CN108373990B - Culture medium for inducing differentiation from human amniotic epithelial stem cells to melanocytes and method thereof - Google Patents
Culture medium for inducing differentiation from human amniotic epithelial stem cells to melanocytes and method thereof Download PDFInfo
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Abstract
The invention relates to a medium for inducing differentiation from human amniotic epithelial stem cells to melanocytes and a method thereof. Endothelin 3, dexamethasone, stem cell factor, basic fibroblast growth factor, insulin iron selenium transfer protein and fetal calf serum are added into an alpha-MEM basic culture medium. The method is that the human amniotic membrane is prepared into a human amniotic epithelial stem cell suspension; placing the cells in an alpha-MEM culture medium added with endothelin 3, dexamethasone, stem cell factors, basic fibroblast growth factors, insulin iron selenium transfer protein and fetal calf serum; at 37 ℃ 5% CO2Culturing the amnion epithelial stem cell adherent monolayer in an incubator, and gradually inducing and differentiating into melanocytes positive for expressing melanocyte marker genes tyrosinase related protein l (trp 1), 2 (trp 2), microphthalmia-related transcription factor (mitf) and tyrosinase (dtc). Has the characteristics of rich cell source, low immunogenicity, no ethical limitation and simple and convenient induction method.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a culture medium for differentiating epithelial stem cells into melanocytes and a method thereof, in particular to a culture medium for inducing differentiation from human amniotic epithelial stem cells into melanocytes and a method thereof.
Background
Skin pigmentation plays a vital role in human health and skin aesthetics, especially in the exposed areas of the face. Because the pigment synthesized by melanocyte can protect the body from the attack of environment and the invasion of various dangerous cells, the loss of skin pigment not only can cause immune diseases such as leucoderma, etc., but also can affect the mental health of patients.
Vitiligo is a common acquired limited or generalized skin depigmentation disease. All parts of the body can occur due to the disappearance of the melanocyte function of the skin, and are commonly seen around the back, wrist, forearm, face, neck and genitals. The mechanism is not clear. Besides the medicine treatment, the decolorization therapy, the physical therapy and the feasible autologous epidermal transplantation operation of patients with no progress in skin lesion, the invention also discloses an autologous melanocyte transplantation treatment method such as a method for directionally differentiating autologous adipose-derived mesenchymal stem cells into melanocytes (patent No. CN 201710169785.9). Not only the adipose-derived mesenchymal stem cells are derived from mesoderm, but also new leucoderma is formed on the part of the wound surface formed by taking materials when the autologous normal skin of a patient is taken to induce melanocyte. The human amniotic epithelial stem cells and the melanocytes are both derived from ectodermal neural crest and are more easily differentiated into the melanocytes. Therefore, allogeneic melanocyte transplantation therapy has become one of the most preferred treatment regimens.
Stem cells are undifferentiated cells and have a multipotent differentiation potential and can be differentiated to form a variety of specific types of target cells. Among many stem cells, the human amniotic epithelial stem cells and the melanocytes are derived from ectodermal neural crest, and have the advantages of low immunogenicity, convenient material acquisition and the like, so the human amniotic epithelial stem cells and the melanocytes must become target cells which are preferentially selected for treating leucoderma by allogeneic melanocyte transplantation.
Disclosure of Invention
The invention provides a culture medium for inducing differentiation from human amniotic epithelial stem cells to melanocytes and a method thereof, which have the problems of wide allogeneic source and no or low immunogenicity.
The technical scheme of the invention is as follows:
the medium for inducing and differentiating the human amniotic epithelial stem cells into the melanocytes comprises:
the method for inducing and differentiating the human amniotic epithelial stem cells into the melanocytes comprises the following steps:
(1) separation of human amniotic epithelial stem cells and preparation of single cell suspension
Mechanically stripping amnion tissue, repeatedly washing with physiological saline to remove surface blood, and collecting human amnion 5 × 5cm2Cutting, and placing at 15 × 15cm2Adding 0.25g/L trypsin into a plate, digesting for 20-35 minutes at 37 ℃, stopping digestion by using 10-20% fetal calf serum, collecting supernatant, digesting for 7-12 minutes by using the same method, stopping digestion, mixing the 2 collected supernatants, sieving by using a 200-mesh cell sieve, centrifuging the filtrate for 8-12 minutes under the conditions of 800 and 1200 revolutions/minute, and discarding the supernatant to obtain cell suspension; filtering the cell suspension to prepare a single cell suspension;
(2) culture and amplification of human amniotic epithelial stem cells
Inoculating the cells obtained in step (1) into DMEM/F12 medium containing 10% fetal bovine serum, and standing at 37 deg.C and saturated humidity with 5% volume fraction of CO2Culturing in an incubator, and obtaining 2-3 generation human amniotic epithelial stem cells through solution change and amplification;
in the above step (2), the cells obtained in the step (1) are cultured at 2X 106L-1-2×107L-1Inoculating the strain in a culture medium at a certain density for culture;
(3) induced differentiation of human amniotic epithelial stem cells
Inoculating 2-3 generation human amniotic epithelial stem cells into a 12-well plate containing DMEM/12 culture medium containing 10-20% fetal calf serum, culturing until the cells are fused to above 80%, replacing melanocyte induced differentiation culture medium, standing at 37 deg.C and saturated humidity, and volume fraction of CO 5%2Culturing in incubator, changing culture solution 1 time every 2-3 days, and continuously culturing for 80-120 days;
the melanocyte induced differentiation culture medium adopts 500ml of alpha-MEM liquid culture medium, wherein the culture medium contains 3100 nM endothelin, 50nM dexamethasone, 50nM stem cell factor, 4nM basic fibroblast growth factor, 1X insulin iron selenium transfer protein and 10% fetal calf serum;
in the above step (3), the cells obtained in the step (2) are cultured at 2X 106L-1-2×107L-1The density is inoculated in a culture medium for culture.
The method for inducing differentiation of the amniotic epithelial stem cells to the melanocytes further comprises the step of deriving the human amniotic epithelial stem cells and the melanocytes from ectoderm.
The invention has the advantages that the human amniotic epithelial cells and the human melanocytes are both derived from ectoderm, the induced differentiation method is simple and easy, and the human amniotic epithelial stem cells are derived from human amnion. The human amniotic membrane is waste after birth of the fetus, is convenient to obtain materials, has wide sources and obvious economic benefit; low immunogenicity, no ethical limitation and the like. The melanocyte obtained after induction can be used for treating allogenic vitiligo diseases, and the treatment effect is very obvious.
Drawings
FIG. 1 is a diagram showing morphological observation (. times.40) of amniotic epithelial stem cells after 24 hours of primary isolation culture.
FIG. 2 is a 2 nd generation amniotic epithelial stem cell morphology observation (x 40) diagram.
FIG. 3 is an inverted microscopic view (X40) of the morphology of human amniotic epithelial stem cells before induction into melanocytes.
FIG. 4 is an inverted microscope (magnification:. times.40) image of the morphology of human amniotic epithelial stem cells after 60 days of induction into melanocytes.
FIG. 5 is a photograph showing the visible pigment particles (x 40) observed by inverted microscope observation of cell morphology after 100 days of induction of human amniotic epithelial stem cells to melanocytes.
FIG. 6 is a Western-blot analysis chart for identifying the expression (P < 0.05) of black pigment cell marker gene trp1 in human amniotic epithelial stem cells, which are not induced, induced and differentiated for 60 days and induced and differentiated for 100 days.
FIG. 7 is a Western-blot analysis chart for identifying the expression (P < 0.05) of a black pigment cell marker gene trp2 in human amniotic epithelial stem cells which are not induced, induced and differentiated for 60 days and induced and differentiated for 100 days.
FIG. 8 is a graph of Real-time PCR identification of the expression of melanocyte marker genes trp1, trp2, mitf and dtc (P < 0.05) in human amniotic epithelial stem cells which are not induced, induced and differentiated for 60 days and induced and differentiated for 100 days.
FIG. 9 is a graph showing immunofluorescence staining expression (X100) of a melanocyte marker gene trp1 after differentiation induction of human amniotic epithelial stem cells by immunofluorescence assay.
FIG. 10 is a graph showing immunofluorescence staining expression (X100) of a melanocyte marker gene trp2 after differentiation induction of human amniotic epithelial stem cells by immunofluorescence assay.
Detailed Description
The invention is described in detail below with reference to the figures and examples:
example 1
The invention relates to a medium for inducing differentiation from human amniotic epithelial stem cells to melanocytes, which comprises the following components:
an aqueous solution was prepared.
Example 2
The invention relates to a medium for inducing differentiation from human amniotic epithelial stem cells to melanocytes, which comprises the following components:
an aqueous solution was prepared.
Example 3
The invention relates to a medium for inducing differentiation from human amniotic epithelial stem cells to melanocytes, which comprises the following components:
an aqueous solution was prepared.
Example 4
The method for inducing and differentiating the amniotic epithelial stem cells into the melanocytes comprises the following steps:
(1) separation of human amniotic epithelial stem cells and preparation of single cell suspension
Taking human placenta of normal full-term cesarean delivery fetus (male) under aseptic condition; detecting that other related infectious indexes such as hepatitis A antibody, hepatitis B virus surface antigen, hepatitis B virus surface antibody, hepatitis B virus e antigen, hepatitis B virus e antibody, hepatitis B core antibody IgM, hepatitis C antibody, hepatitis E antibody, AIDS virus antibody, treponema pallidum antibody and the like are negative; blunt separating amnion on umbilical cord surface of placenta on sterile super clean bench, repeatedly washing with physiological saline, removing surface blood stain, and collecting human amnion 5 × 5cm2Cutting, and placing at 15 × 15cm2Adding 0.25g/L trypsin into a plate, digesting for 20-35 minutes at 37 ℃, stopping digestion by using 10-20% fetal calf serum, collecting supernatant, digesting for 7-12 minutes by using the same method, stopping digestion, mixing the 2 collected supernatants, filtering by using a 200-mesh cell sieve, centrifuging the filtrate for 8-12 minutes under the condition of 800-1200 rpm, and discarding the supernatant to prepare single cell suspension;
(2) culture and amplification of human amniotic epithelial stem cells
Inoculating the cells obtained in step (1) in DMEM/F12 medium containing 10% FBS, and maintaining at 37 deg.C, saturated humidity and 5% volume fraction CO2Culturing in an incubator for 48-72 hours, replacing culture solution, discarding non-adherent cells, replacing the culture solution once every 2-4 days according to the growth condition of the cells, and when the cells reach 80-90% fusion, carrying out passage according to the proportion of 1:2 or 1:3, and recording as P1 generation. Repeating the above operations for passage, wherein the passage culture is marked as P2 generation; the above procedure was repeated again for passage, and this passage was designated as P3. Obtaining 2-3 generation human amniotic epithelial stem cells through the liquid change and amplification;
in the above step (2), the cells obtained in the step (1) are cultured at 2X 106L-1-2×107L-1The density is inoculated in a culture medium for culture.
(3) Induced differentiation of human amniotic epithelial stem cells
Inoculating 2-3 generation human amniotic epithelial stem cells into a 12-well plate containing 10-20% FBS DMEM/12 culture medium, culturing until the cells are 80% -90% fused, replacing melanocyte induced differentiation culture medium, placing at 37 deg.C, saturation humidity, and volume fraction of 5% CO2Culturing in incubator, changing culture solution 1 time every 2-3 days, and continuously culturing for 80-120 days.
The melanocyte induction differentiation culture medium comprises 400ml of alpha-MEM liquid culture medium, wherein the alpha-MEM liquid culture medium contains 370 nM of endothelin, 30nM of dexamethasone, 30nM of stem cell factor, 1X of insulin iron selenium transfer protein, 3nM of basic fibroblast growth factor and 10% of fetal calf serum.
In the above step (3), the cells obtained in the step (2) are cultured at 2X 106L-1-2×107L-1The density is inoculated in a culture medium for culture.
Example 5
The invention adopts Western-blot to identify the expression of melanocyte marker genes trp1 and trp2, and comprises the following steps:
and respectively inducing the cells of the uninduced group and the melanocyte differentiation induction culture medium for 60 days and inducing the cells of the group for 100 days by using a Western-blot method to detect the melanocyte marker genes trp1 and trp 2.
1. The group without induction and the group with 100 days of induction of the melanocyte induced differentiation medium are respectively 3 multiplied by 106The density of individual cells/well was seeded into 6-well plates and replaced 1 time every 3 days after adherence. The induction period was 14 days.
2. Identification of melanocytes induced to form: protein was extracted using RIPA and protein quantification was performed by BCA method. Preparing 10% separation gel and 4% concentrated gel, loading, performing electrophoresis, performing membrane transfer at 100V for 1 hour, hybridizing trp1, trp2 and GAPDH primary antibody overnight at 4 ℃, incubating with corresponding secondary antibody for 1 hour, and developing with ECL. Expression of trp1 and trp2 was observed in the uninduced and induced groups.
Example 6
The invention adopts Real-time PCR to identify the expression of melanocyte marker genes trp1, trp2, mitf and dtc genes, and comprises the following steps:
respectively inducing the cells of the uninduced group and the melanocyte induced differentiation medium for 60 days and inducing the cells of the group for 100 days, and detecting the melanocyte marker genes trp1, trp2, mitf and dtc of the cells by adopting a Real-time PCR method.
1. The group without induction and the group with 100 days of induction of the melanocyte induced differentiation medium are respectively 3 multiplied by 106The density of individual cells/well was seeded into 6-well plates and replaced 1 time every 3 days after adherence. The induction period was 14 days.
2. Identification of induced melanocytes the RNA of the cells was extracted by Trizol method. The extracted RNA is reverse transcribed to synthesize the first strand cDNA. Reaction conditions are as follows: after melting at 95 ℃ for 20 seconds, 45 cycles were performed at 95 ℃ for 5 seconds and 60 ℃ for 20 seconds. Using the obtained cDNA as a template, Real-time PCR was performed on a fluorescent quantitative PCR apparatus to detect the expression levels of trp1, trp2, mitf, dtc and gapdh genes. The gapdh expression is used as an internal reference to calibrate the expression quantity of each experimental index, and the calculation method is 2–ΔΔCTRelative quantification method.
Example 7
The invention adopts an immunofluorescence method to identify the expression of melanocyte marker genes trp1 and trp2, and comprises the following steps:
after fixation of cells in uninduced group and induced group with 4% paraformaldehyde, respectively, cells were incubated overnight at 4 ℃ with primary antibody of trp1 and trp2 genes, and then incubated with FITC-labeled secondary antibody for 1 hour, washed with PBS, and observed with a fluorescence microscope.
1. Cell inoculation: cells of the uninduced group and the group cultured in a melanocyte-induced differentiation medium for 60 days and 100 days were cultured at 3X 105Inoculating into six-hole plate at density, and placing in 5% CO at 37 deg.C2Culturing in a saturated humidity incubator;
2. fixing: when the cells grow to 70-80% and are fused, removing the culture solution, washing twice with 1 XPBS, adding 4% paraformaldehyde solution, and fixing for 20 minutes at room temperature;
3. washing: absorbing paraformaldehyde completely, adding 1 × PBS, washing at 50rpm/min for 5 min, and repeating for three times;
4. and (3) sealing: adding blocking solution (1% calf serum albumin (BSA) in PBS), and blocking at room temperature for 30 min;
5. primary antibody incubation: primary anti-trp 1, trp2, diluted 1: 100 in 500. mu.l antibody dilution buffer (1 XPBS, 1% BSA) at 4 ℃ overnight;
6. washing: absorbing paraformaldehyde completely, adding 1 × PBS 1ml, shaking and washing on a shaking table for 5 minutes, and repeating for three times;
7. and (3) secondary antibody incubation: diluting the secondary antibody FITC-conjugated donkey anti-mouse and the tetramethyl isocetyl rhodamine (TRITC) -conjugated donkey anti-goat with 500. mu.l of antibody dilution buffer (1 XPBS, 1% BSA) 1: 500, and incubating for 1 hour at room temperature;
8. washing: absorbing paraformaldehyde completely, adding 1ml of 1 XPBS (phosphate buffer solution), washing at 50rpm/min for 5 minutes, and repeating for three times;
9. nuclear staining: with deionized water (ddH)2O) Dilute Diamidinophenylindole (DAPI) stock solutions at a ratio of 1: 1000, working solution concentration 1. mu.g/ml, stain for 1 min, using ddH2Washing for three times for 5 minutes each time;
10. and (4) observation: and (5) observing under a fluorescence microscope.
Example 8
The identification result of the directional induced differentiation of the human amniotic epithelial stem cells into the melanocytes is as follows:
1. as shown in figures 1, 2, 3, 4 and 5, during the induction and differentiation process of the human amniotic epithelial stem cells, the cell morphology of the human amniotic epithelial stem cells changes after 60 days of induction, the cells gradually become slender and in a long fusiform shape, and the transparency is reduced; after 100 days of cell induction, the cells exhibited a dendritic, multi-polarized morphology of melanocytes.
2. Western-blot detection of the expression of melanocyte markers trp1 and trp2 as shown in FIGS. 6 and 7: the results show that the expression of trp1 and trp2 increased with time after induction compared to the control group, P < 0.05.
3. As shown in FIG. 8, the results of Real-time PCR detection show that the expression of the melanocyte marker genes trp1, trp2, mitf and dtc is higher than that of the control group, and the expression of the induced trp1, trp2, mitf and dtc genes is higher with the time, and P is less than 0.05.
4. The immunofluorescence assay shows that the expression results of the melanocyte marker genes trp1 and trp2 show that the inducible groups trp1 and trp2 are positively expressed and the uninduced group is negatively expressed as shown in FIGS. 9 and 10.
The experimental identification results prove that the culture medium can directionally induce and differentiate the human amniotic epithelial stem cells into the melanocytes.
Example 9
The following table shows the comparison between the directional induced differentiation of human amniotic epithelial stem cells into melanocytes and the directional induced differentiation of adipose derived mesenchymal stem cells into melanocytes (patent number CN 201710169785.9)):
Claims (1)
1. a method for inducing differentiation from human amniotic epithelial stem cells to melanocytes is characterized by comprising the following steps:
(1) separation of human amniotic epithelial stem cells and preparation of single cell suspension
Taking human amniotic membrane, cutting into pieces, adding 0.25g/L trypsin, digesting for 20-35 minutes at 37 ℃, stopping digestion with 10-20% fetal calf serum, collecting supernatant, digesting for 7-12 minutes by the same method, stopping digestion, mixing the 2 collected supernatants, sieving with a 200-mesh cell sieve, centrifuging the filtrate for 8-12 minutes under the conditions of 800-1200 rpm, and removing the supernatant to obtain single cell suspension;
(2) culture and amplification of human amniotic epithelial stem cells
Inoculating the cells obtained in step (1) into Darbeck's modified eagle's/F12 medium containing 10% fetal bovine serum, and standing at 37 deg.C and saturated humidity with 5% volume fraction of CO2Culturing in an incubator, and obtaining 2-3 generation human amniotic epithelial stem cells through solution change and amplification;
in the above step (2), the cells obtained in the step (1) are cultured at 2X 106L-1-2×107L-1Density ofInoculating into a culture medium for culturing;
(3) induced differentiation of human amniotic epithelial stem cells
Inoculating 2-3 generation human amniotic epithelial stem cells into 12-well plate containing DMEM/F12 culture medium containing 10-20% fetal calf serum, culturing until cells are fused more than 80%, replacing melanocyte induced differentiation culture medium, standing at 37 deg.C and saturated humidity, and volume fraction of CO 5%2Culturing in incubator, changing culture solution 1 time every 2-3 days, and continuously culturing for 80-120 days;
the melanocyte induction differentiation culture medium adopts 500ml of alpha-MEM liquid culture medium 400-;
in the above step (3), the cells obtained in the step (2) are cultured at 2X 106L-1-2×107L-1Inoculating the strain in a culture medium at a certain density for culture;
the human amniotic epithelial stem cells and the melanocytes are both derived from ectoderm.
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| CN112481194B (en) * | 2020-12-02 | 2023-03-21 | 深圳清华大学研究院 | Method for preparing cell suspension with melanocyte activity |
| CN114181894A (en) * | 2021-12-09 | 2022-03-15 | 益诺思生物技术南通有限公司 | A kind of89Method for marking human amniotic epithelial stem cells by Zr |
| CN117646052A (en) * | 2023-12-04 | 2024-03-05 | 安徽科门生物科技有限公司 | Extraction and preparation method of stem cell active factor preparation for turning white hair into black hair |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102858955A (en) * | 2010-02-23 | 2013-01-02 | 国家医疗保健研究所 | Methods for preparing human melanocytes from human pluripotent stem cells |
| CN104039953A (en) * | 2011-09-30 | 2014-09-10 | 株式会社爱茉莉太平洋 | Melanocyte or progenitor cell thereof adapted to keratinocyte, and preparation method thereof |
| CN104232574A (en) * | 2014-09-15 | 2014-12-24 | 江苏大学附属医院 | Method for in-vitro directional differentiation inducing of mesenchymal stem cell towards melanocyte |
| CN107254431A (en) * | 2017-05-25 | 2017-10-17 | 武汉珺睿再生医学科技研究有限公司 | A kind of novel tissue engineering skin preparation method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050026279A1 (en) * | 2003-04-28 | 2005-02-03 | Tseng Scheffer C.G. | Surgical grafts and methods of preparation |
-
2018
- 2018-03-19 CN CN201810223838.5A patent/CN108373990B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102858955A (en) * | 2010-02-23 | 2013-01-02 | 国家医疗保健研究所 | Methods for preparing human melanocytes from human pluripotent stem cells |
| CN104039953A (en) * | 2011-09-30 | 2014-09-10 | 株式会社爱茉莉太平洋 | Melanocyte or progenitor cell thereof adapted to keratinocyte, and preparation method thereof |
| CN104232574A (en) * | 2014-09-15 | 2014-12-24 | 江苏大学附属医院 | Method for in-vitro directional differentiation inducing of mesenchymal stem cell towards melanocyte |
| CN107254431A (en) * | 2017-05-25 | 2017-10-17 | 武汉珺睿再生医学科技研究有限公司 | A kind of novel tissue engineering skin preparation method |
Non-Patent Citations (3)
| Title |
|---|
| Generation of Human Melanocytes from Induced Pluripotent Stem Cells;Shigeki Ohta et al.;《PLoS ONE》;20110113;第6卷(第1期);第1-10页 * |
| 人羊膜应用的研究进展;冯国纹等;《生物医学工程学杂志》;20140831;第31卷(第4期);第930-934页 * |
| 外胚间充质干细胞在构建含有黑色素细胞和血管样结构组织工程皮肤中的应用;刘源等;《中国科协第五届青年学术年会文集：科技、工程与经济社会协调发展》;20041031;第192-193页 * |
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