Rescuing mesenchymal stem cell regenerative properties on hydrogel substrates post serial expansion

Abstract

The use of human mesenchymal stem/stromal cells (hMSCs) in most clinical trials requires millions of cells/kg, necessitating ex vivo expansion typically on stiff substrates (tissue culture polystyrene [TCPS]), which induces osteogenesis and replicative senescence. Here, we quantified how serial expansion on TCPS influences proliferation, expression of hMSC‐specific surface markers, mechanosensing, and secretome. Results show decreased proliferation and surface marker expression after five passages (P5) and decreased mechanosensing ability and cytokine production at later passages (P11‐P12). Next, we investigated the capacity of poly(ethylene glycol) hydrogel matrices (E ~ 1 kPa) to rescue hMSC regenerative properties. Hydrogels reversed the reduction in cell surface marker expression observed at P5 on TCPS and increased secretion of cytokines for P11 hMSCs. Collectively, these results show that TCPS expansion significantly changes functional properties of hMSCs. However, some changes can be rescued by using hydrogels, suggesting that tailoring material properties could improve in vitro expansion methods.

Nuclear mechanosensing controls MSC osteogenic potential through HDAC epigenetic remodeling

“…This mechanical desensitization has also been seen in MSCs from older donors, where YAP nuclear translocation is decreased and RUNX2 expression is down-regulated on stiff gels compared to young MSCs (46,47). Even with passaging, this dysregulation has been observed (6). Furthermore, the blocked nuclear mechanosensing might have induced a feedback loop to the cytoplasm; a possible reason why we observed a decrease in cell spreading with stiffening compared to the mCherry control (SI Appendix, Fig.…”

Shattering barriers toward clinically meaningful MSC therapies

“…Several biomaterial strategies have been explored to maintain more homogeneous MSCs during the expansion phase of MSC manufacturing. For example, Rao et al (41) showed that expanding hMSCs on soft poly(ethylene glycol) hydrogel matrices was able to avoid reductions in cell surface marker expression and cytokine expression that was observed when hMSCs were serially passaged on polystyrene. Growing MSCs in three-dimensional (3D) culturing systems has also been beneficial in maintaining early-passage MSC phenotype during expansion (42,43).…”

Hydrogels in the clinic

“…Due to this high water content, hydrogels exhibit favorable biocompatibility and as such have been developed for and used in a variety of medical applications 1‐3 . Since hydrogel properties can be tuned through a variety of chemical approaches, their rational design and engineering has enabled new modalities for delivery of small molecules, 4‐7 proteins, 8‐12 and cells 13,14 and as tissue engineering scaffolds for directing cell fate/lineage, 15,16 stem cell expansion, 17‐20 and tissue regeneration 21‐25 . Research efforts to develop hydrogels for biomedical applications represents one of the most studied areas at the interface of engineering and medicine 26 .…”