Modulation of stem cell shape and fate A: the role of density and seeding protocol on nucleus shape and gene...

Mesenchymal stem cell shape and fate are intrinsic manifestations of form and function at the cellular level. We hypothesize that cell seeding density and initial seeding protocol influence stem cell shape and fate. Nucleus shape and early (within days of seeding) expression of genes typical for pre-, peri-, and postcondensation events were compared between groups of cells after seeding at or proliferating to target density (low density [LD], 16,500 cells/cm2; high density [HD], 35,000 cells/cm2; very high density [VHD], 86,500 cells/cm2). Significant differences in nuclear shape could be attributed to seeding protocol in the VHD group, where nuclei from cells that proliferated to VHD were significantly rounder than nuclei from cells seeded at target VHD. Furthermore, cells that proliferated to VHD exhibited significantly rounder nuclei than nuclei from all other cell density and seeding protocol groups. In contrast, nuclei from cells that were seeded at the VHD were flatter than nuclei from cells of all other groups. Furthermore, the significant rounding of nuclei in the cells that proliferated to VHD was accompanied by a two-, six-, and ninefold increase from baseline in Runx2, Sox9, and Aggrecan (AGC) expression, markers indicative of precondensation, peri-, and post-condensation events, respectively. None of the other groups showed significant changes in gene expression over baseline. Finally, seeding at target density results in greater overlap of cells compared to groups in which cells proliferate to target density, conferring increased thickness to multicellular culture aggregates seeded at target density. These data suggest that seeding protocols can be exploited to modulate mesenchymal stem cell shape and early gene expression typical for condensation events in development, which occur over an approximately 12-h period at E11.5 in the mouse limb bud. Follow-on studies will delineate longer-term effects of density and seeding protocol on modulation of stem cell fate and cell assembly to form tissues.