UNRAVELLING THE CROSS TALK BETWEEN CELL SHEETS OF HUMAN ADIPOSE STEM CELLS AND KERATINOCYTES

We have previously shown that 3D constructs of human adipose stem cells (hASCs) cell

sheets led to the formation of de novo hair follicles and rete-ridges like structures. An upregulation

of keratinocyte growth factor (KGF) was also observed in the experimental condition

in relation to the control groups. We hypothesized that the natural adhesive character of the

cell sheets promoted the direct interaction between the host and the transplanted cells1. In this

sense, the present work aims at elucidating this communication between hASCs and human

keratinocytes (hKC) and determining the extent of its mediation by KGF. In an in vitro scratch

assays we showed that the secretome of hASCs in contact with hKC promotes cell migration

and closure of the scrape. Moreover, when KGF-antibody was added diminished hKC

migration was observed, suggesting that KGF might be one of the key cytokines involved in the

interaction with hASCs. Furthermore, in order to assess the communication via gap junctions

(GJ), a calcein-AM transfer assay was carried out in the presence/absence of a GJ inhibitor.

The transference of the dye from hASCs to adjacent hKC, confirmed both by fluorescence

microscopy and flow cytometry, showed that these cells also communicate via GJ. Moreover

while hKCs expressed connexin (cx)43 and cx26, highly expressed at the wound margins,

hASCs were only positive for cx43 as shown by immunocytochemistry and flow cytometry.

Finally, the direct communication between transplanted hASCs cell sheets and hKC at the

wound margins is being addressed in a human ex-vivo skin model with an artificial wound, to

better mimic the previous in vivo conditions and confirm our hypothesis. So far we were able to

demonstrate that hASCs and hKCs communicate directly through cx43 and indirectly via KGF

secreted by hASCs, which promote KCs migration.