Human skin precursors (hSKP), isolated from the dermis, are neural-crest derived adult precursors growing as floating spheres. These cells harbor stem cell-like properties due to the capacity to differentiate in vitro into multiple lineages.
Traditionally, primary spheres are dissociated and cultured in a conventional two-dimensional (2D) monolayer set up. Monolayer cultures are used for expansion and generation of large batches of hSKP, derived from several donors, which are stored until use. Preliminary results show that the expression of few hSKP markers involved in multipotency (SOX10, SNAI1, SNAI2, OCT4, SOX2) is downregulated when cells are sub-cultured in 2D-monolayers.
Hence, the aim of this study is to investigate whether native hSKP spheroids possess higher degree of stemness compared to a standard 2D-culture and how the cell culture configuration could affect intrinsic cellular properties of hSKP.
Genome-wide analysis reveals a drastic change in gene expression modulation between spheroids and 2D-culture with a downregulation of 1,189 genes and an upregulation of 359 genes in 2D. Gene ontology analysis indicates that gene sets associated with embryonic development and differentiation potential are downregulated in 2D. Compared to spheroids, hSKP in monolayer show an increased expression of markers characteristics of committed cell types. In addition, while cell maintenance, DNA replication and survival are suppressed, pathways involved in cell death are activated in 2D.
Overall, these results suggest that hSKP monolayers loose stemness characteristics, display lower potency as well as a decline in cell maintenance processes compared to primary spheroids. In contrast, the native phenotype of hSKP is appropriately preserved within the spheroid configuration.
Next, transcriptomic analysis of reaggregated hSKP generated from 2D-monolayers, will elucidate whether stemness properties can be restored, or if are permanently lost, in comparison to the 2D counterpart and primary spheroids.