIPTO (TDGF1) TGCCCAAGAAGTGTTCCCTGT GCAGCAGCCTTTACTGGTCAT 60 33 REX1 CGCTGACACCATCCTCATCGG GGCGTCATCGCTTGGTCTTGG 55 33 KRT1 AGGATGTGGATGGTGCTTAT GCTTTGCTCTTCTGGGCTAT 58 33 KRT5 CTGGACACCAAGTGGACCCT GCTCCGCATCAAAGAACATC 58 33 KRT10 TGATAATGCCAACATCCTGC CCTCCTCGTGGTTTCTTCTTC 68 33 KRT14 GGAGATGATTGGCAGCGTGGA GGACCTGCTCGTGGGTGGACA 62 33 KRT15 AGCCTACCTGAAGAAGAACCACG TGGCATAGCGGCACTCTGTCT 58 33 KRT19 GCGACTACAGCCACTACTACACGAC CGACCTCCCGGTTCAATTCTT 58 33

MITF GACAGAAAGAAACTGGAGCACG ATCAGTGACACCGACGGGAGA 62 33 TYR TAGTCCACTTACTGGGATAGCG AGTCTGGGTCTGAATCTTGTAG 62 33 β-actin AAAGACCTGTACGCCAACAC GTCATACTCCTGCTTGCTGAT 62 32

*The conditions (number of cycles) of the PCR reaction are very different for different gene fragments, so we need to find out the best conditions during the experiment.

1.1.4 Detection of amplified fragments by agarose gel electrophoresis

(1) Weigh 1g electrophoresis grade agarose, add 100ml 1xTBE, and heat in a microwave oven to completely dissolve the agarose;

(2) After the gel is cooled to 50-60°C, add EB dye solution with a final concentration of 0.5ug/ml and shake well;

(3) Seal the rubber plate with glue strips, and pour the gel that has been cooled to 30-40°C;

(4) After the gel is solidified, add a certain amount of electrophoresis liquid to the electrophoresis tank, remove the strip, and put the gel tank into the electrophoresis tank so that the electrophoresis liquid does not pass through the gel tank. Gently pull out the comb;

(5) Take 5ul of Marker to load the sample, and take 8ul of the amplified PCR product for electrophoresis. At the beginning, the voltage is 100V, and when the sample runs out of the gel hole, 60V. Observe and take pictures under the light.

1.2 Immunofluorescence

(1) Take IPS clone cells in good condition, wash the cells twice with PBS, add 1ml of 4% paraformaldehyde to fix for 30min

(2) Discard the fixative, wash the cells 3 times with PBS, 5 min each time

(3) Add 1ml 0.05% permeate solution for 30min

(4) Discard the permeate, wash the cells with PBS 3 times, 5 min each time

(5) Add 500ul sheep serum blocking solution, 1h. Primary Antibody Diluent Dilute the primary antibody in the desired ratio

(6) Add the diluted primary antibody, put it in a wet box, and refrigerate overnight at 4°C

(7) Take out the cells incubated with the primary antibody the next day, wash once with PBS, and then wash 3 times with PBS, 10min each time

(8) Prepare the secondary antibody in PBS, discard the PBS, add 500ul of the diluted secondary antibody to the cells (after taking out the secondary antibody, the operation should be protected from light!) and incubate for 1h45min.

(9) Dilute the DAPI staining solution with PBS, add it to the staining solution containing the secondary antibody at a ratio of 1:2500, and continue to incubate for 30 minutes

(10) Discard the liquid in the petri dish, rinse once with PBS, and wash 3 times with PBS, 10min each time

(11) Add a small amount of PBS and observe under a fluorescent microscope

1.3 Detection by flow cytometry

(1) Digest the epidermal stem cells selected by type IV collagen attachment (when the medium is replaced after 1 hour of attachment), digest the cells with TE, stop the digestion with DTI, and use pre-cooled PBS containing 10% FBS to digest the cells Wash the cells three times, 12000rpm, 5min.

(2) Resuspend the cells with the above-mentioned PBS, and count the number of diluted cells to 5×105.

(3) Suspend the cells in 100ul PBS containing 3% BSA for each sample, add the corresponding antibodies: 10ul CD29, 10ul CD90, 10ul CD24, and incubate at 4°C for 30min.

(4) Wash the cells three times with cold PBS, 12000rpm, 5min.

(5) Dilute the secondary antibody with 3% PBS containing BSA, rabbit anti-(1:400), mouse anti-(1:200)

(6) Incubate in a 4-degree refrigerator for 20-30 minutes, repeat step 4, resuspend the cells in 500ul of cold PBS containing BSA, and detect by flow cytometry.

2. Identification results:

As shown in Figure 1, CD90, CD24, and CD29, the characteristic markers of epidermal stem cells, were detected by immunofluorescence, wherein the positive rate of CD29 was greater than 15%, the positive rate of CD24 was greater than 80%, and the positive rate of CD29 was greater than 90%. As shown in Figure 2, undifferentiated keratinocytes derived from the basal layer of human foreskin were detected by flow cytometry. CD90, CD24, and β1integrin are the surface markers of undifferentiated keratinocytes. The left picture of each group is the control group, and the right picture For the detected epidermal stem cell group, the results show that the obtained keratinocytes have strong stemness. As shown in Figure 3, RT-PCR identified stemness genes, OCT4, SOX2, Klf4, Nanog, c-myc, CRIPTO, REX1, KRT14, and differentiation markers KRT1, KRT5, KRT10, KRT19 in epidermal stem cells at various time points in culture , the expression of other cell markers MTTF, TYR, c-kit.

The current technology for isolating stem cells is not yet able to achieve 100% purity of the isolated cells. The results detected by flow cytometry show that the cells we have isolated are of high purity, and the detection rate of CD29 is as high as 99%. At the same time, we used immunofluorescence to detect the molecular markers of other cells, and the detection results showed that there were very few other cells.

Example 2 Directed induction and domestication of epidermal stem cells into nerve cells

1. Culture medium composition

(1) The components and dosage of nerve induction medium are:

DMEM/F12 (Invitrogen, cat.no.11330-32); 2% FBS (Gibco, cat.no.10437010) 45ng/ml b-FGF (Invitrogen, cat.no.13256-029); 20ng/ml EGF ( invitrogen, cat.no.); 0.5mM retinoic acid (RA) (sigmaR2625); 0.5mM valproic acid (Sigma, cat.no.T8552); 2000U/ml leukemia inhibitory factor (Chemicon, cat.lif1010); 100mM sodium pyruvate ( sigma, cat.no.P2256); 0.5mM β-mercaptoethanol (sigma cat.no.m7522);

(2) The two components and dosage of nerve induction medium are:

DMEM/F12 (Invitrogen, cat.no.11330-32) 2% FBS (Gibco, cat.no.10437010); 20ng/ml b-FGF (Invitrogen, cat.no.13256-029); 20ng/ml EGF ( invitrogen, cat.no.); 0.5uM RA (sigma R2625); 1×B27 (invitrogen Cat.No.17504); 100ug/ml butylated hydroxyanisole (sigma, cat.no.B1253).

(3) The composition and dosage of dry maintenance medium are: 98% Epilife (Cascade Biologics, cat.no.M-EPI-500-CA), 1% PS double antibody (double antibody Invitrogen, cat.no.15070) and 1% HKGS additive (HKGS, Cascade Biologics, cat. no. S-001-5).

2. Induced domestication method:

(1) After the epidermal stem cells are cultured, take the epidermal stem cells with a density greater than 80% to 95%, wash the cells twice with PBS, replace with fresh Eplife medium, and after 20 to 30 hours, dry them in 2% neural induction medium and 98% Cultured in the mixed medium of sex maintenance medium for 36h;

(2) The medium was replaced with 20% neural induction medium 2 and 80% dryness maintenance medium to continue culturing for 2 days;

(3) The medium was replaced every 2 days thereafter; the second component of the neural induction medium was increased by 20% each time, until the second component of the neural induction medium was 100% without dry maintenance medium.

Identification and result analysis of the target cell of embodiment 3

1. Immunofluorescence identification of induced differentiation results

1. Identification method: In the experimental verification of induction of nerve cells, the identification method of the nerve cell marker (a-SMA) detected by immunofluorescence is as follows:

(1) Cells three weeks after induction and epidermal stem cells were taken as controls, and cells were washed twice with PBS.

(2) Add 1ml of 4°C pre-cooled Tribule Lysis Solution to each six-well plate, and see that the cells are all lysed, about 5min

(3) Use a nuclease-free gun tip to suck the cell lysate into a 1.5ml centrifuge tube

(4) Add 200ul chloroform, shake vigorously up and down four to five times to make it fully mixed

(5) 12000rpm, 5min 4°C. The liquid is divided into three layers, and the upper transparent liquid layer is sucked into a new 1.5ml centrifuge tube

(6) Add an equal volume of isopropanol, mix upside down four or five times, and let stand at room temperature for 10 minutes

(7) 12000rpm, 5min, 4°C, RNA precipitation can be seen at this time, discard the supernatant, and be careful not to suck out the precipitate

(8) Add 700ul of pre-cooled 70% ethanol, 12000, 2min, 4°C, discard the supernatant, repeat the operation once

(9) Dry the remaining liquid in the RNA, and add nuclease-free water to dissolve the RNA depending on the volume of the precipitate (note that the RNA is too dry and difficult to dissolve)

2. Identification results:

Epidermal cell-specific markers (involucrin positive) and nerve cell-specific markers (nestin) were detected by immunofluorescence at 10 days, 2 weeks, and 3 weeks after acclimation induction, respectively. As shown in Figure 5, neural cell lines were generated for domestication and induction of keratinocytes, A represents keratinocytes before induction and cells two days after induction; B arrows indicate Nestin-positive cells derived from keratinization two weeks after induction; C Arrows indicate the immunofluorescence results of the nerve-specific marker β3-tublin after three weeks of induction; D arrows indicate GFAP-positive cells, and the right picture shows the typical glial cell differentiation morphology of 6FAP-positive cells induced by keratinocytes; E shows nerve induction Three weeks later, RT-PCR analysis of gene expression, M, marker; K, differentiated keratinocytes; S, sample;

Figure 4 shows the initial state of epidermal cells and the state after two days of induction. The left picture shows the growth state of epidermal stem cells induced initially; the right picture shows the growth state of epidermal stem cells after two days of acclimation induction (can be used as a reference index)

Figure 6 shows the positive staining of neural (nestin) specificity after directional induction and domestication.

2. RT-PCR Identification of Induced Differentiation Results

1. RNA extraction

(1) Cells three weeks after induction and epidermal stem cells were taken as controls, and cells were washed twice with PBS.

(2) Add 1ml of 4°C pre-cooled Tribule Lysis Solution to each six-well plate, and see that the cells are all lysed, about 5min

(3) Use a nuclease-free gun tip to suck the cell lysate into a 1.5ml centrifuge tube

(4) Add 200ul chloroform, shake vigorously up and down four to five times to make it fully mixed

(5) 12000rpm, 5min 4°C. The liquid is divided into three layers, and the upper transparent liquid layer is sucked into a new 1.5ml centrifuge tube

(6) Add an equal volume of isopropanol, mix upside down four or five times, and let stand at room temperature for 10 minutes

(7) 12000rpm, 5min, 4°C, RNA precipitation can be seen at this time, discard the supernatant, and be careful not to suck out the precipitate

(8) Add 700ul of pre-cooled 70% ethanol, 12000, 2min, 4°C, discard the supernatant, repeat the operation once

(9) Dry the remaining liquid in the RNA, and add nuclease-free water to dissolve the RNA depending on the volume of the precipitate (note that the RNA is too dry and difficult to dissolve)

2. Reverse transcription of RNA to cDNA

(10) Configure the reverse transcription system according to the reverse transcription kit, see Table 3:

table 3

*The amount of RNA added should depend on the amount of precipitation, and the amount of nuclease-free water should be supplemented to 10ul

After adding according to the above table, 37°C, 15min, 85°C, 5s. -20 Save for later use.

3.PCR amplification

(1) The PCR reaction system is as follows

PCR mixture 12.5ul

Primer 1 1ul

Primer 2 1ul

cDNA 1ul

dd H2O 9.5ul

(2) PCR reaction conditions are shown in Table 4

*The conditions (number of cycles) of the PCR reaction are very different for different gene fragments, so we need to find out the best conditions during the experiment

Table 4:

4. Detection of amplified fragments by agarose gel electrophoresis

(1) Weigh 1g electrophoresis grade agarose, add 100ml 1xTBE, heat in a microwave oven to completely dissolve the agarose

(2) After the gel is cooled to 50-60°C, add EB dye solution with a final concentration of 0.5ug/ml and shake well

(3) Seal the rubber plate with glue strips, and pour the gel that has been cooled to 30-40°C and prepared

(4) After the gel is solidified, add a certain amount of electrophoresis liquid to the electrophoresis tank, remove the strip, and put the gel tank into the electrophoresis tank so that the electrophoresis liquid does not pass through the gel tank. Gently pull out the comb

(5) Take 5ul of Marker to load the sample, and take 8ul of the amplified PCR product for electrophoresis. At the beginning, the voltage is 100V, and when the sample runs out of the gel hole, 60V. Observe and take pictures under the light. The results shown in Figure 5E are RT-PCR analysis of gene expression three weeks after neural induction, M, marker; K, differentiated keratinocytes; S, sample.

3. Induction results:

We prepared 133 specimens of epidermal stem cells, 15 cases of directional induction, 100% success. After the above directed induction, we proved that epidermal stem cells can successfully differentiate into neural cells.

Four, purposes of the present invention:

Epidermal stem cells can differentiate into nerve cells in a non-directed culture system, which proves that epidermal stem cells are not unipotent stem cells, and they have a clear potential for multidirectional differentiation. Epidermal stem cells are widely distributed in the human body, easy to obtain materials, stem cell activity is not limited by age, and have good in vitro expansion ability, which will add advantages to the application field of adult stem cells.

Claims (2)

1. A method for inducing and acclimating epidermal stem cells into neurons, characterized in that the specific steps of the method are: (1) After the epidermal stem cells are cultured, take the epidermal stem cells with a density greater than 80% to 95%, wash the cells twice with PBS, replace with fresh Epilife medium, and after 20 to 30 hours, mix in 1% to 5% neural induction medium Cultivate in the mixed medium of 95%-99% dry maintenance medium for 30-40 hours; (2) Replace the culture medium with 15%-25% neural induction medium 2 and 75%-85% dryness maintenance medium and continue culturing for 3-5 days; (3) The medium is replaced every 3 to 5 days thereafter; the second component of the neural induction medium is increased by 15% to 25% each time, until the second component of the neural induction medium is 100% and there is no dry maintenance medium; One component and consumption of described nerve induction medium are: DMEM/F12 2% FBS; 40～45ng/ml basic fibroblast growth factor b-FGF; 15～25ng/ml epidermal growth factor EGF; 0.3～0.5mM retinoic acid; 0.3～0.5mM valproic acid; 1500～ 2000U/ml leukemia inhibitory factor; 80~100mM sodium pyruvate; 0.5mM β-mercaptoethanol; The two components and dosages of the nerve induction medium are: DMEM/F12 2% FBS; 15~20ng/ml basic fibroblast growth factor b-FGF; 15~20ng/ml epidermal growth factor EGF; 0.5uM retinoic acid; 1×B27; 100ug/ml butylated hydroxyanisole; The composition and consumption of described dry maintenance medium are: 94-98% Epilife, 1% PS double antibody and 1-5% HKGS additive. 2. the method for inducing and domesticating epidermal stem cells according to claim 1, is characterized in that, the preparation steps of the epidermal stem cells used in the step (1) are as follows: (1) Take the human epidermal tissue that has been isolated for 1-4 hours and stored at 4°C, soak it in a dilute iodophor solution containing 20% iodophor for 1 min, wash it with PBS/normal saline for 3 times, remove the fat tissue, and trim it into ^1cm2 tissue piece; (2) Place the tissue block in a neutral protease-containing solution for 14-16 hours at 4°C or directly in a 37°C incubator for more than 3 hours; (3) Rinse the specimen 3 times with PBS at 4°C; separate the epidermis; (4) After chopping, add an equal volume of 0.25% Typsin-EDTA 3-8ml, treat at 37°C for 30-90min, stop digestion with 3-8ml MEFs medium, and then layer by vigorous shaking; (5) Transfer the cell suspension in the middle layer to another centrifuge tube, and centrifuge at 600-1000rpm for 3-8min; (6) Discard the supernatant and resuspend with human epidermal stem cell culture medium; (7) Transfer to a culture bottle, let stand for 30-90 minutes, discard the culture supernatant and rinse with PBS 2-3 times, replace with fresh human epidermal stem cell culture medium; place in a 37°C, 5% CO2 _incubator to continue culturing; The human epidermal stem cell culture medium consists of 98% Epilife, 1% PS double antibody, and 1% HKGS additive.

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