Higuera GA, Fernandes H, Spitters TW, van de Peppel J, Aufferman N, Truckenmueller R, Escalante M, Stoop R, van Leeuwen JP, de Boer J, Subramaniam V, Karperien M, van Blitterswijk C, van Boxtel A, Moroni L.
Cells and tissues are intrinsically adapted to molecular gradients and use them to maintain or change their activity. The effect of such gradients is particularly important for cell populations that have an intrinsic capacity to differentiate into multiple cell lineages, such as bone marrow derived mesenchymal stromal cells (MSCs). Our results showed that nutrient gradients prompt the spatiotemporal organization of MSCs in 3D culture. Cells adapted to their 3D environment without significant cell death or cell differentiation. Kinetics data and whole-genome gene expression analysis suggest that a low proliferation activity phenotype predominates in stromal cells cultured in 3D, likely due to increasing nutrient limitation. These differences implied that despite similar surface areas available for cell attachment, higher cell concentrations in 3D reduced MSCs proliferation, while activating hypoxia related-pathways. To further understand the in vivo effects of both proliferation and cell concentrations, we increased cell concentrations in small (1.8 μl) implantable wells. We found that MSCs accumulation and conditioning by nutrient competition in small volumes leads to an ideal threshold of cell-concentration for the induction of blood vessel formation, possibly signaled by the hypoxia-related stanniocalcin-1 gene.