3D soft hydrogels induce human mesenchymal stem cells “deep” quiescence

Abstract

Human mesenchymal stem/stromal cells (hMSCs) present a great opportunity for tissue regeneration due to their multipotent capacity. However, when cultured on 2D tissue culture polystyrene (TCPS) plates, hMSCs lose their differentiation capacity and clinical potential. It has been reported that cells need a more physiologically relevant micro-environment that allows them to maintain their phenotype. Here, we have developed a 3D alginate hydrogel functionalized with the Arg-Gly-Asp (RGD) sequence and having low mechanical stiffness that mimics the mechanical properties (>5 KPa) of bone marrow. hMSCs cultured in these hydrogels appeared to be halted in G1 phase of the cell cycle and to be 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 TCPS. Comparative studies in 3D hydrogels of alginate-RGD presenting higher concentration of the peptide or in collagen hydrogels revealed that independently of the concentration of RGD or the chemistry of the adhesion motives, hMSCs cultured in 3D presented a similar phenotype. This quiescent phenotype was exclusive of 3D cultures. In 2D, even when cells were starved of fetal bovine serum (FBS) and became also 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 to be in deep quiescence and be kept alive ex vivo for a long period of time. Our results represent a step forward towards understanding hMSCs quiescence and its molecular pathways, providing more insight for hMSCs cell therapies.

A comparative study of mesenchymal stem cells cultured as cell‐only aggregates and in encapsulated hydrogels

“…The final product was dialyzed (MWCO 3500, Spectra/Por, VWR) against decreasing concentrations of NaCl (7.50, 6.25, 5.00, 3.75, 2.50, 1.25 mg) in 4 L of ultrapure water for 3 days at 4°C , freeze-dried, and stored at −20°C until use. The RGD concentration was 35 μM, as reported in a previous study (Gomes et al, 2021).…”

Anisotropic Hydrogel Degradation Enhances 3D Collective Cell Alignment, Mechanotransduction and Osteogenic Differentiation

“…It was shown that some cells undergo adipogenic differentiation; however, it is still unclear what happens with the rest of the cells in the noDeg regions. The stromal cell niche suggests that the non-dynamic surroundings can contribute to preserving the naivety of stromal cells [57][58]. Furthermore, the noDeg sections also have a notable contribution to the mechanical and structural stability of the patterned material.…”