Method for reprogramming hair follicle stem cells into induced pluripotent stem cells

Abstract

The invention belongs to the technical field of cell reprogramming, and particularly relates to a method for reprogramming hair follicle stem cells into induced pluripotent stem cells. The method for reprogramming the hair follicle stem cells into the induced pluripotent stem cells comprises the following steps: (1) adding a tetrahydrate 1CoMiP plasmid into a cell suspension of the hair follicle stem cells, mixing, and carrying out electrotransformation; (2) inoculating the cells obtained after the electrotransfection treatment in the step (1) into a culture container, and culturing by adopting a first culture medium; (3) sequentially replacing the culture medium with a second culture medium, a third culture medium and a fourth culture medium, and continuously culturing; (4) selecting monoclonal cells, and culturing by adopting a fifth culture medium; and (5) replacing the fifth culture medium with a sixth culture medium to obtain the induced pluripotent stem cells. The induced pluripotent stem cells obtained by reprogramming through the method are identified to meet the requirements of the induced pluripotent stem cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell reprogramming, and in particular relates to a method for reprogramming hair follicle stem cells into induced pluripotent stem cells. Background Art

[0002] Induced pluripotent stem cells (iPSCs) are cells that have been transformed from non-stem cells into cells with stem cell characteristics through genetic manipulation. They are very similar to natural stem cells in terms of gene expression, protein synthesis, chromatin methylation, cell proliferation, embryonic development, teratoma formation, chimera formation, and differentiation potential.

[0003] Unlike natural stem cells, induced pluripotent stem cells are not directly extracted from human organs or tissues, but are created through genetic engineering technology. For example, Shinya Yamanaka reprogrammed cells by introducing four key transcription factors (Oct4, Sox2, Klf4 and c-Myc) into differentiated somatic cells through viral vectors, thereby obtaining iPSCs similar to embryonic stem cells. Scientists around the world have also discovered a variety of ways to prepare such cells, and they only need to choose the appropriate preparation strategy according to the cell type.

[0004] Minicircle vectors are non-viral DNA vectors designed to improve transfection efficiency. They are circular DNA molecules that contain key stem cell genes, such as Oct3 / 4, SOX2, Nanog, LIN28, etc. The genes encoded by these minicircle vectors are not easily degraded or silenced in cells, and can be efficiently expressed in both dividing and non-dividing cells, so they can produce high levels of target proteins. This makes them an ideal choice in the reprogramming process of iPSCs.

[0005] Despite this, research on iPSCs reprogramming is still relatively limited.

[0006] Therefore, it is necessary to develop a new technical solution to overcome the limitations of existing technologies. Summary of the invention

[0007] The present invention aims to develop a new method for converting hair follicle stem cells into induced pluripotent stem cells to overcome the shortcomings of the prior art.

[0008] To achieve this goal, the present invention proposes the following technical solution: a method for converting hair follicle stem cells into induced pluripotent stem cells, the specific steps are as follows: the hair follicle stem cell suspension is mixed with a 4-in-1 CoMiP plasmid, then transferred to an electroporation cup, and an electroporation reagent is added, and electroporation is performed by an electroporator. The cells treated with electroporation are inoculated into a culture container and cultured using BRSC complete medium (first culture medium). The culture medium is replaced in sequence, first using BRSC complete medium containing sodium butyrate and ascorbic acid (second culture medium), then a mixture of the second culture medium and mTeSR-E7 culture medium (third culture medium), and finally using mTeSR-E7 culture medium containing sodium butyrate and ascorbic acid (fourth culture medium) to continue culture. Monoclonal cells are selected and transferred to a new culture container, and cultured using E8 culture medium (fifth culture medium) containing ROCK inhibitor. The fifth culture medium is replaced with E8 culture medium (sixth culture medium), and induced pluripotent stem cells are finally obtained. In a preferred embodiment, the culture time of the first, second and third culture media are all 2 days; the culture time of the fourth culture media is determined according to the degree of obvious clone emergence, usually 20 to 30 days, and the culture medium is replaced every two days; the fifth culture media is used on the day of cell passage or recovery.

[0009] Preferably, in step (1), the density of the hair follicle stem cells in the cell suspension is 10 million / ml, and the dosage ratio of the 4-in-1 CoMiP plasmid is 12 μg / 100 μL of the cell suspension.

[0010] Preferably, in step (2), the inoculation density is (0.5-2)×10 5 cells / well (six-well plate).

[0011] Preferably, in step (2), the culture container is a culture plate pre-coated with Matrigel or Cellstart.

[0012] Preferably, the ROCK inhibitor is Y-27632.

[0013] Beneficial effects:

[0014] The method of reprogramming hair follicle stem cells into induced pluripotent stem cells of the present invention uses hair follicle stem cells derived from hair follicles as a cell source, has a simple separation process, does no harm to the body, obtains a large amount, and can be rapidly expanded.

[0015] The transfection process of the method for reprogramming hair follicle stem cells into induced pluripotent stem cells of the present invention is simple, and a minicircle vector is used. Only one vector needs to be transfected, and a sufficient amount of induced pluripotent stem cells can be obtained by transfecting once. The induced pluripotent stem cells obtained by reprogramming using the method of the present invention are identified to meet the requirements of induced pluripotent stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. Among them:

[0017] Figure 1 A diagram showing the separation of primary hair follicle stem cell tissue from hair follicles and the generation of cell passages after attachment provided by an embodiment of the present invention;

[0018] Figure 2 This is a typical clonal region observed at 30 days of reprogramming in Example 1 (4X);

[0019] Figure 3 This is a morphological picture of cells after passage of the induced pluripotent stem cells obtained by reprogramming in Example 1;

[0020] Figure 4 This is a flow cytometry result diagram of the induced pluripotent stem cells obtained by reprogramming in Example 1;

[0021] Figure 5 This is a diagram showing the immunofluorescence detection results of the induced pluripotent stem cells obtained by reprogramming in Example 1; DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0023] The present invention will be described in detail below in conjunction with embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0024] The inventors found in their research that human hair follicle stem cells (BRSC cells; cells isolated from normal human scalp hair follicles) have a simple isolation process, do not damage the body, are large in number, are not prone to mutation, and are easy to culture on a large scale in vitro, so they can be used as a raw material cell for reprogramming iPSC cells. How to reprogram human hair follicle stem cells into induced pluripotent stem cells is an urgent problem to be solved in this field.

[0025] The method for reprogramming hair follicle stem cells into induced pluripotent stem cells according to the embodiment of the present invention comprises the following steps: (1) adding a 4-in-1 CoMIP plasmid (i.e., MIP 247CoMiP 4in1withshRNA) to a cell suspension of hair follicle stem cells; p53 plasmid), after mixing the cells and plasmid, transfer them to an electroporation cup, add electroporation reagent, and place them in an electroporator for electroporation; (2) inoculate the cells obtained after the electroporation treatment in step (1) in a culture container and culture them with a first culture medium; (3) replace the culture medium with a second culture medium, a third culture medium, and a fourth culture medium in sequence, and continue culturing; (4) pick up monoclonal cells and inoculate them into a new culture container and culture them with a fifth culture medium; (5) replace the fifth culture medium with a sixth culture medium to obtain induced pluripotent stem cells; the first culture medium is BRSC complete culture medium; the second culture medium is BRSC complete culture medium containing sodium butyrate and ascorbic acid; the third culture medium is a mixture of the second culture medium and mTeSR-E7 culture medium; the fourth culture medium is mTeSR-E7 culture medium containing sodium butyrate and ascorbic acid; the fifth culture medium is E8 culture medium containing ROCK inhibitor; the sixth culture medium is E8 culture medium (i.e., Essential 8TM culture medium).

[0026] Among them, MIP 247CoMiP 4in1 with shRNA p53 plasmid is a mini-intron plasmid (MIP) vector, which is an alternative, robust, non-silencing DNA vector for efficient transgene expression in vitro and in vivo. The mini-intron plasmid features a pUC replication origin and RNA-OUT antibiotic selection, with free selection markers in designated non-coding exons. MIP vectors overcome transgene silencing observed with plasmids and provide at least 2-10 times higher transgene expression levels in vivo and in vitro. The 4-in-1 CoMIP plasmid expresses four reprogramming factors of Oct4, Klf4, Sox2 and c-Myc, which are separated by different self-cleaving 2A peptide sequences under the control of a promoter and integrated into the MIP vector. 4-in-1 CoMIP plasmid is a new single-plasmid reprogramming system with the advantages of efficient integration, no insertion risk, and high cost performance.

[0027] The method for reprogramming hair follicle stem cells into induced pluripotent stem cells of the present invention comprises the following steps: firstly electrotransferring 4-in-1 CoMIP plasmids to hair follicle stem cells, and then adopting BRSC complete medium to culture the hair follicle stem cells, so as to facilitate the adhesion and proliferation of the hair follicle stem cells; adopting BRSC complete medium containing sodium butyrate and ascorbic acid as the second medium, so as to facilitate improving the efficiency of reprogramming; adopting the third medium for culture, so as to play a certain transitional role, and to avoid or reduce the death of some cells due to the inability to adapt to the sudden change of the medium; the reprogramming of the hair follicle stem cells mainly occurs during the culture process of the fourth medium; adopting the fifth medium to culture the hair follicle stem cells, so as to facilitate the expansion culture of the induced pluripotent stem cells obtained by reprogramming, and facilitate the survival and adhesion of the cells; and then adopting the sixth medium to culture the induced pluripotent stem cells obtained by reprogramming.

[0028] The method of reprogramming hair follicle stem cells into induced pluripotent stem cells of the present invention has a simple transfection process, and only one vector needs to be transfected, and a sufficient amount of induced pluripotent stem cells can be obtained by transfecting once. The induced pluripotent stem cells obtained by the method of the present invention meet the requirements of induced pluripotent stem cells.

[0029] In a preferred embodiment of the present invention, the first culture medium, the second culture medium and the third culture medium are cultured for 2 days; the fourth culture medium is cultured until obvious clones appear, and the culture time is 20-30 days, and the medium is replaced every two days during the culture process; the fifth culture medium is used on the day of cell passage or recovery.

[0030] In a preferred embodiment of the present invention, in the third culture medium, the mass ratio of the second culture medium to the mTeSR-E7 culture medium is 1:1; the concentration of the ROCK inhibitor in the fifth culture medium is 10 μM; in the second culture medium and / or the fourth culture medium, the concentration of sodium butyrate is 0.2 mM, and the concentration of ascorbic acid is 50 μg / mL.

[0031] In a preferred embodiment of the present invention, in step (1), the density of hair follicle stem cells in the cell suspension is 10 million / ml, and the dosage ratio of 4-in-1 CoMIP plasmid is 12 μg / 100 μL cell suspension.

[0032] In a preferred embodiment of the present invention, in step (2), the inoculation density is (0.25-4)×10 5 cells / well (six-well plate).

[0033] In a preferred embodiment of the present invention, in step (2), the culture container is a culture plate pre-coated with Matrigel or Cellstart.

[0034] In a preferred embodiment of the present invention, the ROCK inhibitor is Y-27632.

[0035] The method for reprogramming hair follicle stem cells into induced pluripotent stem cells of the present invention is described in detail below through specific examples.

[0036] Sources of some of the reagents used in the following examples (other reagents not specified can be purchased commercially):

[0037] Table 1: Sources of culture media

[0038]

[0039] Table 2: Sources and specifications of some reagents

[0040]

[0041]

[0042] Table 3: Antibody sources

[0043]

[0044] Example 1

[0045] 1. Isolation and culture of hair follicle stem cells:

[0046] (A) Collection, storage and transportation of hair follicles: Normal adult hair follicle units are legally obtained from hair transplant surgery patients (age 25-35 years old).

[0047] (B) Isolation of hair follicle stem cells: The hair follicles obtained by surgery were repeatedly rinsed with PBS solution containing 5% penicillin-streptomycin at 4°C ( Figure 1 a) Remove the blood clots on the surface of the hair follicles. Use forceps to remove the epidermal tissue. Hold the proximal end of the hair shaft with one forceps to stabilize the hair follicle. Hold the epidermal tissue with the other forceps and peel off the epidermal tissue against the growth direction of the hair shaft. Rinse the treated hair follicles with PBS three times. Under a stereoscope, remove the hair papilla with a surgical scalpel. Wash the hair follicles obtained from the hair papilla with washing solution continuously, and transplant the hair follicles to the transwell-6 membrane with 8-12 hair follicles per well ( Figure 1 b), in the initial stage, the culture medium is added below the membrane and the liquid surface just contacts the membrane with a volume of about 1 mL. The culture medium is used for static culture at 37°C, 5% O2, 5% CO2, and 90% N2 partial pressure. The cells migrate out after about 3 days ( Figure 1c) As the number of culture days increases, cells continue to migrate out, and the lower liquid increases continuously, usually increasing to 1.5mL in about a week, and the upper liquid 0.5mL. When the cell density is about 50%, the lower liquid is 2mL and the upper liquid is 1mL to maintain the cell density to 80%. The frequency of changing the liquid during this culture process is once every 2-3 days. The cells on the Transwell-6 membrane are tightly attached and difficult to digest. The cells are harvested by two-step digestion, that is, after normal digestion and collection of cells, secondary digestion is used to collect cells still attached to the membrane, so as to maximize the benefits while avoiding the damage of trypsin to the cells.

[0048] (C) Primary harvesting of hair follicle stem cells: The cells in step (B) are cultured for 10-12 days. When the confluence reaches 80%-95%, the culture medium in the culture dish is discarded. 1mL PBS is used to submerge the bottom of the culture dish, and the PBS is discarded after rinsing twice. An appropriate amount of digestive enzyme is added to the bottom of the culture dish to digest the hair follicle stem cells. The digestion is complete when the cells appear as bright circles under the microscope. After the digestion is complete, an appropriate amount of PBS is added to terminate the digestion. The supernatant is taken out and placed in a 15mL centrifuge tube. 3mL of culture medium is added to rinse the bottom of the dish once, and the rinsing solution is added to the centrifuge tube. The supernatant is centrifuged at 1500rpm for 5min in a centrifuge, the supernatant is discarded, 500μL of culture medium (DMEM / F12 culture medium) is added to resuspend, the cells are counted and inoculated as P1 cells. P1 cells can be passaged when the growth confluence reaches 80%-95% (see Figure 1 (d)).

[0049] (D) Cell harvesting of hair follicle stem cells: The cells harvested in step (C) were inoculated at a density of 8000 / cm2 and cultured for 4-5 days. After the confluence reached 80%-95%, the culture supernatant was discarded, washed once with PBS, and then digested at room temperature for 3 minutes with an appropriate amount of TrypLE (GIBCO, catalog number: 12604013; concentration 1X) digestive enzyme. After digestion, PBS was added to terminate the digestion, and the supernatant after digestion was collected, centrifuged at 1500rpm for 5min, and counted. The cells were subcultured to the P2 generation. The P2 generation cells can be subcultured when the confluence reaches 80%-95%.

[0050] 2. BRSC cell recovery and transfection:

[0051] (1) Resuscitate P2 cells and culture them in T75 culture flasks at a density of 10,000 cells per square centimeter.

[0052] (2) Wash the cells with PBS solution first, and then treat the cells with Tryple select enzyme for 5 to 7 minutes.

[0053] (3) Add BRSC complete medium to stop enzyme treatment, gently pipette to evenly disperse the cells, and transfer the cell suspension to a 15 mL centrifuge tube.

[0054] (4) Centrifuge at 200 g for 5 minutes.

[0055] (5) Remove the supernatant, add an appropriate amount of culture medium to the cell pellet to resuspend the cell pellet, and take a sample for counting.

[0056] (6) The cells were seeded into a T175 culture flask at a density of 6,000 to 8,000 cells per square centimeter and cultured in a 37°C incubator for 3 days.

[0057] (7) After 3 days, when the cell confluence reached 70-90%, cell digestion was performed.

[0058] (8) BRSC cells were resuspended in buffer at a density of 10 million cells / ml, and then 12 μg of 4-in-1 CoMIP plasmid was added per 100 μl. The mixture was then transferred to a 100 μl electroporation cuvette and an appropriate amount of electroporation reagent (using R buffer provided with the Thermo Neon electroporator) was added. Finally, the cells were electroporated using the Invitrogen Neon system (1600 volts, 10 ms, 3 pulses) (following the instructions of the electroporator).

[0059] (9) After electroporation, cells were seeded into Matrigel-pretreated six-well plates at different densities (e.g., 0.25×10^5 cells, 0.5×10^5 cells, 1×10^5 cells, 2×10^5 cells, 3×10^5 cells, and 4×10^5 cells per well) and cultured for 2 days using BRSC complete medium. The experimental data clearly indicated that different cell culture densities are critical for the growth balance of BRSC cells and newly induced iPS cells. Specifically, when the cell density was set at 3×10^5 and 4×10^5 per well, the proliferation rate of BRSC cells (hair follicle stem cells) was too fast, causing them to occupy the space required for iPS cell clone formation, thus failing to form ideal iPS cell clones. On the other hand, when the seeding concentration of BRSC cells was reduced to 0.25×10^5 per well, the probability of iPS cell generation was reduced due to the small number of cells, and iPS cell clones were also not detected. However, when the seeding concentration of BRSC cells was adjusted to 0.5×10^5, 1×10^5, and 2×10^5 per well, these densities were considered to be better choices. The iPS cell clones observed in the experiment were mainly derived from seeding at these densities. This suggests that under the current electroporation conditions, cell densities of 0.5×10^5, 1×10^5, and 2×10^5 may be more conducive to the formation of iPS cell clones than other densities.

[0060] (10) The cell culture medium was replaced with BRSC complete medium containing 0.2 mM sodium butyrate and 50 μg / mL ascorbic acid and the culture was continued for 48 h.

[0061] (11) The culture medium was replaced with an equal ratio mixture (1:1) of BRSC complete medium and mTeSR-E7 medium containing 0.2 mM sodium butyrate and 50 μg / mL ascorbic acid, and the culture was continued for 48 h.

[0062] (12) The culture medium was replaced with mTeSR-E7 medium containing 0.2 mM sodium butyrate and 50 μg / mL ascorbic acid and the culture was continued.

[0063] (13) The culture medium was replaced every 48 h using mTeSR-E7 medium containing 0.2 mM sodium butyrate and 50 μg / mL ascorbic acid.

[0064] (14) Use a 10 μL pipette tip to transfer 6 clones to 6 Matrigel-pretreated culture wells and culture them in Essential 8TM medium containing 10 μM ROCK inhibitor (Y-27632). Note that Y-27632 is only used on the day of cell passaging or recovery. After that, only E8 medium (Essential 8TM medium) is used during medium changes.

[0065] 3. Passaging and identification of iPSC cells:

[0066] Cell digestion steps:

[0067] Wipe the outer surface of the six-well plate with an alcohol-containing lint-free cloth and transfer it to a biosafety cabinet. Gently shake the culture flask to loosen the cells, and then use a pipette to remove the old culture medium. Wash the cell surface once with an appropriate amount of PBS solution. Add 1 mL of ReleSR digestion enzyme to each well, incubate at room temperature for 1 minute, and then remove the digestion solution. Place the plate in a 37°C incubator and incubate for 5 to 7 minutes. Add an appropriate amount of E8 complete medium to terminate the digestion process. Fix the culture plate with one hand and tap the side of the culture plate with the other hand for about 30 to 60 seconds. Observe under a microscope and confirm that most of the clone edges begin to detach from the bottom of the culture plate, and most of the cells inside the clones fall off in clumps.

[0068] Cell seeding and culture steps:

[0069] Transfer cell aggregates at the desired density into wells pre-treated with Matrigel and containing E8 complete medium.

[0070] Add ROCK inhibitor Y27632 to a final concentration of 10 μM. Observe the cells under a microscope to ensure that they form clumps of 4 to 10 cells. Gently shake the culture plate to evenly distribute the cells and record information such as cell lot number, seeding density, and seeding date. Place the culture plate in an incubator at 37°C with 5.0% carbon dioxide for culture.

[0071] Experimental Case

[0072] 1. Isolation of hair follicle stem cell tissue, observation of primary hair follicle stem cells and passaged hair follicle stem cells, such as Figure 1 shown.

[0073] Figure 1 Middle: (a) is a picture of hair follicle samples extracted by a hair follicle extraction machine; (b) is a picture of primary hair follicle stem cells crawling out on day 3; (c) is a picture of primary hair follicle stem cells crawling out on day 14. (d) Cell morphology of hair follicle stem cells before harvesting at the P1 generation.

[0074] 2. Typical areas begin to appear around 20 days after reprogramming (see below) Figure 2 ), and typically after 35-40 days, typical areas can be selected for cloning. 6-7 clones can appear in each well.

[0075] Figure 2 Shown are two representative clonal regions observed after 30 days of culture in mTeSR-E7 medium containing 0.2 mM sodium butyrate and 50 μg / mL ascorbic acid.

[0076] 3. Cell morphology: Figure 3 As shown (the left and right pictures are cell morphology pictures of P15 cells cultured for 3 days and 4 days, respectively);

[0077] Depend on Figure 3 It can be observed that after passage, the cells showed typical iPSC cell morphology and formed compact multicellular colonies with clear boundaries. The cells were tightly stacked, with a high nuclear-cytoplasmic ratio and prominent nucleoli.

[0078] 4. Flow cytometry:

[0079] The steps include:

[0080] (1) Cell collection: For the iPSCs cells to be tested, wash each 10 cm culture dish twice with 8 mL of PBS solution. Add 5 mL of TrypLE Select enzyme to each culture dish and place it in the incubator for digestion for 7 minutes. Add 5 mL of basal culture medium to terminate the digestion process, gently tap the edge of the culture plate to dissociate the cells, and transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge at 1250 rpm for 4 minutes at room temperature, discard the supernatant, and resuspend the cell clumps with basal culture medium. Filter the suspension with a 40 μm filter to obtain iPSCs single cell suspension, and use a 1000 μL pipette tip to gently pipette 35 times to obtain a single cell suspension. Take 20 μL of the single cell suspension and mix it evenly with 20 μL of AO / PI dye solution, then count and record the live cell concentration and cell viability.

[0081] (2) Cell fixation: According to the counting results, add 1 mL of 4% paraformaldehyde for every 1×10^6 living cells, mix well and let stand for 10 minutes. Centrifuge at 500g for 4 minutes and discard the supernatant. Resuspend every 1×10^6 cells in 1 mL of PBS and centrifuge at 500g for 4 minutes. Repeat three times.

[0082] (3) Perforation: When the detection target is located inside the cell, the fixed cells need to be permeabilized. Add 0.5 mL of cell membrane perforation solution for every 1×10^6 fixed cells, mix well and let stand for 10 minutes. Centrifuge at 500g for 4 minutes, discard the supernatant, resuspend with 1 mL of PBS for every 1×10^6 cells, centrifuge at 500g for 4 minutes, and repeat twice.

[0083] (4) Primary antibody staining: Add 0.5 mL of blocking solution for every 1×10^6 cells, resuspend and dispense into 1.5 mL centrifuge tubes.

[0084] According to the experimental design and antibody dilution factor, add the corresponding volume of primary antibody to each tube. Mix well and stain overnight at 4°C.

[0085] (5) Flow cytometry: After staining, vortex the sample to mix well and centrifuge at 500g for 4 minutes. Discard the supernatant and add 500μL of washing solution to each tube to resuspend the cells and let stand for 5 minutes. Centrifuge at 500g for 4 minutes and repeat three times. After completion, add 250μL of PBS to each tube to resuspend the cells and detect them on a flow cytometer. The flow cytometry results are shown in Figure 2. Figure 4

[0086] Combination Figure 4 It can be seen that:

[0087] Through flow cytometry experiments, the marker genes OCT4 and SSEA4 of P15 iPS cells were detected. After removing dead cells and adherent cells through gating, a high proportion of single living cells expressing iPS marker genes (OCT4 95.88%, SSEA4 99.96%) were detected, proving that the iPS obtained by the above screening method has the characteristics of pluripotency gene expression.

[0088] 5. Immunofluorescence detection:

[0089] The steps include:

[0090] (1) Cell collection:

[0091] For the iPSCs cells to be tested, wash each 10 cm dish twice with 8 mL of PBS solution. Add 5 mL of TrypLE Select enzyme to each dish for cell digestion and incubate in the incubator for 7 minutes. Terminate the digestion process, add 5 mL of basal medium, and gently tap the edge of the culture plate to dissociate the cells. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1250 rpm for 4 minutes at room temperature. Discard the supernatant, resuspend the cell clumps with basal medium, and filter through a 40 μm filter to obtain a single cell suspension. Use a 1000 μL pipette tip to gently pipette 35 times to obtain a uniform single cell suspension. Take 20 μL of the single cell suspension and mix it evenly with 20 μL of AO / PI staining solution, count it, and record the concentration of live cells and cell viability.

[0092] (2) Cytological smear:

[0093] Use a pipette tip to spread the cells evenly on the slide.

[0094] (3) Cell fixation:

[0095] According to the counting results, add 1 mL of 4% paraformaldehyde for every 1×10^6 living cells, mix well and let stand for 10 minutes. Centrifuge at 500g for 4 minutes and discard the supernatant. Resuspend every 1×10^6 cells with 1 mL of PBS and centrifuge at 500g for 4 minutes. Repeat three times.

[0096] (4) Perforation:

[0097] When the detection target is located inside the cell, perform cell membrane permeabilization on the fixed cells. Add 0.5 mL of cell membrane permeabilization solution for every 1×10^6 fixed cells, mix well and let stand for 10 minutes. Centrifuge at 500g for 4 minutes, discard the supernatant, resuspend with 1 mL of PBS for every 1×10^6 cells, centrifuge at 500g for 4 minutes, and repeat twice.

[0098] (5) Primary antibody staining:

[0099] Add 0.5 mL of blocking solution to each 1×10^6 cells for resuspending and aliquot into 1.5 mL centrifuge tubes. Add the corresponding volume of primary antibody to each tube according to the experimental design and antibody dilution factor. Mix well and stain overnight at 4°C.

[0100] (6) Secondary antibody staining:

[0101] After the primary antibody staining is completed, remove the sample, shake and mix, and centrifuge at 500g for 4 minutes. Discard the supernatant, add 500μL of washing solution to each tube to resuspend the cells, let it stand for 5 minutes, centrifuge at 500g for 4 minutes, and repeat three times. After completion, add 250μL of blocking solution to each tube to resuspend the cells, and add the corresponding volume of secondary antibody to each tube according to the experimental design and antibody dilution factor. After mixing, place it in a dark environment at room temperature for 2 hours.

[0102] (7) DAPI staining:

[0103] After secondary antibody staining, shake and mix, centrifuge at 500g for 4 minutes. Discard the supernatant, add 500μL of washing solution to each tube to resuspend the cells, let stand for 5 minutes, centrifuge at 500g for 4 minutes, repeat three times. After completion, add 200-250μL of DAPI (diluted 1:10 with blocking solution) to each tube and incubate at room temperature for 10 minutes.

[0104] (8) Immunofluorescence detection:

[0105] After staining, shake and mix, centrifuge at 500g for 4 minutes. Discard the supernatant, add 500μL washing solution to each tube to resuspend the cells, let stand for 5 minutes, centrifuge at 500g for 4 minutes, repeat three times. After completion, add 100μL anti-fluorescence quenching mounting agent to each slide and seal with a coverslip. Observe under a confocal microscope.

[0106] Test results:

[0107] like Figure 5 As shown, the immunofluorescence labeling method visually shows that nearly 100% of the P21 iPS cells express the iPS cell marker genes OCT4, TRA-1-60 and TRA-1-81, further proving that the iPS obtained by the above screening method has the characteristics of pluripotency gene expression.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for reprogramming hair follicle stem cells into induced pluripotent stem cells, characterized in that: The steps include: 1) Add 4-in-1 CoMiP plasmid to the cell suspension of hair follicle stem cells, mix, transfer to an electroporation cup, add electroporation reagent, and place in an electroporator for electroporation; 2) inoculating the cells obtained after the electroporation treatment in step 1) into a culture container and culturing them using a first culture medium; 3) replacing the culture medium with the second culture medium, the third culture medium and the fourth culture medium in sequence, and continuing the culture; 4) picking up monoclonal cells and inoculating them into a new culture container, and culturing them using the fifth culture medium; 5) replacing the fifth culture medium with the sixth culture medium to obtain induced pluripotent stem cells; The first culture medium is BRSC complete culture medium; The second culture medium is a BRSC complete culture medium containing sodium butyrate and ascorbic acid; The third culture medium is a mixture of the second culture medium and mTeSR-E7 culture medium; The fourth culture medium is mTeSR-E7 culture medium containing sodium butyrate and ascorbic acid; The fifth culture medium is E8 culture medium containing ROCK inhibitor; The sixth culture medium is E8 culture medium.

2. The method for reprogramming hair follicle stem cells into induced pluripotent stem cells according to claim 1, characterized in that: The first culture medium, the second culture medium and the third culture medium are cultured for 2 days; The fourth culture medium is cultured until obvious clones appear, the culture time is 20-30 days, and the medium is changed every two days during the culture process; The fifth culture medium is used on the day of cell passaging or recovery.

3. The method for reprogramming hair follicle stem cells into induced pluripotent stem cells according to claim 1, characterized in that: In the third culture medium, the mass ratio of the second culture medium to the mTeSR-E7 culture medium is 1:1; The concentration of ROCK inhibitor in the fifth culture medium is 10 μM; In the second culture medium and / or the fourth culture medium, the concentration of sodium butyrate is 0.2 mM, and the concentration of ascorbic acid is 50 μg / mL.

4. The method for reprogramming hair follicle stem cells into induced pluripotent stem cells according to claim 1, characterized in that: In step 1), the density of the hair follicle stem cells in the cell suspension is 10 million / ml, and the dosage ratio of the 4-in-1 CoMiP plasmid is 12 μg / 100 μL of the cell suspension.

5. The method for reprogramming hair follicle stem cells into induced pluripotent stem cells according to claim 1, characterized in that: In step 2), the inoculation density is 0.5×10 5 ~2×10 5 cells / well.

6. The method for reprogramming hair follicle stem cells into induced pluripotent stem cells according to claim 1, characterized in that: In step 2), the culture container is a culture plate pre-coated with Matrigel or Cellstart.

7. The method for reprogramming hair follicle stem cells into induced pluripotent stem cells according to claim 1, characterized in that: The ROCK inhibitor is Y-27632.

Images