Assessment of transformed properties in vitro and of tumorigenicity in vivo in primary keratinocytes cultured for epidermal sheet transplantation

Abstract

Epidermal keratinocytes are used as a cell source for autologous and allogenic cell transplant therapy for skin burns. The question addressed here is to determine whether the culture process may induce cellular, molecular, or genetic alterations that might increase the risk of cellular transformation. Keratinocytes from four different human donors were investigated for molecular and cellular parameters indicative of transformation status, including (i) karyotype, (ii) telomere length, (iii) proliferation rate, (iv) epithelial-mesenchymal transition, (v) anchorage-independent growth potential, and (vi) tumorigenicity in nude mice. Results show that, despite increased cell survival in one keratinocyte strain, none of the cultures displayed characteristics of cell transformations, implying that the culture protocol does not generate artefacts leading to the selection of transformed cells. We conclude that the current protocol does not result in an increased risk of tumorigenicity of transplanted cells.

Figure 1

Growth rates of keratinocytes in function of passages. (a): growth rate of KAL 0501 from P1.

Figure 2

Pictures of KAL0501 in culture at different passages (x100). Primary keratinocytes KAL0501 are culture in green medium on human feeder layer. Their morphology has changed during the culture from passage P1 to P25.

Figure 3

Karyotype of KAL0501 at passage P20. At this passage, two abnormalities are observed: an unbalanced translocation 4q32 and duplication 1q, or a translocation 1q with another chromosome.

Figure 4

Expression of vimentin and E-cadherin in KAL0501 at different passages in western blot and immunofluorescence. (a): E-cadherin expression level of KAL 0501 decrease from P3 to P16 on contrary to vimentin expression which increase. (b): picture (confocal microscope x25) of KAL0501 at P3, P11, and P15 cultured on monolayer. E-cadherin disappears progressively with the passage and vimentin appear at P11.

Figure 5

Picture of soft agar assay (Microscope x10). The 4 cell strains are not able to grow in soft agar after 21 days of culture on contrary to the positive control, the transformed human embryonic kidney 293T cells.