Conversion of human fibroblasts to endothelial-like cells via dedifferentiation to mesodermal progenitors in 3D condensates

We have previously shown that human mesenchymal stromal cells dedifferentiate to early mesoderm when cultured in defined three-dimensional (3D) in vitro conditions, to mimic mesenchymal condensation, through a controlled autophagy response. Here it was determined if human dermal fibroblasts (HDFs), a terminally differentiated cell, could similarly undergo dedifferentiation. By varying initiating cell number and culture duration, we identified optimised conditions for HDF dedifferentiation in 3D spheroids. This was revealed by QPCR which identified low level expression of pluripotency factors, Oct4, Sox2 and Nanog, and a 6 to 1200-fold increase in mesodermal markers, Brachyury, Goosecoid and CXCR4, compared to adherent 2D HDFs, whilst expression of the CXCR4 ligand and stromal marker CXCL12 decreased. CXCR4 protein expression was confirmed by western blot analysis and immunostaining, which showed positive distribution across the spheroid. These changes occurred with a concomitant increase in autophagic features (increased TFEB mRNA expression, elevated LAMP-1 protein and the accumulation of autophagosome-like structures). We next tested the redifferentiation potential of 3D HDFs by exposing the disaggregated cells to endothelial growth conditions on Matrigel. When exposed to EGF and FGF-2, endothelial-like networks formed within 7 days, which was reduced to 24h following the addition of IGFI, VEGF and BMP4. These cells were also shown to express low levels of endothelial cell markers, VE-Cadherin, Nrp1, Nrp2 and ALK1 and higher levels of EphB4 and KDR by QPCR. These findings suggest that human cell differentiation can be efficiently reversed by physiological means and then re-differentiated into a new cell type.