Limiting dilution analysis of murine epidermal stem cells using an in vivo regeneration assay

Abstract

Epidermal stem cells are of major importance for tissue homeostasis, wound repair, tumor initiation, and gene therapy. Here we describe an in vivo regeneration assay to test for the ability of keratinocyte progenitors to maintain an epidermis over the long-term in vivo. Limiting dilution analysis of epidermal repopulating units in this in vivo regeneration assay at sequential time points allows the frequency of short-term (transit amplifying cell) and long-term (stem cell) repopulating cells to be quantified.

Figure 1

Limiting dilution analysis for the determination of short term (TA) and long term (stem) repopulating cell frequency. (A) Diagram of the 6 mm diameter silicone chamber implanted onto the dorsal fascia of a NODSCID mouse into which the keratinocyte and fibroblast populations are seeded. (B) Schematic of epidermal layers where GFP positive units derived from short term repopulating cells (TA cells) disappear after 3 to 5 weeks (top row) or GFP positive cells derived from long term repopulating cells (stem cells) persist for 9 to 30 weeks. (C) Maintenance of GFP positive repopulating units up to 14 weeks. Top panels show bright-field images of regenerated skin, middle panels show epifluorescence images taken at the same time, and bottom panels show an overlay of the GFP positive repopulating units on the regenerated skin. (D) Hematoxylin and eosin staining of regenerated skin in cross section (10x, 20x). d: dermis, e: epidermis, hf: hair follicle.