Isolation Of Epithelial Stem Cells Research Articles

Isolation of epithelial stem cells from dermis by a three-dimensional culture system

Skin is a representative self-renewing tissue containing stem cells. Although many attempts have been made to define and isolate skin-derived stem cells, establishment of a simple and reliable isolation procedure remains a goal to be achieved. Here, we report the isolation of cells having stem cell properties from mouse embryonic skin using a simple selection method based on an assumption that stem cells may grow in an anchorage-independent manner. We inoculated single cell suspensions prepared from mouse embryonic dermis into a temperature-sensitive gel and propagated the resulting colonies in a monolayer culture. The cells named dermis-derived epithelial progenitor-1 (DEEP) showed epithelial morphology and grew rapidly to a more than 200 population doubling level over a period of 250 days. When the cells were kept confluent, they spontaneously formed spheroids and continuously grew even in spheroids. Immunostaining revealed that all of the clones were positive for the expression of cytokeratin-8, -18, -19, and E-cadherin and negative for the expression of cytokeratin-1, -5, -6, -14, -20, vimentin, nestin, a ckit. Furthermore, they expressed epithelial stem cell markers such as p63, integrin beta1, and S100A6. On exposure to TGFbeta in culture, some of DEEP-1 cells expressed alpha-smooth muscle actin. When the cells were transplanted into various organs of adult SCID mice, a part of the inoculated cell population acquired neural, hepatic, and renal cell properties. These results indicate that the cells we isolated were of epithelial stem cell origin and that our new approach is useful for isolation of multipotent stem cells from skin tissues.

Enrichment for murine keratinocyte stem cells based on cell surface phenotype.

The identification and physical isolation of epithelial stem cells is critical to our understanding of their growth regulation during homeostasis, wound healing, and carcinogenesis. These stem cells remain poorly characterized because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying (TA) cells, which have a more restricted proliferative potential. Cell kinetic analyses have permitted the identification of murine keratinocyte stem cells (KSCs) as slowly cycling cells that retain [(3)H]thymidine ([(3)H]Tdr) label, termed label-retaining cells (LRCs), whereas TA cells are visualized as rapidly cycling cells after a single pulse of [(3)H]Tdr, termed pulse-labeled cells (PLCs). Here, we report on the successful separation of KSCs from TA cells through the combined use of in vivo cell kinetic analysis and fluorescence-activated cell sorting. Specifically, we demonstrate that murine dorsal keratinocytes characterized by their high levels of alpha(6) integrin and low to undetectable expression of the transferrin receptor (CD71) termed alpha(6)(bri)CD71(dim) cells, are enriched for epithelial stem cells because they represent a minor ( approximately 8%) and quiescent subpopulation of small blast-like cells, with a high nuclear:cytoplasmic ratio, containing approximately 70% of label-retaining cells, the latter being a well documented characteristic of stem cells. Conversely, TA cells could be enriched in a phenotypically distinct subpopulation termed alpha(6)(bri)CD71(bri), representing the majority ( approximately 60%) of basal keratinocytes that are actively cycling, and importantly contain approximately 70% of [(3)H]Tdr pulse-labeled cells. Importantly, immunostaining of dorsal skin revealed the presence of CD71(dim) cells in the hair follicle bulge region, a well documented location for KSCs.