CN111662864A - Method for differentiating umbilical cord mesenchymal stem cells into dermal stem cells through in-vitro induction - Google Patents
Method for differentiating umbilical cord mesenchymal stem cells into dermal stem cells through in-vitro induction Download PDFInfo
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Abstract
A method for inducing umbilical cord mesenchymal stem cells to differentiate into dermal stem cells in vitro relates to the field of stem cells and cell culture. It is used for promoting hair follicle regeneration and improving alopecia, and can be combined with other stem cells and 3-D tissue engineering material to replace skin transplantation and repair injury. The method comprises the following steps: firstly, obtaining human umbilical cord mesenchymal stem cells, carrying out primary culture, digestion passage, amplification and cryopreservation; and secondly, inducing the hUC-MSCs to differentiate into SKPs cell suspension spheres, thus completing the process. The invention efficiently induces MSCs to be differentiated and induced into SKPs (mesenchymal stem cells) to replace dermal stem cells in skin with extremely limited sources, provides a new way for solving the problem of difficult collection of the dermal stem cells in clinic, and is clinically used for promoting hair follicle regeneration and improving alopecia. And combined with other stem cells and 3-D tissue engineering materials to replace skin transplantation and repair damage. The method is applied to the differentiation of the umbilical cord mesenchymal stem cells into the dermal stem cells in vitro.
Description
Technical Field
The invention belongs to the field of stem cell and cell culture, and particularly relates to a method for differentiating umbilical cord mesenchymal stem cells into dermal stem cells by in vitro induction.
Background
The skin is the largest organ of the human body and is the first barrier of the human body to contact the outside world. The large-area defect and deep injury caused by burns, scalds, wounds and chronic diseases such as diabetic complications cannot be repaired spontaneously, and artificial intervention treatment such as skin repair and skin grafting is needed. Because of the multiple limitations on the source and application of transplanted skin, there has been a growing search for practical skin substitutes.
On the other hand, alopecia, which is the most common one of skin diseases, causes great troubles in daily life for many patients. Pathologic hair loss refers to abnormal or excessive hair loss. It is essentially a manifestation of hair follicle cells being in a resting or catagen phase for a long period of time. With the increasing working pressure of life, alopecia is younger and younger.
Of course, the treatment of hair loss is initiated primarily by the cause, but many causes of hair loss are not completely removed. Local symptomatic medicine and systemic medicine, including blood circulation promotion and traditional Chinese medicine body resistance strengthening and constitution consolidating therapy, have limited curative effects. Hair transplantation is expensive and the transplanted hair is difficult to survive for a long period of time because the survival conditions of hair follicles are not sufficiently improved. There are many different stem cells present in the skin. Hair Follicle Stem Cells (HFSCs), which are present in the bulge region of the upper section of the hair follicle, are capable of differentiating into all the different types of cells of the follicle structure, thereby forming new hair. The hair follicle stem cells are mainly derived from Epidermal stem cells (Epidermal stem cells) distributed in the Epidermal basal layer, and the number of the hair follicle stem cells decreases with the age; the most important step of inducing human epidermal stem cells to differentiate into hair follicle stem cells is dermal stem cells, also called skin precursor cells (SKPs). SKPs are widely present in the papillary layer of the dermis and the perivascular region of the dermis, and the cells can grow in a suspension cell ball in a serum-free culture medium.
Researchers at the university of pennsylvania, america analyzed hair and scalp tissue from men aged 40 to 65 years. As a result, it was found that the number of Hair Follicle Stem Cells (HFSCs) was the same in the scalp tissue with or without hair loss. In contrast, hair follicle stem cells in the hair-losing scalp tissue are not transformed into hair follicle precursor cells, and lack of SKPs cell-inducing activity may be the cause thereof.
Therefore, the SKPs cell transplantation is expected to become an effective way for treating alopecia. Meanwhile, the artificial skin prepared by transplanting skin stem cells with different functions and combining the 3-D tissue engineering technology can replace skin grafting to treat severe skin injury.
However, SKPs derived from human cells are limited, especially in the elderly. Furthermore, in vitro culture, the expression of SKPs cell-associated markers and the ability to promote hair follicle regeneration rapidly decline with the number of passages. Therefore, a rich source of SKPs with strong activity is found, and the method has extremely great clinical application value.
Disclosure of Invention
The invention aims to provide a method for inducing umbilical cord mesenchymal stem cells to be differentiated into dermal stem cells in vitro, which is used for promoting hair follicle regeneration and improving alopecia, and further combined with other stem cells and 3-D tissue engineering materials to replace skin transplantation and repair damage.
A method for inducing umbilical cord mesenchymal stem cells to be differentiated into dermal stem cells in vitro is realized according to the following steps:
firstly, obtaining human umbilical cord mesenchymal stem cells, and performing primary culture, digestion passage, amplification and cryopreservation;
secondly, inducing the human umbilical cord mesenchymal stem cells to differentiate into dermal stem cell suspension balls, namely suspending the human umbilical cord mesenchymal stem cells in a dermal stem cell culture medium after the above digestion and passage, counting and diluting to the density of 1 × 105The single cell suspension is inoculated in a low-adsorption 6-hole culture plate according to 1.5 ml/hole, 10mM Y-27632 with the volume percentage of 1 per mill is added into the dermal stem cell culture medium, the liquid is changed once every 2 to 3 days, the culture is carried out for 6 days,collecting the dermal stem cell suspension ball, centrifuging at 300rpm for 2min, and suspending in a dermal stem cell culture medium to complete the in vitro induction of the differentiation of the umbilical cord mesenchymal stem cells into the dermal stem cells.
The invention has the advantages that:
1. the invention adopts a cell suspension culture mode for the first time, efficiently induces MSCs to differentiate and induce into SKPs cells, and provides a new way for clinically solving the problem of difficult collection of dermal stem cells. The method for inducing SKPs cells is expected to solve the problem that autologous SKPs cells of old people and alopecia patients have limited amplification capacity and cannot achieve treatment effect in the future.
2. The expression quantity of the characteristic marker genes Oct4, Nestin, SOX9 and Vimentin of the cell expression SKPs obtained by the invention in the cell suspension of induced differentiation SKPs is respectively 51, 21, 47 and 3 times of that of human umbilical cord Mesenchymal Stem Cells (MSCs).
3. The co-culture result of the SKPs and the human epidermal cells shows that the SKPs induced by differentiation can promote the transformation of the skin-worn cells to the direction of the epidermal stem cells/hair follicle stem cells, and the cultured cells express epidermal stem cell/hair follicle stem cell characteristic marker genes CD200, Sox9 and keratinocyte 19 which are respectively 3.8, 1.5 and 4.5 times of the individually cultured human skin cells. Therefore, the effect of promoting the differentiation of the hair follicle stem cells is successfully verified.
4. The invention fully utilizes abundant sources of human umbilical cord mesenchymal stem cells and the potential of various tissue cells differentiation, especially various cells in dermal connective tissues, to induce the differentiation into SKPs cells for replacing dermal stem cells in skin with extremely limited sources; is clinically used for promoting the regeneration of hair follicles and improving alopecia. And further combined with other stem cells and 3-D tissue engineering materials to replace skin transplantation to repair the injury.
The method is applied to the differentiation of the umbilical cord mesenchymal stem cells into the dermal stem cells in vitro.
Drawings
FIG. 1 is a microscope photograph of adherent culture of human umbilical cord Mesenchymal Stem Cells (MSCs) before induced differentiation in the examples;
FIG. 2 is a micrograph of single cells of MSCs at the time of inoculation in the example;
FIG. 3 is a microscope photograph of SKPs cell suspension spheres formed on day 3 after inoculation of single cells from MSCs in the examples;
FIG. 4 is a microscope photograph of SKPs cell suspension spheres formed on day 6 after inoculation of single cells from MSCs in the examples;
FIG. 5 is an immunofluorescence staining chart of the characteristic marker Nestin of SKPs, wherein the SKPs cell suspension balls induced to differentiate for 6 days in the example show strong expression;
FIG. 6 is an immunofluorescence staining pattern of a SKPs cell suspension ball with strong SKPs characteristic marker Vimentin, wherein the SKPs cell suspension ball is induced to differentiate for 6 days in the embodiment;
FIG. 7 is an immunofluorescence staining pattern of Nestin of human umbilical cord Mesenchymal Stem Cells (MSCs) before induction of differentiation in examples;
FIG. 8 is a photograph of immunofluorescence staining of Vimentin of human umbilical cord Mesenchymal Stem Cells (MSCs) before induced differentiation in the examples;
FIG. 9 is a bar graph of the expression levels of the genes involved in the real-time quantitative PCR examination of the examples;
FIG. 10 is a microscope photograph of human skin cells co-cultured with SKPs cell suspensions in the examples to generate the characteristic marker CD200 of epidermal stem cells/hair follicle stem cells;
FIG. 11 is a microscopic picture of the positive cell of Sox9, a characteristic marker for producing epidermal stem cells/hair follicle stem cells, of human skin cells co-cultured with SKPs cell suspension spheres in the examples;
FIG. 12 is a microscopic image of human skin cells cultured alone in the example to generate the characteristic marker CD200 of epidermal stem cells/hair follicle stem cells;
FIG. 13 is a microscopic image of the cultured human skin cells alone in the example producing cells positive for the characteristic marker Sox9 for epidermal stem cells/hair follicle stem cells;
fig. 14 is a bar graph of real-time quantitative PCR examination of expression levels of the epidermal stem cell/hair follicle stem cell characteristic markers CD200, Sox9 and keratin 19 in examples.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
The first embodiment is as follows: the embodiment provides a method for inducing umbilical cord mesenchymal stem cells to be differentiated into dermal stem cells in vitro, which is realized by the following steps:
firstly, obtaining human umbilical cord mesenchymal stem cells, and performing primary culture, digestion passage, amplification and cryopreservation;
secondly, inducing the human umbilical cord mesenchymal stem cells to differentiate into dermal stem cell suspension balls, namely suspending the human umbilical cord mesenchymal stem cells in a dermal stem cell culture medium after the above digestion and passage, counting and diluting to the density of 1 × 105And/ml, inoculating the single cell suspension into a low-adsorption 6-hole culture plate according to 1.5 ml/hole, adding 10mM Y-27632 with the volume percentage content of 1 per thousand into a dermal stem cell culture medium, changing the liquid once every 2-3 days, culturing for 6 days, collecting a dermal stem cell suspension ball, centrifuging at 300rpm for 2min, and suspending in the dermal stem cell culture medium to complete the in-vitro induction of the differentiation of the umbilical cord mesenchymal stem cells into the dermal stem cells.
In the first step of the embodiment, the primary culture of the human umbilical cord mesenchymal stem cells can be performed with cryopreservation and recovery culture if necessary, and the process comprises the steps of taking the human umbilical cord mesenchymal stem cells out of liquid nitrogen, thawing in water bath at 37 ℃, suspending in MSCs complete culture medium, centrifuging at 1000rpm for 5min, suspending in MSCs complete culture medium, and performing 1 × 104/cm2Inoculating at a density of 5% CO at 37 deg.C2Culturing in an incubator.
The second embodiment is as follows: the difference between the first embodiment and the second embodiment is that the specific operations of obtaining the human umbilical cord mesenchymal stem cells, performing primary culture, digestion and passage, amplification and cryopreservation in the step one are as follows:
a. the umbilical cord of the newborn is disinfected by alcohol with the volume percentage of 75 percent and then is cleaned for 2 times by normal saline;
b. cutting the cleaned umbilical cord into sections with the length of 2-3cm, and stripping the waffle gel in a plate;
c. placing the Huachuang gum in a centrifugal tube, and cutting into pieces of 1-2mm with surgical scissors3The block shape of (1);
d. inoculating to umbilical cord mesenchymal stem cell complete culture medium according to 2g/T75 flask, and culturing at 37 deg.C with 5% CO2Culturing in an incubator, changing the culture solution after 4-5 days, and changing the culture solution once every 3 days, and culturing until the cell fusion degree reaches more than 70 percent to complete primary culture;
e. digesting, passaging and amplifying human umbilical cord mesenchymal stem cells, namely sucking out a culture medium, washing with PBS once, then digesting with pancreatin with the volume percentage concentration of 0.25 percent for 4-6min, blowing and suspending, washing with DMEM/F12, centrifuging at 1000rpm for 3min, and then centrifuging at 1 × 104/cm2Suspending the mixture in a complete culture medium of umbilical cord mesenchymal stem cells in a density manner for passage and amplification culture;
f. freezing and storing: adding DMSO into the cell suspension to 10%, mixing, subpackaging in a freezing tube, freezing overnight at-80 deg.C by using a programmed cooling box, and transferring to a liquid nitrogen tank. Other steps and parameters are the same as those in the first embodiment.
The third concrete implementation mode: this embodiment differs from the second embodiment in that the umbilical cord mesenchymal stem cell complete medium (i.e., MSCs complete medium) has the following composition: 95% alphaMEM medium, 5% platelet lysate and 5IU/ml heparin sodium. Other steps and parameters are the same as those in the second embodiment.
The fourth concrete implementation mode: this embodiment differs from the first to third embodiments in that the composition of the dermal stem cell medium (i.e., SKPs medium) in step two: DMEM F12 ═ 1:1 basal medium supplemented with 1/50B27, 20ng/ml EGF, 20ng/ml bFGF, 0.2mM ascorbic acid and 2 μ g/ml gentamicin. Other steps and parameters are the same as those in one of the first to third embodiments. The fifth concrete implementation mode: this embodiment is different from the first to fourth embodiments in that the conditions for the culture in the second step are 37 ℃ and 5% CO2. Other steps and parameters are the same as in one of the first to fourth embodiments.
The beneficial effects of the present invention are demonstrated by the following examples:
example (b):
a method for inducing umbilical cord mesenchymal stem cells to be differentiated into dermal stem cells in vitro is realized according to the following steps:
firstly, obtaining human umbilical cord mesenchymal stem cells, and performing primary culture, digestion passage, amplification and cryopreservation;
secondly, inducing the human umbilical cord mesenchymal stem cells to differentiate into dermal stem cell suspension balls, namely suspending the human umbilical cord mesenchymal stem cells in a dermal stem cell culture medium after the above digestion and passage, counting and diluting to the density of 1 × 105And/ml, inoculating the single cell suspension into a low-adsorption 6-hole culture plate according to 1.5 ml/hole, adding 10mM Y-27632 with the volume percentage content of 1 per thousand into a dermal stem cell culture medium, changing the liquid once every 2-3 days, culturing for 6 days, collecting a dermal stem cell suspension ball, centrifuging at 300rpm for 2min, and suspending in the dermal stem cell culture medium to complete the in-vitro induction of the differentiation of the umbilical cord mesenchymal stem cells into the dermal stem cells.
In the first step of this example, the human umbilical cord mesenchymal stem cells may be cryopreserved after primary culture and then resuscitated if necessary by removing human umbilical cord mesenchymal stem cells from liquid nitrogen, thawing in a 37 ℃ water bath, suspending in MSCs complete medium, centrifuging at 1000rpm for 5min, suspending in MSCs complete medium, and performing 1 × 104/cm2Inoculating at a density of 5% CO at 37 deg.C2Culturing in an incubator.
In this embodiment, the method for obtaining human umbilical cord mesenchymal stem cells in step one includes the following specific operations of primary culture, digestion, passage, amplification and cryopreservation:
a. the umbilical cord of the newborn is disinfected by alcohol with the volume percentage of 75 percent and then is cleaned for 2 times by normal saline;
b. cutting the cleaned umbilical cord into sections with the length of 2-3cm, and stripping the waffle gel in a plate;
c. placing the Huachuang gum in a centrifugal tube, and cutting into pieces of 1-2mm with surgical scissors3The block shape of (1);
d. inoculating to umbilical cord mesenchymal stem cell complete culture medium according to 2g/T75 flask, and culturing at 37 deg.C with 5% CO2Culturing in an incubator, changing the culture solution after 4-5 days, and changing the culture solution once every 3 days, and culturing until the cell fusion degree reaches more than 70 percent to complete primary culture;
e. human umbilical cord mesenchymal stemCell digestion, passage and amplification, wherein the culture medium is aspirated, PBS is washed once, pancreatin with the volume percentage concentration of 0.25% is used for digestion for 4-6min, the suspension is blown, DMEM/F12 is washed, centrifugation is carried out for 3min at 1000rpm, and then 1 × 104/cm2Suspending the mixture in a complete culture medium of umbilical cord mesenchymal stem cells in a density manner for passage and amplification culture;
f. freezing and storing: adding DMSO into the cell suspension to 10%, mixing, subpackaging in a freezing tube, freezing overnight at-80 deg.C by using a programmed cooling box, and transferring to a liquid nitrogen tank;
wherein the composition of the umbilical cord mesenchymal stem cell complete culture medium (namely the MSCs complete culture medium) is as follows: 95% alphaMEM medium, 5% platelet lysate and 5IU/ml heparin sodium.
The composition of the dermal stem cell culture medium (i.e., SKPs culture medium) in step two of this example: DMEM F12 ═ 1:1 basal medium supplemented with 1/50B27, 20ng/ml EGF, 20ng/ml bFGF, 0.2mM ascorbic acid and 2 μ g/ml gentamicin.
The conditions for the culture in step two of this example were 37 ℃ and 5% CO2。
In the second step of this embodiment, the in vitro induction of differentiation of the umbilical cord mesenchymal stem cells into dermal stem cells can be performed as the first generation dermal stem cells for induced differentiation, and further passaging can be performed.
After the second step of this example, the inoculation of human skin cells was performed:
collecting human skin cells after recovery or digestion passage according to the ratio of 1.5 × 105Inoculation in/mlInsert nested type12 holesCultivation method Support boardAdding 10mM Y-27632 with volume percentage content of 1 per thousand into a skin cell culture medium, culturing for 4h, then inoculating 300 mul of the dermal stem cell suspension balls suspended in the dermal stem cell culture medium obtained in the step two, carrying out co-culture, changing liquid once every 2 days, after culturing for 6 days, respectively collecting dermal stem cells and human skin cells in the co-culture, extracting RNA, carrying out real-time quantitative PCR analysis, or carrying out immunofluorescence staining after 4% paraformaldehyde is fixed. To identify the effect of dermal stem cells on human skin cells.
The components of the skin cell culture medium are as follows: DMEM: F12 ═ 1:1 basal medium plus 5% fetal bovine serum, 1/100B27, 1/200ITS, 2mM glutamine, 10ng/ml EGF and 10ng/ml bFGF.
The CO-culture conditions were 37 ℃ and 5% CO2。
RNA, cDNA preparation and real-time quantitative PCR analysis:
each experiment was performed according to the instructions of each kit.
1) The Trizel method purifies RNA. Determining the RNA content;
2) and preparing cDNA by using the Toyo Boseki reverse transcription kit. Measuring the content of cDNA;
3) real-time quantitative PCR analysis was performed using TaKaRa TB Green Fast qPCR Mix.
Performing immunofluorescence staining on SKPs suspension culture specimens:
1) poly ornithine (PLO) coating 24 holes (with slide) >2hr, washing with PBS;
2) inoculating SKPs cell suspension balls in PLO holes;
3) the cell pellet was attached well after one day. PBS was washed once and fixed with 4% Paraformaldehyde (PFA). Freezing and slicing the SKPs after partial SKPs cell suspension ball embedding;
4) PBS washed X1. Rupture of the membrane for 30min by 0.1 percent Triton X-100;
5) blocking with 5% normal sheep serum (NGS) -PBS for 30 min;
6) add various primary antibodies (dissolved in 0.3% BSA-PBS). Incubating overnight at 4 ℃;
7) washing with PBS X1, washing with PBS (shaking table) for 15min X2;
8) secondary antibodies (Alexa Fluor 594-goat anti-mouse, Alexa Fluor 488 goat anti-rabbit, 1:500) were added. Incubating for 2h at room temperature;
9) x1 was washed with PBS and stained for 10min for DAPI nuclei. Washing with PBS (shaking table) for 15 min;
10) prolonggold coversheet;
11) observing under a microscope and taking a picture;
12) photographic image analysis, cell counting, statistical analysis.
As a result:
1 successfully inducing human umbilical cord mesenchymal stem cells (hUC-MSCs) to differentiate into cell suspension balls highly expressing the characteristic markers of SKPs. FIGS. 1 to 4 show umbilical cord Mesenchymal Stem Cells (MSCs) in culture, and SKPs cell suspensoids formed from single cells of MSCs (SKPs 0 days) at the time of inoculation, to days 3 and 6. The SKPs cell suspension ball on the sixth day is used for immunofluorescence staining and real-time quantitative PCR analysis; co-culturing with skin cells; and animal transplantation experiments. Scale bar 200 μm.
SKPs cell suspensions induced to differentiate for 6 days showed strong immunofluorescent staining for markers characteristic of SKPs (Nestin, Vimentin) (see FIGS. 5 and 6). Whereas the immunofluorescent staining for Nestin and Vimentin induced to differentiate into pre-human umbilical cord Mesenchymal Stem Cells (MSCs) was negative (see fig. 7 and 8, same exposure conditions). Scale bar 100 μm. The Vimentin counting result shows that more than 90% of the cells in the SKP cell suspension ball are positive.
Detecting related gene expression by real-time quantitative PCR: as shown in FIG. 9, the expression levels of Oct4, Nestin, SOX9, and Vimentin, which are markers characteristic of SKPs, in cell suspensions of induced-differentiation SKPs, were 51, 21, 47, and 3 times as high as those of human umbilical cord Mesenchymal Stem Cells (MSCs), respectively.
2. The SKPs cell suspension balls promote the generation of epidermal stem cells/hair follicle stem cells by the human skin cells in the co-culture, and compared with the human skin cells cultured alone, the human skin cells co-cultured with the SKPs cell suspension balls generate more epidermal stem cells/hair follicle stem cells.
It was shown that human skin cells co-cultured with SKPs cell suspension (see fig. 10 and 11) produced more cells positive for the markers CD200 and Sox9 characteristic of epidermal/hair follicle stem cells than human skin cells cultured alone (see fig. 12 and 13). Scale bar 200 μm. The percentage of positive cells is shown in table 1.
TABLE 1 immunohistochemical analysis Positive expression Rate of Hair follicle Stem cell marker protein after Co-culture
| Co-culture of skin cells and SKPs | Skin cell culture alone | |
| CD200 | 8.7％ | 0％ |
| SOX9 | 12.2％ | 4.1％ |
The real-time quantitative PCR results are shown in fig. 14, and the human skin cells cultured with SKPs cell suspension beads expressed larger amounts of the epidermal stem cell/hair follicle stem cell characteristic markers CD200, Sox9, and keratin 19 than the human skin cells cultured alone. The ratios are 3.8, 1.5 and 4.5 times, respectively.
Claims (5)
1. A method for inducing umbilical cord mesenchymal stem cells to be differentiated into dermal stem cells in vitro is characterized by comprising the following steps:
firstly, obtaining human umbilical cord mesenchymal stem cells, and performing primary culture, digestion passage, amplification and cryopreservation;
secondly, inducing the human umbilical cord mesenchymal stem cells to differentiate into dermal stem cell suspension balls, namely suspending the human umbilical cord mesenchymal stem cells in a dermal stem cell culture medium after the above digestion and passage, counting and diluting to the density of 1 × 105And/ml, inoculating the single cell suspension into a low-adsorption 6-hole culture plate according to 1.5 ml/hole, adding 10mMY-27632 with the volume percentage of 1 per thousand into a dermal stem cell culture medium, changing the liquid once every 2-3 days, culturing for 6 days, collecting a dermal stem cell suspension ball, centrifuging at 300rpm for 2min, and suspending in the dermal stem cell culture medium to complete the in-vitro induction of the differentiation of the umbilical cord mesenchymal stem cells into the dermal stem cells.
2. The method for inducing differentiation of umbilical cord mesenchymal stem cells into dermal stem cells in vitro according to claim 1, wherein the human umbilical cord mesenchymal stem cells are obtained in step one, and the specific operations of primary culture, digestion passage, amplification and cryopreservation are as follows:
a. the umbilical cord of the newborn is disinfected by alcohol with the volume percentage of 75 percent and then is cleaned for 2 times by normal saline;
b. cutting the cleaned umbilical cord into sections with the length of 2-3cm, and stripping the waffle gel in a plate;
c. placing the Huachuang gum in a centrifugal tube, and cutting into pieces of 1-2mm with surgical scissors3The block shape of (1);
d. inoculating to umbilical cord mesenchymal stem cell complete culture medium according to 2g/T75 flask, and culturing at 37 deg.C with 5% CO2Culturing in an incubator, changing the culture solution after 4-5 days, and changing the culture solution once every 3 days, and culturing until the cell fusion degree reaches more than 70 percent to complete primary culture;
e. digesting, passaging and amplifying human umbilical cord mesenchymal stem cells, namely sucking out a culture medium, washing with PBS once, then digesting with pancreatin with the volume percentage concentration of 0.25 percent for 4-6min, blowing and suspending, washing with DMEM/F12, centrifuging at 1000rpm for 3min, and then centrifuging at 1 × 104/cm2Suspending the mixture in a complete culture medium of umbilical cord mesenchymal stem cells in a density manner for passage and amplification culture;
f. freezing and storing: adding DMSO into the cell suspension to 10%, mixing, subpackaging in a freezing tube, freezing overnight at-80 deg.C by using a programmed cooling box, and transferring to a liquid nitrogen tank.
3. The method for inducing differentiation of umbilical cord mesenchymal stem cells into dermal stem cells in vitro according to claim 2, wherein the components of the umbilical cord mesenchymal stem cell complete medium are as follows: 95% alphaMEM medium, 5% platelet lysate and 5IU/ml heparin sodium.
4. The method for inducing differentiation of umbilical cord mesenchymal stem cells into dermal stem cells in vitro according to claim 1, wherein the components of the culture medium of dermal stem cells in step two are as follows: DMEM F12 ═ 1:1 basal medium supplemented with 1/50B27, 20ng/ml EGF, 20ng/ml bFGF, 0.2mM ascorbic acid and 2 μ g/ml gentamicin.
5. The method for inducing differentiation of umbilical cord mesenchymal stem cells into dermal stem cells in vitro according to claim 1, wherein the culturing conditions in the second step are 37 ℃ and 5% CO2。
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