Isolation of small-sized human epidermal progenitor/stem cells by Gravity Assisted Cell Sorting (GACS).

Background: Small diameter characterizes epidermal progenitor/stem cells. We have developed Gravity Assisted Cell Sorting (GACS) to simply enrich small-sized epidermal progenitor/stem cells.

Objective: The cells sorted by GACS were characterized by fluorescence-activated cell sorting analysis, and cultured for up to 7 weeks. The cultured cells were then used for reconstruction of skin equivalent.

Methods: GACS was performed on primary cultures (primary cell) and passage 6-7 cultures (cultured cell) of keratinocytes. A keratinocyte suspension was sized into two groups: cells trapped by a 20 microm filter (trapped cells), and cells flowing through both a 20 and 11 microm filter (non-trapped cells).

Results: In the primary cell groups, viability of the trapped cells was 62.5+/-7.2% compared to 77.0+/-3.7% for the non-trapped cells. In the cultured cell groups, viability of the trapped cells was 64.3+/-14.9%, compared to the non-trapped cells (93.1+/-2.0%). Flow cytometric analysis showed better discrimination by cell size between trapped and non-trapped cells in culture than in the primary cell suspension. Non-trapped cells contained a larger number of cells with high levels of alpha6 integrin and low levels of CD71 (alpha6 integrin(bri)CD71(dim)), indicating an enriched progenitor/stem cell population. The difference in these markers between the non-trapped and trapped cells was seen in both the primary and cultured cell groups although this difference was more distinct in cultured cells. Culture of both groups showed that cultures originating from the trapped cells senesced after approximately 15 days while the non-trapped keratinocytes grew for up to 40 days. Manufacture of an epidermis/dermal device (artificial skin) showed that non-trapped cells formed a significantly thicker epithelial layer than the trapped cells, demonstrating the enhanced regenerative capability of the smaller diameter, alpha6 integrin(bri)CD71(dim) cells separated by GACS.

Conclusion: These results indicate that GACS is simple and useful technique to enrich for epidermal progenitor/stem cell populations, and is more efficient when used on cells in culture.