Analysis of three dimensional culture mechanisms to induce de-differentiation and reprogramming in human multipotent stromal cells

Cells undergo reprogramming/de-differentiation through nuclear and cytoplasmic remodelling. Autophagy is a physiological stress response, in which cytoplasmic contents including mitochondria are recycled to aid cell survival in response to nutrient starvation. It has recently been demonstrated that a functional autophagy response is required for factor-based reprogramming to pluripotency, but there is little work explicitly linking the stimulation of autophagy with the mechanism of cytoplasmic clearance and metabolic remodelling. Human multipotent stromal cells/mesenchymal stem cells (MSCs) are cells of mesenchymal origin, which are cultured as adherent monolayers in vitro. MSCs can also be cultured as 3D cell aggregates or spheroids. Variations in spheroid size should result in variations in nutrient availability. I hypothesised that in 3D spheroids, controlled autophagy, stimulated by nutrient deprivation, and balanced in favour of its pro-survival/anti-apoptotic effects, would be sufficient to drive cytoplasmic remodelling and de-differentiation towards a more primitive state. When MSCs were cultured under optimal conditions, they had a multi-lobed irregular nuclear morphology, and reduced staining for the heterochromatin marker H3K9me3. 3D spheroids increased expression of Oct4, Nanog, Sox2 and telomerase. Under optimal conditions markers of increased autophagy were observed along with indicators of a shift to anaerobic metabolism. Mitochondria underwent remodelling to a small, rounded morphology, highly similar to mitochondria observed in pluripotent cells. Furthermore, oxygen consumption rate reduced significantly in 3D culture, and genes associated with oxidative metabolism were down-regulated. 3D MSCs increased expression of early mesendoderm markers including Brachyury (T), Goosecoid, KDR, Mixl1 and CXCR4. In vitro haematopoietic induction stimulated disaggregated 3D MSCs to form blast-like colonies, whilst 2D MSCs were unresponsive to haematopoietic stimulation. On implantation into nude mice, 3D MSCs formed organised mesodermal tissues, indicating enhanced lineage-restricted regenerative capacity, but did not form teratomas, so avoiding associated risks. Strikingly optimal 3D culture restored proliferative capacity and reversed senescence-associated hypertrophy in culture-aged MSCs, suggestive of cellular rejuvenation. Together these results suggest that a scaled autophagy response could play a fundamental role in reprogramming/de-differentiation and tissue regeneration in human cells