The Discovery Of The Multipotency Of Epidermal Stem Cells And Related Research

Objective:1:To explore the potential subpopulation of epidermal stem cells (ESCs) and analyze its characteristic of anatomical distribution; 2. To establish a method for transdifferentiation of ESCS, and to expand the multi-lineage differentiation potency of ESCS; 3. To identify the expression profile of cultured ESCs and set up an experimental system for producing induced pluripotent stem cells (iPSCs) from ESCs by non-transgenetic reprogramming.Methods:1. Immunohistochemical assay was used to detect the expressions of CD34 in skin of different anatomical sites. RT-PCR and immunofluorescence were adopted to detect the expression of CD34 molecule of foreskin-derived epidermal basal-layer cells in the mRNA and protein levels, double-label immunofluorescence was used to detect the expression of CD34 and either of the two well documented ESC markersβ1 integrin and p63. The samples were divided into groups according to the age and flow cytometry was adopted to compare expression differences of CD34 epitopes of each group.2. Foreskin-derived undifferentiated keratinocytes (UKs) were isolated by classic assay. Flow cytometry and immunofluorescence were used to detect the specific markers of UKs in order to assess the role of Epifile culture system in the growth and proliferation of UKs. Fibronectin, nestin, c-kit were detected in isolated UKs for excluding the contamination by other kinds of cells. UKs at 80-95% confluence were used for induction. UKs were induced by either serum or lineage-committed medium or acclimatized induction. Adipogenesis-related genes were detected and Oil Red O staining was employed to confirm adipocyte lineage transdifferentiation; myogenesis-related genes and SMA were detected to confirm the myogenic lineage transdifferentiation; neurogenesis related-genes, nestin,β3-tubulin and GFAP were detected to confirm the neurogenic lineage transdifferentiation. Serum induction of foreskin-derived skin (including epidermis and dermis) fragments or epidermis was performed to imitate the microenviroment of wound site, and transdifferentiation of keratinocytes was detected by immunofluorescence. Skin injury model in mice was established and different time points during wound healing were selected to detect the transdifferentiation of keratinocytes in wound region by immunofluorescence.3. The dynamic changes of expression profile of cultured ESCs were detected in mRNA level, especially the crucial transcription factor of transcriptional regulatory network for pluripotency. ESCs were reprogrammed by means of transgene through transfection of OCT4, SOX2, KLF4 and c-Myc, or induced for reprogramming directly under embryonic stem cells culture condition. Alkaline phosphatase staining was used to calculate the efficency of reprogramming. Immunofluorescence were adopted to detect pluripotent transcription factor--OCT4, SSEA1 and NANOG to verify the generation of iPSCs, and EB formation was performed to detect the pluripotency of iPSCs.Results:1. In this study, we observed the expression of CD34 with all three epitope types specifically at the epidermal basal layers derived from human scalp and foreskin, but not trunk skin, which revealed the differential distribution of CD34 at different skin sites. Noticeably, CD34-positive cells clustered at the bottom of the rete ridge, which is a well known location of epidermal stem cells. The results of double immunofluorescence studies demonstrate that almost all CD34-positive cells are also positive for either of the two well documented epidermal stem cell markersβ1 integrin and p63, although the former number is markedly less than those of the later. Althoughβ1 integrin-, p63-, CD34-positive cells gradually decayed with passaging, CD34-positive cells were not detected when cultured to passage 6. Furthermore, no significant difference of CD34 expression was observed among different age groups. 2. Using our recently developed acclimatization induction strategy, we demonstrated the multipotency of adult human UKs. The UKs were isolated from the basal layer of adult human foreskin and cultured in Epilife medium, which allows for the growth of only keratin-positive keratinocytes, promotes high proliferation of UKs, and prevents their differentiation. Induction of the UKs by either serum or lineage-committed medium only produced differentiated epidermal cells. Hence, serum or lineage-committed medium was added to Epilife to acclimate UKs to differentiate to other cell types. Unexpectedly, serum acclimatization can induce UKs to produce a large number of smooth muscle cells and myofibroblasts and fewer of adipocytes and neurocytes within 3 weeks. In contrast, except for the terminally differentiated epidermal cells, committed acclimatization could induce UKs to differentiate exclusively into the adipocytic, myogenic, or neurogenic lineages. Cultured the foreskin fragments in serum for 48hrs, a few of the keratinocytes of epidermis were transdifferentiated into vimentin-and FSP-positive cells. However, cultured the epidermis in serum for 48hrs, the most of the basal layer cells showed SMA positive, no detection of vimentin-and FSP-positive cells. In skin wound model of mice, we found that keratinocytes at the edge of wound could transdifferentiated into vimentin-and FSP-positive cells during wound repair; while no SMA-positive cells were observed.3. There were expression of pluripotent transcription factors-OCT4, SOX2, KLF4, c-Myc and NANOG in cultured ESCs, and these molecules simultaneously reached peak on 3-5d, which was consistent with one of the iPSC production theoriestranscription noise theory. Flow cytometry results showed that there existed OCT4-positive cells in ESCs after 3d culture in vitro, which further confirmed another theory for iPSC production--elite cell theory. ESCs after 3-5d cultured in vitro were selected for transgenetic or non-transgenetic reprogramming. We found that iPSCs-like clones appeared on day 6 after induction by transgenetic reprogramming; while iPSCs-like clones occurred on day 14 after induction by non-transgenetic reprogramming. Immunofluorescence showed that these clones were positive for pluripotent transcription factors--OCT4, SSEA1 and NANOG, and simultaneously expressed CD29, a marker specific to ESCs. The reprogramming efficiency of transgenetic (0.3%) was significantly higher than that of non-transgenetic (0.00002-0.00015%) by alkaline phosphatase staining. iPSCs could form EB in vitro suggested that these iPSCs own capability of development.Conclusion:1. We initially made a systemic study on the expressions of three epitopes of CD34 in different anatomical regions. The study shows that CD34 may represent a subpopulation of ESCs in scalp and foreskin tissues, and the unbalanced expression of CD34 further confirmed the heterogeneity of stem cells.2. The data of this study shows that human UKs possess multipotency in vitro, In addition, it is confirmed by mice injury model that keratinocytes transdifferentiate into mesenchymal-like cells during injury repair. These results indicate that the local microenvironment is significant for cells to transdifferentiatied. Meanwhile, these results are also theoretical basis for the application of UKs in regenerative medicine.3. The characteristics of expression profile of cultured ESCs are consistent with the two fundamental principles for producing iPSC. In this study, we obtain iPSCs-like clones by means of transgenetic or non-transgenetic. These results may make ESCs preferential cells for production of iPSCs by non-transgenetic reprogramming. This method may point out an alternative way to eliminate oncogenicity of iPSCs which derive from transgenetic ways.