Rapid isolation and expansion of skin-derived precursor cells from human primary fibroblast cultures

Leithe Budel,Karima Djabali

doi:10.1242/bio.025130

Abstract

ABSTRACTSkin-derived precursor (SKP) cells have self-renewal and multipotent abilities and are found in the dermis. SKP cells have been isolated previously from pre-established dermal fibroblast cultures. In these procedures, long-term culture and low yield remain the crucial aspects requiring improvement. In this study, we exposed pre-established dermal fibroblasts to 30-min acid stress prior to isolating SKP cells (termed pH-SKP) and compared the yield to the previously published trypsin- and no-stress methods. Spheroid formation was confirmed and analyzed at days 3, 5 and 7. Stemness was investigated by immunohistochemistry for the stem cell markers Nestin, CD9, vimentin and NG2. Multipotency was investigated by differentiation into adipocytes, smooth muscle cells and fibroblasts. The pH-SKP spheroid yield at day 5 was four- and threefold higher than those obtained using trypsin- and no-stress methods, respectively. The expression of stem cell markers Nestin, CD9, vimentin and NG2 were significantly expressed in pH-SKPs compared to the fibroblast origin. Successful pH-SKP spheroid formation and differentiation were achieved and validated in 11 distinct human primary fibroblast lines. These results demonstrate that acute acidic stress treatment of dermal fibroblast cultures greatly improves SKP isolation, growth, yield and multipotency compared to previous methods.

Highlights

There is increasing interest in human skin-derived precursor (SKP) stem cells
Defining the acidic pH stress for the SKP isolation method To determine the optimal acidic stress pH for our method, we first investigated the effect of low acidic stress on primary dermal fibroblasts using a pH range from 7 to 5 in Hank’s balanced salt solution (HBSS)
We evaluated the potency of Smooth muscle cells (SMCs) differentiation of no-stress SKP (NS-SKP) and fibroblast cultures relative to pH-SKPs (Fig. S4A)

Summary

Introduction

There is increasing interest in human skin-derived precursor (SKP) stem cells. These types of stem cells are multipotent and are present throughout adulthood (Fernandes et al, 2004; Toma et al, 2005, 2001). SKP cells reside within the dermis, express stem cell markers (Avilion et al, 2003; Lendahl et al, 1990; Mitsui et al, 2003; Pesce and Schöler, 2001), and can be readily isolated and expanded from skin biopsy, independent of the subject’s age, site of biopsy, or disease (Joannides et al, 2004; Uchida et al, 2000) These factors, together with the ability of self-renewal (Toma et al, 2005), have made SKPs a topic of great interest as studies continue to find increasing potential in their multipotent ability.

Methods

Results

Conclusion