Isolation,purification,culture and identification of epidermal stem cells
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The isolation and culture of putative epidermal stem cells by way of enzyme-digesting and direct-tissue-culturing from mouse,goat,milk cow and pig were conducted in this study.Results indicated that enzyme-digesting was more suitable for the isolation of mouse epidermal stem cells which self-differentiated into neural cells after two passages of in vitro culturing while cells from goat and cow lived more vigorously and maintained longer characteristics of epidermal stem cells than those from mouse and pig either by enzyme-digesting or tissue-culturing.Epidermal stem cells from goat were selected by collagen Ⅳ,cultured in serum-containing medium,identified by immunocytochemical staining and could be subcultured to at least passage nine but the cells grew weaker as the passage continued.Keywords:
Isolation
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Objective To investigate the isolation and culture of mouse epidermal stem cells. Methods Putative epidermal stem cells were isolated and selected by rapid adherence on type IV collagen matrix,and the cells were identified by immunohistochemistry staining mehod. Results The cells showed a spherical and polygonal appearance characteristics of typical epidermal cells. Cell pattern can be observed clearly and cytoplasm is homogeneous. The heteromorphism and bigger neuclear- cytoplamic ratio were also observed. The expression of K15 and Beta1 integrin were positive in cytoplasm of cells and the stained positive rates were 100%. In contrast,the expression of CD 34 was negative. Conclusion The isolated epidermal stem cells using a rapid adherence method have stem cell- like properties and offers a potential route to their clinical application.
Matrix (chemical analysis)
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This study was aimed to explore the suitable conditions for the isolation,culture of pig male germline stem cells,and establish the in vitro culture system.The cells were isolated from the neonatal pig test by the two step enzyme digestion method,and cultured for further identification,compared the enrichment efficiency of cells on laminin and gelatin due to the different adherence velocity.And then the cells were identified for the alkaline phosphatase activity and the expression of stem cell marker protein OCT-4.The results showed that laminin was more suitable for the enrichment and growth of porcine germline stem cells.The enrichment efficiency and proliferation rate of cells using laminin method was obviously superior to which using gelatin sorting.The cultured mGSCs had the similar morphology and proliferation characteristics to mGSCs of mouse.The expression of OCT-4was positive in target cells,while it was negative in feeder cells-sertoli cells.The target cells showed strong expression of alkaline phosphatase,while nothing was detected in feeder cells-sertoli cells.The results showed that,we had established the preliminary culture system of porcine germline stem cells,with well maintained stem cell activity,normal replication function and differentiation potentials.
Stem cell marker
Cell Sorting
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The study was to explore a feasible and efficient method for isolating rat epidermal stem cells.Direct-tissue culturing and enzyme-digesting were used to isolate and culture rat epidermal stem cells.Then,the epidermal stem cells were chosen by type IV collagen.The expressions of CK15,P63,β1-integrin and α6-integrin in ESCs were detected by immunohistochemistry staining.Meanwhile,the clone forming efficiency and the growth curve of epidermal stem cells were also detected.The epidermal stem cells were got by both methods,and the epidermal stem cells had higher colony forming efficiency.The cells got by direct-tissue culturing could be passaged to the 6th generation,while the cells got by enzyme digestion were differentiated after passaged to 3-4th generation.Immuno histochemistry staining showed that CK15,P63,β1-integrin and α6-integrin were strongly expressed in the cultured epidermal stem cells.Both methods could isolate,purify and culture rat epidermal stem cells successfully,but direct-tissue culturing is more suitable for the isolation of rat epidermal stem cells.
clone (Java method)
Amniotic stem cells
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Objective To explore a new method to isolate human epidermal stem cells and provide seed cells for the constitution of tissue engineering in skin. Methods Epidermal basal cells were cultured with common cell culture technique. Cell sorting technology was used based on the properties of epidermal stem cells identified with S P method of immunocytochemistry. Results Basal cells could be obtained by digestion with dispase and trypsin. Basal cells with density of (1-2)×10 4/ml were cultured on 3T3 feeder layer in DMEM containing epidermal growth factor(EGF) etc. Cell proliferation started 2 days later. Feeder layer was removed after a week and keratinocytes could be passaged at 10-12th day. A few round cells of similar size could be obtained after the basal cells marked with FITC and sorted by flow cytometry and these sorted cells were adhered by type IV collagen for 20 minutes. Monoclonal antibody to human K19 serving as first antibody, S P method of immunocytochemistry showed that the isolated cells positively stained with grown yellow were epidermal stem cells. Conclusion Human epidermal stem cells could be isolated from basal cells by fluorescence activated cell sorting and adhesion to type IV collagen, which provides the basis for further study of tissue engineering in skin.
Dispase
Cell Sorting
Stem cell marker
Epidermis (zoology)
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Objective:To confirm the location of epidermal stem cells of rats and to investigate the methods to isolate and culture epidermal stem cells in vitro.Methods:Immnohistochemical methods were used to confirm the location of rats' epidermal stem cells.Collagenase and trypsin solution were used to dissociate neonatal rats' skin into single cells.The rapidly adherent cells to rats' tail collagen were cultured to be investigated their clone forming rate and subcultured,while the others were cultured as controlled cells.And immunocytochemical staining was used to identify epidermal stem cells.Results:K15 was expressed in the cytoplasm of rats' hair follicle bulge cells.Then K15 positive cells were also found in hair matrix and the basal layer of epidermis with ages.The cells cultured in vitro formed large clones,and their clone forming rate was higher than that of controlled cells.They remained their morphologic features cultured after four passages with K15 expressed in their cytoplasm.Conclusion:Epidermal stem cells locate in hair follicle bulge regions.This method we used to isolate and culture epidermal stem cells is feasible.
Epidermis (zoology)
clone (Java method)
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Objective:To study the localization and in -vitro culture of epidermal stem cells in normal adult skin. Meth ods:The expressions of β1 integrin and keratin 19(K19) in normal adult skin were detected with immunocytochemical methods. The epidermal stem cells were isolated by adhering to collagen type IV and cultured in vitro in conditional medium prepared according to that for human fibroblasts. Then, the cultured cells were identified by immunohistochemistry staining of (3, integrin and K19, and the colony forming efficiency was also studied. Gliocytes were taken as control. Results: The cells expressing integrin β1 and K19 in epidermal layer were less. The epidermal stem cells selected by rapid adherence to type Ⅳ collagen formed large colonies after 10 d. The clone forming rate of epidermal stem cells was 17. 17% , which was much higher than that in control group (6. 83% ,P0. 05). Conclusion: The adult epidermal stem cells can be isolated in vitro by means of rapid adherence to type Ⅳ collagen, and can be cultured with conditional medium.
clone (Java method)
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Abstract Background Continuously renewing epithelia are maintained by stem cells that slowly proliferate and remain in the tissues for life. It has been known for decades that mouse epithelial stem cells can be selected by adherence to specific integrins. Methods The adherence of cashmere goat epidermal cells to collagen type IV for 10 min was used to obtain enriched epidermal stem cells. The characteristics of the rapidly adherent epidermal cells were determined. Results The rapidly adherent epidermal cells exhibited the stem cell characteristics of immaturity, were quiescent, showed a high colony formation efficiency, and expressed candidate surface markers for epidermal stem cells (keratin 15, keratin 19, p63, CD34, and β1‐integrin). Conclusions The rapidly adherent epidermal cells represented the epidermal stem cell population.
Stem cell marker
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The epidermal stem cells were isolated from goat ear skin by epidermal explant and cultured in DMEM/F12 and F12 two different groups.The resultant cells were observed and identified by cell growth curve,immnohistochemical staining and colony-forming efficiency.The results showed that DMEM/F12 was a good cell culture medium for the epidermal stem cells and the epidermal stem cells can survive to the 11 subculture in vitro.
Subculture (biology)
Isolation
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AIM: Our study was designed to look for an easy and feasible approach to isolate and culture rhesus epidermal stem cells.METHODS: Skin specimens were cut into strips and immersed into 0.25% trypsin overnight.Then transparent cuticular layeres were striped off with ophthalmic microscopic forceps.The epithelial layers were scratched,harvested and transferred to culture in skin epithelium media.The cells in 2nd-4th passages were harvested by 0.25% trypsin and relayed in IV collagen plate at 37 ℃,5% CO_2 for 20 min to harvest rapid attaching cells.Flow cytometric analysis,immunohistology and RT-PCR were conducted to identify rapid attaching cells.RESULTS: The specific protein and mRNA of epidermal stem cells(α6 integrin,K15 and β1 integrin) were identified in rapid attaching cells.No K1/K10(marker of terminally differentiated cells) and CD71 expression were found.CONCLUSION: Our method of isolation and culture can apply for rhesus epidermal stem cells.
Epidermis (zoology)
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