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 distribution, isolation and culture of the epidermal stem cells from rats. Methods Immnohistochemical methods were used to confirm the location of the epidermal stem cells. The skin of neonatal rats were dissociated into single cells by dispaseⅡ and trypsin solution,the rapidly adherent cells to collgenⅣ were cultured with KSFM,and those no rapidly adherent cells were regarded as control. Immunohistology and flow cytometry were conducted to identify the epidermal stem cells. Results α 6-integrin and K15 were expressed in the basal layer cells and hair follicle bulge cells, while the CD71 was negative negatively expressed. CD34 were expressed in the hair follicle bulge cells while not in the basal layer cells. The epidermal stem cells isolated by collgenⅣ had higher colony forming efficiency. Immunocytochemical staining showed that α 6-integrin and K15 were strongly expressed in the cultured epidermal stem cells. Flow cytometry indicated that 84% cultured epidermal stem cells were expressed α 6-integrin. Conclusion The epidermal stem cells of rats are located at the basal layer of epidermis and the hair follicle bulge.
Dispase
Amniotic stem cells
Epidermis (zoology)
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Epidermis (zoology)
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Objective:To explore a method for quickly isolating,culturing and identifying epidermal stem cells of human. Methods:Epidermis was obtained by digesting human foreskin with DispaseⅡand Trypsin-EDTA,with observation of the cell growth by inverted microscope and examination of cell cloning efficiency and duration of clone sustaining. Immunocytochemistry was used to observe the expression of β1-integrin and keratin19(K19). Keratinocytes were served as controls. Results:Histological findings showed that colonies were formed 24 hours after inoculation. The efficiency of isolated and cultured cell cloning was higher than that of the control group. Positive expression was detected in β1-integrin and K19 of cultured cells by immunocytochemistry. Conclusion:Adult epidermal stem cells were successfully isolated and cultured in vitro.
Dispase
clone (Java method)
Foreskin
Cloning (programming)
Epidermis (zoology)
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Objective To study epidermal stem cell isolation and culture in vitro in order to provide experimental basis for tissue engineering of skin.Methods With heat-cool alternative method and trypsin digestion of tissue mass,the epidermal stem cells were cultured and observed continuously in vitro.The isolated cells were investigated on molecule β1 integrin,K15 and p63 by immunohistochemistry staining and scanning microscope.Results The positive expressions of molecule β1 integrin,K15,and p63 and the spindle-and angular like cells in cluster growth were observed with scanning microscope.The culture system was stable for the growth and classification of the stem cells and there existed affiliated organ of the skin in the culture system.Conclusion With the culture system developed,the epidermal stem cells isolated presents obvious biologic features and differentiative trend with the formation of affiliated skin organ.
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Background and Objective: Epidermis is the outer layer of skin, regenerating continuously. Epidermal stem cells play important roles in tissue regeneration, scar regeneration and neoplasm formation.This study was displayed for the isolation and culture of interfollicul ar epidermal stem cells from newborn mouse skin without feeder layer. Materials and Methods: This experimental study was displayed on 0 -3 old -day newborn NMRI mouse skin 60-70 gr weight. The epidermal keratinocytes were separated mechanically and enzymatically from 0-3 old day newborn mice skin (NMRI strain) and seeded on fibronectin-collagen culture substrates. Putative epidermal stem cells were selected by rapid adherence for 10 minutes on this composite matrix of type 1 collagen and fibronectin and the unattached cells were discarded and attached cells were cultured in essential minimal eagle medium (EMEM) (ca+2-free culture medium containing 0.05 mM Ca+2, 9% FBS, 50% conditioned medium, EGF (epidermal growth factor) and Cholera Toxin. The immunocytochemistry of β1-integrin analysis used to indicate their stemness nature. Results: The results indicated that rapid adherence yields 50% purity. By using this method, the stem cells have been subcultured continuously without any change in the cell properties. The isolated interfollicular epidermal stem cells, expressed epidermal stem cells special marker (β1-integrin) in high levels, which indicates stem cell nature. Conclusion: This new method yields pure viable epidermal stem cells that can be used in regenerative medicine and cell therapy.
Epidermis (zoology)
Amniotic stem cells
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【Objective】 To explore a method for isolating culture of human pidermal stem cells in vitro and identify human pidermal stem cells.【Methods】Epdermis and single epidermis cells was obtained by digesting human for skin with Dispase Ⅱ and Trypsin, then suspension of single epidermis cells on the epidermal stem cell medium (ESCM). These single epidermis cells were inoculated on human collagen Ⅳ coated flasks and cultured on the ESCM. The cell growth was observed through inverted microscope. Immunocytochemistry was used to observe the expression of β1 integrin, keratin 19 (CK19), CK10 on pidermal stem cells.【Results】It was revealed by histological observation that colonies of the isolated and cultured cells were formed after inoculation, the time of clone sustain was longer. Positive expression of β1 integrin and K19 of the most cultured cells was detected by immunocytochemistry and CK10 was negative in the experimental group.【Conclusion】Epidermal stem cells could be successfully isolated and cultured by adhension with type Ⅳ collagen and culture with ESCM.
Dispase
Epidermis (zoology)
clone (Java method)
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Direct-tissue culturing was used to isolate and culture rat epidermal stem cells.Then,observe the appearance of the culturing cells,and the expression of CK15,P63 and α6-integrin in ESCs were detected by immunohistochemistry staining.By the way,the ESCs marked by α6-integrin were detected by flow cytometry(FCM).Meanwhile,the growth curve of epidermal stem cells was also detected.We could isolate,purify and culture rat epidermal stem cells successfully by direct-tissue culturing method and these cells grew well.Immuno-histochemistry staining showed that CK15,P63 and α6-integrin were strongly expressed in the cultured epidermal stem cells.According to the flow cytometry,the probability of sticky cells marked by α6-integrin was 80%,while the probability of unsticky cells marked by α6-integrin was only 2%.
Stem cell marker
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Objective:To isolate and culture the mouse epidermal stem cells and analyze its capability of clone formation for further studies.Methods:The neonate mice epidermal basal cells were digested by dispase and trypsin and then seeded directly and cultivated in culture flasks without any feeder cells.The epidermal stem cells were identified by K15 and α6-integrin staining and their clone formation abilities were evaluated when they were co-cultured with mouse embryonic fibrolasts feeder cells in a cell differentiation condition.Results:The clones of neonate mice epidermal stem cells were successfully formated after 2～3 day's cultivation characterized by low nucleo-cytoplasma ratio and tiny and round shape.Those cells could be specialized marked with K15 and α6-integrin after cell passage.Conclusion:The cultivation and passage of mouse epidermal stem cells can be achieved using this kind of method.
Dispase
clone (Java method)
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Objective:To explore a method for isolation and culture of human epidermal stem cells. Methods:The Epidermal stem cell were isolated by adhering to collagen typeⅣafter obtained by digesting human foreskin with DispaseⅡand Tryp and culture in vitro in K-SFM. The expressions ofβ1-integrin and keratin 19 (K19) in epidermal stem cell were detected with immunocytochemical methods,and the colony forming efficiency was also studied. Keratinocytes were served also detected. Results:It was revealed by histologieal observation that colonieswere formed 24 hours after inoculation. The isolated and cultured cell cloning efficiency was higher than that of the control group. Positive expression ofβ1-integrin and K19 of cultured cells was detected by immunocytochemistry. Conclusion:Adult epidermal stem cells could be successfully isolated and cultured by adhension with typeⅣcollagen and culture with K-SFM
Dispase
Foreskin
Cloning (programming)
Isolation
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