Evidence for keratinocyte stem cells in vitro: long term engraftment and persistence of transgene expression from retrovirus-transduced keratinocytes

Abstract

Epidermis is renewed by a population of stem cells that have been defined in vivo by slow turnover, label retention, position in the epidermis, and enrichment in beta1 integrin, and in vitro by clonogenic growth, prolonged serial passage, and rapid adherence to extracellular matrix. The goal of this study is to determine whether clonogenic cells with long-term growth potential in vitro persist in vivo and give rise to a fully differentiated epidermis. Human keratinocytes were genetically labeled in culture by transduction with a retrovirus encoding the lacZ gene and grafted to athymic mice. Analysis of the cultures before grafting showed that 21.1-27.8% of clonogenic cells with the capacity for >30 generations were successfully transduced. In vivo, beta-galactosidase (beta-gal) positive cells participated in the formation of a fully differentiated epithelium and were detected throughout the 40-week postgraft period, initially as loosely scattered clusters and later as distinct vertical columns. Viable cells recovered from excised grafts were seeded at clonal densities and 23.3-33.3% of the colonies thus formed were beta-gal positive. In addition, no evidence of transgene inactivation was obtained: all keratinocyte colonies recovered from grafted tissue that were beta-gal negative also lacked the lacZ transgene. These results show that cells with long-term growth properties in vitro do indeed persist in vivo and form a fully differentiated epidermis, thereby exhibiting the properties of stem cells.

Figure 1

Histochemical staining of grafted tissue for transgene expression. Organotypic raft cultures constructed with MFGlacZ-transduced keratinocytes were grafted to athymic mice. At the times noted, the tissue was excised and examined for β-gal expression by staining with X-Gal. β-gal positive cells are present in all grafts. (A) Organotypic raft culture before grafting. (B) One week after grafting. (C) Ten weeks after grafting. (D) Twenty weeks after grafting. (E) Forty weeks after grafting. (F) A 10-week graft stained with an antibody for β-gal (green) and an antibody for filaggrin (red). Note dual expression of β-gal and filaggrin (orange) in the column of cells on the left. (A–E, ×240.)

Figure 2

Sites of integration of retrovirus transgene DNA isolated from various β-gal positive colonies was digested with HindIII enzyme and analyzed by Southern blotting using lacZ DNA as a probe. HindIII does not cut within the retrovirus genome and different sizes of restricted fragments indicate different sites of provirus integration. Arrows indicate the position of labeled fragments that did not appear in the photo reproduction, but were evident in the autoradiograph. The background, but not the bands, has been digitally enhanced to improve appearance.

Figure 3

Consequences of transduction of stem cells and transient amplifying cells. (A) EPU is illustrated by a polygonal figure. The stem cell is a square located at the left side of the polygon and sequential cycles of transient amplifying cells are depicted by areas that expand to the right. Terminal differentiation is not shown in the figure, but would occur after the last round of amplification division. (B) When an EPU is infected with a retrovirus, the stem cell or transient amplifying cells are transduced depending on the position of each in the cell cycle. (C) Transduced cells and their progeny are noted by shaded areas. Cell replication is indicated by an arrowhead. When the stem cell is transduced, all cells in the EPU become and remain labeled with repeated cycles of cell replication (Upper). When transient amplifying cells are transduced, there is an initial rise in the number of transgene-positive cells; however, with further replication, the EPU becomes depleted of transduced cells (Lower).