Isolation And Purification Of Goat Epidermal Stem Cells

Epidermal stem cells (EpiSCs) are a group of cells that continuously produce functional cells through asymmetric or symmetric division to maintain the stability of epidermis. There are two possible places that EpiSCs locate, basal layer of epidermis and follicular bulge. It's the combination of niche and the intrinsic characteristics of EpiSCs that shapes their properties and defines their potential. During these years, EpiSCs show great potential in skin wound healing, artificial skin explant, cellular therapy. This experiment aims to harvest goat EpiSCs with long-term regenerative ability, investigate their relative characteristics, and prepare plenty of seed cells for artificial skin or cornea. The experiment includes the following aspects. 1. Isolating goat EpiSCs through explant culture method. After washing the goat skin piece, dissect epidermis from dermis. Separately dry the epidermis for 5 min, 10 min, 20 min, 30 min, in order to identify the appropriate time for best adhering effects to the plastic dish. The best result is 10 min with the ratio of 90.00±0.0816%. After culturing the explant, there are epidermal cells outgrows from it, and there appear PGCs colony-like cell groups on the single cell layer. Original cells are cultured in M199 containing 20%NBS, 5 μg/mL Insulin, 0.5 μg/mL Hydrocortisone. When cells are confluent, passage them with .025% trypsin, and seed them in M199 containing 20% conditioned medium, 10% NBS, 0.1% BSA, 5 μg/mL Insulin, 0.5 μg/mL Hydrocortisone, 20 ng/mL EGF. We analyze some relative data of explant culture method: time when original epidermal cells outgrows, 4.9±1.5d;, time needed to passage original cells, 16.4±5.7d; passaged times, 3.8±1.5. 2. Isolating EpiSCs through trypsinizing method. Keep the dissected skin pieces in Dispase II, then separate epidermis through surgical tool, and trypsinize the epidermis into cell suspensionl. Isolate EpiSCs by rapidly adhering on Collagen IV. We've tested the adhering efficiency at 5 min, 10 min, 20 min, 60 min, finding that 10 min is the best time for choosing EpiSCs and the ratio of adhering cells to the whole basal cells is 10.8%. Selected cells are cultured in D/F containing 10%NBS, 5 μg/mL Insulin, 0.5 μg/mL Hydrocortisone, 20 ng/mL EGF. Passaged cells form many clones during the process of regeneration, ultimately shape a single cell layer of classic epidermal cells. The final number of EpiSCs take up almost 2.5% of the whole basal cells. We also sum the relative data of this method: time when clones appear, 3.6±1.0d; time need to passage the original cells, 9.5±1.1d; passaged times, 6.9±2.7. Comparing relative statistics of the two methods, we found there exist obvious difference. Here we conclude that trpsinizing method is much more effective than explant method in isolating EpiSCs. 3. Effects of conditioned medium (CM) and EGF to EpiSCs growth. In order to evaluate the effects, we designed the experimental group as 50% (group 1) and 20% (group 2), contrast group 0%(group 3). Using MTT method to picture growth curve, and contrast the differences of OD between every two groups. The results show that the differences in group1, 2, and 3 are all statistically significant (P<0.01). This demonstrates that CM has important impact on EpiSCs growth. The same method is also used to testify the effects of EGF. The density of experimental group is respectively 50 ng/mL, 40 ng/mL, 30 ng/mL, 20 ng/mL, 10 ng/mL, the contrast group is 0 ng/mL. The results show that there is no difference between experimental groups and contrast group, demonstrating that EGF has no obvious effect on EpiSCs in serum containing medium. 4. Identification of EpiSCs. To achieve this, the experiment combined three means. 1) immunocytochemical staining using diverse EpiSCs markers, including integrin a6, ?1, OCT-4, K19, P63, CD71. The results show that the selected cells express all of the positive markers, but not negative. 2) Colony forming efficiency. The CFE of P1, P3, P8 are respectively 24.52±1.71%, 28.59±1.99%, 29.89±.090%. 3) Electron-microscope observation. The results show that most of the cells have a high nuclear to cytoplasmic ratio. All these conclusions support that the cells identified are EpiSCs.