Rapid adhesion to collagen isolates murine keratinocytes with limited long-term repopulating ability in vivo despite high clonogenicity in vitro

¹ Department of Dermatology, University of California San Francisco and San Francisco Veterans Affairs Medical Center, San Francisco, CA 94121
² Department of Medicine, Division of Rheumatology, University of California San Francisco and San Francisco Veterans Affairs Medical Center, San Francisco, CA 94121
³ Department of Medicine, University of California San Francisco, School of Medicine; Medical Service, San Francisco Veterans Affairs Medical Center, CA 94121

^* To whom correspondence should be addressed. E-mail: ghadiallyr{at}derm.ucsf.edu.

	Abstract

A prevalent belief in epidermal biology is that stem cells are highly clonogenic, i.e. have the ability to produce many large colonies in vitro. However, it has been well established in hematology, and recently suggested in epithelial biology, that short-term in vitro clonogenic assays may not be reliable predictors of long-term in vivo repopulating ability. Numerous groups have shown that rapid adhesion to collagen selects for highly clonogenic keratinocytes, but it has not been demonstrated whether this subpopulation is enriched in stem cells as defined by long-term repopulating ability in vivo. We found that while rapid adhesion to collagen (within 5 minutes) selected for cells with increased short-term colony forming ability in vitro, these cells were not enriched in long-term proliferative ability in vitro, or repopulating ability in vivo after nine weeks. Conversely, keratinocytes that did not adhere to collagen (after 20 minutes) were less clonogenic in short-term assays, but possessed equivalent long-term proliferative ability in vitro, and superior long-term repopulating ability in vivo. Both the rapidly adherent cell (RAC) and not rapidly adherent cell (NAC) populations contained small, non-complex basaloid cells, expressed integrin 2 (a collagen IV receptor), and expressed the putative epidermal stem cell phenotype, integrin 6^hiCD71^lo. Our results indicate that the superior short-term colony forming ability of collagen-adherent murine keratinocytes does not correlate with long-term repopulating ability in vitro or in vivo, and that proliferation in vitro is not a reliable surrogate for stem cell behavior in vivo.