3D Soft Hydrogels Induce Human Mesenchymal Stem Cells "Deep" Quiescence

It has been reported that cells need a more physiologically relevant micro-environment that allows them to maintain their phenotype. When cultured on 2D tissue culture plates, human mesenchymal stem cells (hMSCs) lose their differentiation capacity and clinical potential. Here, we developed a 3D alginate hydrogel functionalized with the Arg-Gly-Asp (RGD) sequence and having mechanical stiffness mimicking the mechanical properties (<5 kPa) of bone marrow. hMSCs cultured in these hydrogels were halted in G₁ phase of the cell cycle and non-proliferative, as shown by flow cytometry and 5-Ethynyl-2'-deoxyuridine (EdU) staining, respectively. Their quiescent state was characterized by an upregulation of enhancer of zeste homolog 1 (EZH1) at the gene level, forkhead box O3 (FoxO3) and cyclin-dependent kinase inhibitor 1B (p27) at the gene and protein levels compared to hMSCs grown in 2D. Studies in 3D hydrogels of collagen or alginate-RGD hydrogels presenting a higher concentration of the peptide revealed that, independently of the concentration of RGD or the chemistry of the adhesion motives, hMSCs cultured in 3D presented a similar phenotype. This phenotype was exclusive to 3D cultures. In 2D, even when cells were serum-deprived and became non-proliferative, the expression of these markers was not observed. We propose that this difference may be the result of mammalian target of rapamycin complex 1 (mTORC1) being downregulated in hMSCs cultured in 3D hydrogels, which induces cells in "deep" quiescence. Our results represent a step forward towards understanding hMSCs quiescence and its molecular pathways, providing more insight for hMSCs cell therapies.