Study On Culture Of Cells From Human Epidermis And Hair Follicles And Reprogramming Of Melanocytes

Objective:(1)To observe the growth of epidermal cells in three groups of media and detect the levels of Endothelin-1(ET-1) and stem cell factor( SCF) in the supernatants at different time points;(2)To observe the cells growth and their morphological changes in outer root sheath(ORS) and dermal papilla(DP) of adult scalp fair follicle in culture;(3)To reprogram human epidermal melanocyte cells(MCs) into induced pluripotent stem cells(i PSCs) by Yamanaka described three factors.Methods:(1)Three kinds of cell culture media were used to culture epidermal cells, the supernatants were collected at 24 h, 48 h, 72 h and 96 h after cells seeding respectively. The levels of ET-1 and SCF in these supernatants were detected by the enzyme linked immunosorbent assay(ELISA). Meanwhile, the cells growth in different media were observed and compared;(2)Single hair follicles were completely isolated by collagenase Ⅳ and dispaseⅡ,the acquired ORS and DP cells were cultured in vitro in K-SFM media superinduced human keratinocyte growth supplement(HKGS) and human melanocyte growth supplement( HMGS). Growth and migration of adult scalp hair follicle ORS and DP cells were continuously observed under inverted microscope.(3)Human epidermal MCs were transduced with lentiviral vectors carrying Oct4, Klf-4 and c-Myc respectively. When the clones formed, alkaline phosphatase staining(AP), the detection of methylation levels, immunofluorescence staining by using the antibodies associated with stem cell markers, ultrastructure observation under transmission electron microscope(TEM) were performed. And then the i PSCs were induced to differentiate into the cells representing three embryonic layers in vitro.Results:(1)Among three groups of supernatants, the content of ET-1 in Medium 2(M2)supernatants was the highest; all of the content of ET-1 were increased with the extension of time. There were significant difference among the three groups(F=21.10, 35.61, P <0.01). The contents of SCF in the three groups of supernatants decreasedsignificantly in 24-48 h. There were significant differences among the three groups(F=12.77,12.17,16.52,P<0.01). After renewed the medium, the trends of ET-1 and SCF were not pronounced(F=49.70,30.40,P<0.01). The cell proliferation in M2 was fastest, where the keratinocytes(KCs) grew in colony and the MCs displayed multiple dendrites. In medium 1, less adherent cells, more death cells and slow cell proliferation were observed. The KCs scattered in the medium 1, in which most of MCs showed bipolar. The growth of the cells in Medium 3(M3) between it in M1 and M2.⑵Single hair follicle were put onto the dish that serum-coated in advance cultured for 2 hours, then added a small amount of culture medium, the hair follicle sticking probability would increase,we could soon find some cells migrate from hair follicle ORS and DP cells some hours later, mainly for KCs and fibroblasts(FBs), fewer MCs. Besides, there were decidedly differences in cells growth and migration between hair follicle ORS and DP cells. If added culture medium excessively, most hair follicle would suspend readily, no cells migrated and died finaly. ⑶ We transfected human epidermis MCs with 3 factors, clones begins to form after 6 days. The AP staining for the i PSCs in their first and third passages were positive. Compared with that of its parental MCs, the gene loci of Nanog and Oct4 were partial demethylated. The i PSCs showed actively karyokinesis under TEM, some had three nucleoli. Meanwhile, a few of melanosomes in stages I and II were seen in the cytoplasm of very few cells. The i PSCs at fifth passage was cultured in suspension and produced embryoid bodies, which were then cultured in adherent condition, and generated adherent cells that showing positive staining with stem cell-associated antibodies including Sox2 and Cdy1. Meanwhile, the i PSCs at fifth passage were induced to differentiate into the cells presenting three embryonic layers. After two weeks induction, the cells showed positive oil red staining, which implies adipose cells, and positive albumin staining that meaning liver cells. However, the marks relative to neurons were negative.Conclusions: ⑴High level of ET-1 in medium facilitates adhesion, proliferation and differentiation of epidermal cells. During the process of culture, the epidermal cells may secrete and release ET-1 into supernatant. The epidermal cells may do notsecrete SCF but consume it.⑵Serum-coated in advance and a small amount of culture medium at the very start can promote hair follicle to adhere, then cells started to migrate and grow.⑶ Reprogramming human epidermis MCs with 3 factors could generate incomplete i PSCs, which expressed mainly characteristics of stem cells and can be induced to adipose cells and liver cells in vitro,but failed to generate neurons cells.