CN106834213B - Induction medium and induction method for inducing pluripotent stem cells to form hair papilla cells - Google Patents
Induction medium and induction method for inducing pluripotent stem cells to form hair papilla cells Download PDFInfo
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
Abstract
The invention provides an induction medium for inducing pluripotent stem cells to form hair papilla cells, which comprises the following components in concentration: adenine 150 mmol/l, FGF-24-8 ng/ml, glutamine 1-4mmol/ml, hydrocortisone 0.2-0.5. mu.g/ml, insulin 8-12. mu.g/ml, transferrin 80-150. mu.g/ml and sodium selenite 8-12 ng/ml. The induction culture medium is based on a Williams E culture medium, increases the probability of inducing pluripotent stem cells (ips) to differentiate into hair papilla cells by adding certain specific cytokines and supplements, and provides a seed cell source for skin tissue engineering. The present invention also provides a method for inducing pluripotent stem cells into hair papilla cells, comprising inoculating an embryoid body of the induced pluripotent stem cells into an induction medium, and inducing and culturing the embryoid body to form the hair papilla cells.
Description
Technical Field
The invention relates to an induction culture medium and an induction method, in particular to an induction culture medium and an induction method for inducing pluripotent stem cells to form papilla cells.
Background
Skin tissue engineering is a research hotspot in recent years, but related cells of skin all belong to terminally differentiated cells, large-scale culture and amplification are difficult to perform in vitro, the development of the tissue engineering is hindered due to the source problem of seed cells, the source of autologous seed cells is limited, the rejection reaction exists among allogeneic seed cells, and the ethical problem also exists among stem cells, so that the problem of the source of the seeds of the skin tissue engineering can be solved by collecting urine cells from urine, inducing the urine cells into IPS cells and inducing the IPS cells into keratinocytes, fibroblasts, sweat gland cells, hair papilla cells and the like, cells are obtained from the urine, and the method is convenient and rapid, is not required to be obtained through wounds, and has wide sources.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an induction culture medium and an induction method for inducing pluripotent stem cells to form papilla cells.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: an induction medium for inducing pluripotent stem cells to form hair papilla cells, the induction medium comprising the following concentrations of the components:
wherein FGF-2 is fibroblast growth factor-2.
The induction culture medium for inducing the pluripotent stem cells to form the hair papilla cells can efficiently induce the pluripotent stem cells to be differentiated into the hair papilla cells, and meanwhile, no serum is used in the whole process, so that the risk caused by animal-derived pathogens is effectively avoided, and the safety of clinical application is improved.
Preferably, the medium for inducing pluripotent stem cells to form hair papilla cells comprises the following components in concentration:
the induction culture medium and the induction method for inducing the pluripotent stem cells to form the hair papilla cells can induce the pluripotent stem cells to be differentiated into the hair papilla cells to the greatest extent and most efficiently.
Preferably, the induction medium further comprises a basal medium, and the basal medium is Williams E.
The induction culture medium is based on Williams E culture medium, increases the probability of inducing pluripotent stem cells (ips) to differentiate into hair papilla cells by adding certain specific cytokines and supplements, and provides a seed cell source for skin tissue engineering.
The present invention also provides a method for inducing pluripotent stem cells to form hair papilla cells, comprising inoculating an embryoid body of the induced pluripotent stem cells into the induction medium and culturing the embryoid body.
Preferably, the induction culture temperature is 25-28 ℃.
Preferably, the induction culture time is 6-10 days.
Preferably, the induction culture time is 8 days.
Preferably, the embryoid-like body is obtained by inoculating a single revived induced pluripotent stem cell into a culture medium containing the embryoid-like body, centrifuging the culture medium, and culturing the culture medium in an incubator for 48 hours to form the embryoid-like body.
Preferably, the single resuscitation induced pluripotent stem cell is inoculated in an amount of 1 × 106Individual cells/well.
Preferably, the parameter conditions of the plate centrifugation are: 700r/min, 5 min.
Preferably, the temperature of the incubator is 37 ℃ and the carbon dioxide content is 5% CO2。
Specifically, the method for obtaining the embryoid body comprises the following steps: (1) and recovering the cells: recovering P30 IPS cells, culturing with matrix-free human embryonic stem cell culture medium (mTeSR1), digesting with Accutase cell digestive juice to obtain single cells when the cells reach 80% confluency, and counting; (2) then, on a per AggreWell basisTMAdding 1X 10 to 800 culture plate hole6Adding EB into the cells to form a culture medium, and mixing AggreWell according to the product operating manualTM800 culture plates were centrifuged at 700r/min for 5min and then placed in a chamber containing 5% CO2Culturing in an incubator at 37 ℃ for 48h to form a simple embryoid body.
In another aspect, the present invention provides a use of the medium for inducing pluripotent stem cells to form hair papilla cells, wherein the medium is used for inducing pluripotent stem cells to form hair papilla cells.
The invention has the beneficial effects that: the invention provides an induction culture medium and an induction method for inducing pluripotent stem cells to form hair papilla cells, and the induction culture medium and the induction method can obviously improve the differentiation probability of the induced pluripotent stem cells (ips) to the hair papilla cells and provide a seed cell source for skin tissue engineering.
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FIG. 1 is a histogram of the days required to induce a change in shape of a simple embryoid body in an induction medium of the present invention and a control medium in example 1 of the present invention;
FIG. 2 is a graph showing the chemical staining of hair papilla cells after induction in the induction medium of the present invention and the control medium;
FIG. 3 is a graph comparing the proliferation rate curves of papilla cells in the induction medium of the present invention and the control medium.
Detailed Description
To better illustrate the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
Example 1
An induction medium for inducing pluripotent stem cells to form hair papilla cells, the induction medium comprising the following concentrations of the components:
example 2
An induction medium for inducing pluripotent stem cells to form hair papilla cells, the induction medium comprising the following concentrations of the components:
example 3
An induction medium for inducing pluripotent stem cells to form hair papilla cells, comprising the following components in concentration:
example 4 in vitro Induction of ips differentiation into dermal papilla cells
First, preparation of experimental group culture medium
The culture medium for inducing pluripotent stem cells to form hair papilla cells according to the present invention was prepared by adding 1mol of glutamine, 5mg of insulin, 200. mu.g of hydrocortisone, 50mg of transferrin, 5mg of sodium selenite, 23 mg of FGF-and 100mmol of adenine to 500ml of Williams E medium, and was used as the culture medium in the experimental group in the following experiments.
Second, preparation of control group culture medium
A control medium was prepared by adding 10ml of FBS to 500ml of Williams E medium.
Thirdly, reviving the cells
The IPS cells of P30 were selected for recovery, cultured in matrix-free human embryonic stem cell medium (mTeSR1), and when the cells reached 80% confluence, they were digested into single cells with Accutase cell digest, and counted.
Fourthly, forming an embryoid body: add 1X 10 to each AggreWellTM800 plate well6Adding EB into the cells to form a culture medium, and mixing AggreWell according to the product operating manualTM800 culture plates were centrifuged at 700r/min for 5min and then placed in a chamber containing 5% CO2Culturing in an incubator at 37 ℃ for 48h to form an embryoid body.
Fifthly, inducing differentiation
Transferring the embryoid body to a low-adsorption 24-pore plate, dividing the 24-pore plate into two groups, namely an experimental group and a control group, wherein each group has 12 pores, culturing the experimental group by using an experimental group culture medium, culturing the control group by using a control group culture medium, changing liquid every other day, recording the deformation of cells in the next day, selecting the deformed cells, carrying out cytochemical staining to identify a hair papilla cell phase, determining that the hair papilla cells are subjected to continuous culture after passage, carrying out cell counting every passage, and comparing the cell proliferation speeds of the two groups.
The specific method for identifying the hair papilla cell phase by adopting cytochemical staining comprises the following steps: preparing buffer solution with pH of 0.5-6.0 with 0.2N hydrochloric acid and 0.2M disodium hydrogen phosphate, preparing toluidine blue into 0.1% solution (pH of 0.5-6.0) and 1% solution (pH of 0.5-4.0) with buffer solution every 0.5 gradient), fixing papilla cell slide, washing with distilled water for 2 times, adding the above two staining solutions with different pH values, respectively, allowing to act for 30 min, washing with water, baking, sealing, and observing to compare the number of stained cells in control group and experimental group.
Sixthly, the results
As shown in fig. 1, when ips are induced to form papillary cells by using the control culture medium, the number of days required for cell deformation to occur after induction is 15 days, whereas the number of days required for cell deformation to occur after pluripotent stem cells are induced to form papillary cells by using the induction culture medium of the present invention is only 8 days, which indicates that the time for cell deformation to occur in the experimental group is earlier than that in the control group;
as shown in fig. 2, the number of stained cells in the experimental group is greater than that in the control group, that is, the number of hair papilla cells induced by the induction medium of the present invention is greater than that induced by the control group;
furthermore, as can be seen in fig. 3, the proliferation rate of the cells in the experimental group culture medium was faster than that of the cells in the control group culture medium;
therefore, the experimental results show that the cytokine and the supplement added on the basic culture medium of the control group achieve the composite synergistic effect, the probability of inducing ips cells into hair papilla cells can be improved, the passage can be stabilized, the proliferation speed of the cells can be improved, a seed cell source is provided for skin tissue engineering, and meanwhile, no serum is used in the whole process, so that the risk caused by animal-derived pathogens is effectively avoided, and the safety of clinical application is improved.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (9)
1. An induction medium for inducing pluripotent stem cells to form hair papilla cells, which is characterized by consisting of the following components in concentration and a basic culture solution, wherein the basic culture solution is Williams E; the components are respectively as follows:
2. the induction medium for inducing pluripotent stem cells to form papilla cells according to claim 1, wherein the induction medium comprises the following components in concentrations and a basal medium, wherein the basal medium is Williams E; the components are respectively as follows:
3. an induction method for inducing pluripotent stem cells to form hair papilla cells, comprising inoculating embryoid bodies of the induced pluripotent stem cells into the induction medium according to claim 1, and culturing the embryoid bodies under induction.
4. The induction method according to claim 3, wherein the induction culture time is 6 to 10 days.
5. The induction method according to claim 3, wherein the induction culture time is 8 days.
6. The induction method of claim 3, wherein the embryoid-like bodies are obtained by inoculating a single revived induced pluripotent stem cell into a medium containing a embryoid-like body, centrifuging the cell, and culturing the cell in an incubator for 48 hours to form the embryoid-like bodies.
7. The induction method according to claim 6, wherein the inoculation amount of the induced pluripotent stem cells for single resuscitation is 1 x 106Individual cells/well.
8. The induction method according to claim 6, characterized in that the parameter conditions of the centrifugation are: 700r/min, 5 min.
9. Use of the induction medium for inducing pluripotent stem cells to form hair papilla cells according to claim 1 or 2 for inducing pluripotent stem cells to form hair papilla cells.
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Citations (3)
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| CN103911339A (en) * | 2013-01-06 | 2014-07-09 | 陕西博鸿生物科技有限公司 | Serum-free fibroblast cell culture medium and preparation method thereof |
| CN106244527A (en) * | 2016-08-29 | 2016-12-21 | 广东依浦赛生物科技有限公司 | People source iPS stem cell in vitro directed differentiation is test kit and the method for myocardial cell |
| WO2016210313A1 (en) * | 2015-06-24 | 2016-12-29 | Whitehead Institute For Biomedical Research | Culture medium for generating microglia from pluripotent stem cells and related methods |
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| CN103911339A (en) * | 2013-01-06 | 2014-07-09 | 陕西博鸿生物科技有限公司 | Serum-free fibroblast cell culture medium and preparation method thereof |
| WO2016210313A1 (en) * | 2015-06-24 | 2016-12-29 | Whitehead Institute For Biomedical Research | Culture medium for generating microglia from pluripotent stem cells and related methods |
| CN106244527A (en) * | 2016-08-29 | 2016-12-21 | 广东依浦赛生物科技有限公司 | People source iPS stem cell in vitro directed differentiation is test kit and the method for myocardial cell |
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| Title |
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| 诱导多能干细胞治疗脱发研究新进展;罗雯，等;《医学综述》;20160731;第22卷(第14期);第2745-2747页 * |
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