Characteristics of homogeneously small keratinocytes from newborn rat skin: possible epidermal stem cells

Abstract

The aims of the present study were to characterize the phenotype, growth kinetics, and proliferative activation in culture of a population of poorly differentiated homogeneously small (HS) keratinocytes. These slow-cycling cells were separated by unit gravity sedimentation from a population of actively proliferating basal keratinocytes in newborn rat skin. This population (approximately 1% of the total basal keratinocytes) consisted of extremely small cells with little cytoplasm or RNA. Their positive KL4 staining demonstrates that they were keratinocytes. HS keratinocytes did not, however, contain epidermal calcium binding protein. Acridine orange, bivariate Hoechst, and ethidium bromide flow cytometry of in vitro bromodeoxyuridine-labeled cells as well as Ki67 staining showed that HS keratinocytes were in the G0 stage of the cell cycle and did not actively proliferate in vivo. [3H]thymidine label-retaining cells were found only in the HS cell population, showing that HS cells may originate from a central position in the epidermal proliferative unit. Growth of HS cells in vitro was characterized by a delayed but progressive increase in RNA before entry into the cell cycle. The clonogenic efficiency of HS cells in primary culture was much less than that of larger cells. Subclones of HS cell colonies exceeded primary colonies in their cloning efficiency and proliferative potential, suggesting that HS cells, although normally prevented from dividing, retain a high self-renewal capacity. They also maintain the ability to differentiate. The results are consistent with the concept that HS cell population may represent the epidermal-specific progenitor cells which act as stem cells in this tissue.

CD133 Is a Marker for Long-Term Repopulating Murine Epidermal Stem Cells

“…Culture conditions may favor proliferation of TACs and/or reprogram proliferative ability [for review see (Cotsarelis, 2006)] or in vitro analysis may reflect a wounding response, not homeostasis (Kaur, 2006). These results are consistent with the belief that stem cells have minimal clonogenic ability in vitro and that not all colony forming cells constitute stem cells (Pavlovitch et al, 1991; Budak et al, 2005; Li et al, 1998; Selver et al, 2011; Strachan et al, 2008; Schofield, 1978; Loutit et al, 1981; Louis et al, 2008). …”

Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells

“…These functional features notwithstanding, our SP cells displayed a low (~3%) CFE. A literature search, though, revealed that stem cell-rich fractions exhibiting substantially lower CFEs than the source whole populations have been observed in adult rat epidermis (Pavlovitch et al, 1991) and in neonatal human foreskin (Li et al, 1998). This is remarkable considering that whole populations include a large proportion of suprabasal, differentiated cells.…”

“…Slow or infrequent cycling, is one of the most unique features of adult stem cells (Leblond, 1964;Bickenbach, 1981;Cotsarelis et al, 1989;Pavlovitch et al, 1991;Morris and Potten, 1994;Wei et al, 1995;Fortunel et al, 1998;Lehrer et al, 1998;Braun et al, 2003;Tumbar et al, 2004). Accordingly, we performed experiments aimed at identifying slow-and fast-cycling cells in the conjunctival and limbal epithelia and in their respective flow cytometry cohorts.…”

Epidermal Label-Retaining Cells: Background and Recent Applications

“…Nevertheless an important study demonstrated that LRC isolated from mouse skin do have higher colony-forming potential in vitro than pulse-labelled cells from the same animal (Morris and Potten, 1996). In the rat it is possible not only to generate LRC but also to culture primary keratinocytes with high colony-forming efficiency and these studies also suggest that LRC are clonogenic in vitro (Pavlovitch et al, 1991;Kobayashi et al, 1993).…”