Cultivation Of Multilineage-differentiating Stress-enduring(Muse) Cells And Construction Of A Three-dimensional Epidermal Model

Objective:(1) To observe the tolerance of normal human dermal fibroblasts(NHDFs) to trypsinization in vitro. To isolate and obtain multilineage-differentiating stress-enduring(Muse) cells derived from human dermal tissue.To explore the characteristics of Muse cells in the respects of cell proliferation, self-renewal, pluripotent, multilineage inducing differentiation and methylation level.(2) To observe the morphological characteristics and the biological properties of human epidermal cells when cultured at an air–liquid interface in polycaprolactone(PCL) fibers as a three-dimensional scaffold for tissue engineering.Methods:(1) NHDFs were obtained by 0.25% trypsin/0.02%EDTA. After long-term trypsin incubation(LTT), NHDFs were suspended in methylcellulose(MC) to form Muse cell–derived cell cluster(M-clusters) that were similar to embryonic stem cells(ESCs)derived embryoid bodies.The single M-clusters in suspension culture were detected by pluripotent markers, methylation level detection, transmission electron microscopy and multilineage inducing differentiation in vitro. After 5–7 days of M-clusters incubation in adherent culture, alkaline phosphatase(AP) staining and immunofluorescence staining for pluripotent markers(Oct4, Sox2, Nanog and SSEA3) were performed.(2) Keratinocytes(KCs) and melanocytes(MCs) were obtained from human scalp skin, seeded onto a PCL film, and cocultured for 2 weeks at air–liquid interface to construct a three-dimensional(3D) skin model. The cells were then characterized by hematoxylin and eosin(H&E) staining, by immunocytochemistry staining with antibodies to cytokeratin 15(CK15), Ki-67, CD34, CD200 and HMB45 and by transmission electron microscopy.Results:(1) After exposing cells to LTT, the vast majority of cells were dead and only a few cells were surviving, which were transferred to MC suspension cultures. Within 5-7 days, individual cells began to proliferate and form clusters. Cell clusters were transferred for adherent culture, they quickly attached andexpanded from the clusters. M-clusters and its expanded cells were positive for AP staining and pluripotent markers. The color was deep in the center of each M-cluster, but gradually faded in expanded cells around the M-clusters. Immunofluorescence staining showed that the cell clusters were positive for the surface markers of Oct4, Sox2, Nanog and SSEA3 when in suspension culture. Compared with NHDFs, Muse cells showed a low DNA methylation level. Transmission electron microscopy revealed that Muse cells were more primitive, had a high nuclear-cytoplasmic ratio and a big nucleolus. In the cytoplasm, there were few organelles and most were relatively immature. In adipocyte induction, Muse cells generated cells with lipid droplets that were positive for Oil Red-O staining. Osteocyte induction generated cells positive for Alizarin Red S. Neuronal induction demonstrated cells positive for nestin, neurofilament and b-III tubulin with a neuron-like morphology. Hepatocyte induction resulted in the generation of cells positive for human albumin.(2) KCs and MCs grew well in the co-culture system. H&E staining revealed that KCs layer became a multilayered concentric structure, the surface became distinctly keratinizedand, and incomplete hair follicles-like structure was formed. Immunohistochemical analyses exhibited a scattered distribution of cells expressing CK15, CD34, CD200, Ki-67 and HMB45 respectively. Transmission electron microscopy revealed that KCs contained a number of keratin fibrils(KFs) and membrane-coated granules, some KCs possessed oval or round electron-dense substances.Conclusions:(1) Muse cells are a novel kind of stem cells and capable of self-renewal, expression of stem cell surface markers, production of clusters similar to ESCs derived embryoid bodies in suspension culture, show low level of methylation and pluripotent stem cell like feature in ultrastructure and have the ability to differentiate into cells representative of all three germ layer.(2) The PCL scaffold has excellent biocompatibility and adhesiveness with human epidermal cells, and maybe suitable for constructing 3D skin models for tissue engineering in the future.