Isolation, Characterization And Directional Differentiation Of Skin Stem Cells Derived From Cashmere Goat Fetus

This study has been conducted to isolate epidermal stem cells from Cashmere goat fetus and to characterize them in stem cell nature. Here we also report the isolation of a stem cell population from dermis (also termed as skin derived precursor cells) having multilineage differentiation capacity. This study was categorized into four parts to accomplish the research objectives.Part A: Keratinocyte isolation and culture from goat fetus skinThis part was conducted to isolate keratinocytes from Cashmere goat fetus with the aim to develop suitable conditions for keratinocyte cultivation and propagation. The methods developed for keratinocytes culture include (i) the explant culture, (ii) use of a feeder-layer (iii) use of a substrate such as collagen IV, or (iv) without use of any substrate. Mitotically inactivated fetal skin fibroblasts from goat and Kunming White Mouse (KWM) and embryonic fibroblasts from KWM were used for making feeder layer. Fibroblasts were cultured by either tissue explant culture or enzymatic digestion method. Epidermal cell removal were established by enzymatically separating keratinocytes from 12-16 weeks aged fetal skin tissues treated with 0.125% trypsin solution overnight at 4°C. ORS keratinocytes were cultured by explant culture of plucked hair follicle. Keratinocytes were maintained in all culture conditions with serum containing medium. The results indicated that spindle shaped fibroblast cell monolayer around the tissue explant had formed after 2-3 days of culture in explant culture method and a dispersed fibroblasts monolayer had formed after 24 hours by enzymatic digestion method. Epidermal keratinocytes multiplication and proliferation were comparable in different culture conditions and the best cellular attachment and growth have been obtained in cultures on feeder layer. Colony forming keratinocytes on feeder layer were heterogeneous in their growth potential and were maintained approximately 48 population doublings (8 passages) without showing signs of replicative senescence. Keratinocytes culture in feeder free conditions were limited to subculturing for their poor attachment in culture dishes. Viable fibroblasts often contaminated the keratinocytes in explant cultures. ORS cells outgrowth around the tissue explant after 4-5 days following inoculation and their morphology and proliferative capacity were similar to the epidermal keratinocytes. This part reports the comparative efficacy of different culture conditions for keratinocytes isolation and in vitro propagation.Part B: Putative epidermal stem cell selection, propagation and characterization This part was undertaken to select putative epidermal stem cells from cultured keratinocytes in Cashmere goat fetus. Putative epidermal stem cells were selected by rapid adherence of epidermal keratinocytes on collagen type IV substrate and maintained in three different medium conditions: high Ca2+ concentration, low Ca2+ concentration, and low Ca2+ concentration with 50% conditioned medium. Clonal analysis of keratinocytes revealed the presence of three clonal types: holoclones, meroclones and paraclones. Holoclones are believed to be derived from stem cells. Collagen IV selected cells grew in low Ca2+ as a monolayer without stratification, and individual cells were small with prominent nuclei and few organells. At higher level of Ca2+ concentration, cells grew rapidly, stratified and formed an upper layer of squames as they reached confluency. Selected keratinocytes on collagen IV substrate could be propagated serially in three medium conditions and the population showed high colony formation efficiency, same morphology with a high nuclear to cytoplasmic ratio. This part reports a selected keratinocytes population with the characteristics of stem cells.Part C: Identification of epidermal stem cells by molecular marker expression This part described the identification of epidermal stem cells by using possible keratinocyte stem cell specific markers. Immunofluorescence staining method had been used for the evaluation of p63, Keratin 19, Keratin 15 and CD71 markers expression in collagen-IV selected keratinocytes. The results showed that all these markers were expressed in a proportion of cells and their expression level were different from one another. A greater proportion of cells (51-57%) were positive for p63. On the other hand, keratin 19 and keratin 15 expressions were associated with the population of 19-27% and 16-21%; respectively. A few proportion of cells (<1%) were positive for CD71 marker expression. This part reports a panel of potential molecular markers for identifying epidermal stem cells in Cashmere goat.Part D: Directional differentiation of skin stem cellsThis step has been conducted for differentiation capacity of stem cells originated from two sites of the skin. Epidermal stem cells have been induced by using high Ca2+ condition medium to reconstruct epidermis in three dimensional organotypic culture model. Dermis derived stem cells (also termed SKPs) were induced by specific inducing medium to generate insulin producing pancreatic cells and neurons. For epidermal differentiation, collagen IV enriched population were seeded on either artificial dermal matrix produced by dermal fibroblasts and collagen I or on acellular dermis and cultured for 1 month. The results showed that multilayered epidermis was formed in both type of culture. For insulin producing cell differentiation, dermis derived stem cells (DSCs) were induced by specific medium containing EGF, nicotinamide and ITS. For neuronal differentiation, skin derived precursor cells (SKPs) were transferred into specific inducing medium containing EGF, FGF-2 and B27. Immunofluorescence staining showed that a proportion of cells were positive for insulin (13±4.68%), nestin (7±3.98 %) andβ3 tubulin (9±4.12%). These results revealed that dermis derived stem cells have the ability to undergo multilineage differentiation.