Soft substrate maintains proliferative and adipogenic differentiation potential of human mesenchymal stem cells on long-term expansion by delaying senescence

Abstract

Human mesenchymal stem cells (hMSCs), during in vitro expansion, gradually lose their distinct spindle morphology, self-renewal ability, multi-lineage differentiation potential and enter replicative senescence. This loss of cellular function is a major roadblock for clinical applications which demand cells in large numbers. Here, we demonstrate a novel role of substrate stiffness in the maintenance of hMSCs over long-term expansion. When serially passaged for 45 days from passage 3 to passage 18 on polyacrylamide gel of Young's modulus E =5 kPa, hMSCs maintained their proliferation rate and showed nine times higher population doubling in comparison to their counterparts cultured on plastic Petri-plates. They did not express markers of senescence, maintained their morphology and other mechanical properties such as cell stiffness and cellular traction, and were significantly superior in adipogenic differentiation potential. These results were demonstrated in hMSCs from two different sources, umbilical cord and bone marrow. In summary, our result shows that a soft gel is a suitable substrate to maintain the stemness of mesenchymal stem cells. As preparation of polyacrylamide gel is a well-established, and well-standardized protocol, we propose that this novel system of cell expansion will be useful in therapeutic and research applications of hMSCs.

Interaction of Human Mesenchymal Stem Cells with Soft Nanocomposite Hydrogels Based on Polyethylene Glycol and Dendritic Polyglycerol

“…Surface morphology associated with cell adhesive ligands is beneficial for stem cells spreading. Especially, topography of the surface enhances the differentiation of hMSCs toward osteogenic and adipogenic lineage . Previous studies have suggested that the combined micro‐ and nanoscale topography is favoring hMSCs proliferation and differentiation .…”

“…And yet, an enhanced spreading area is characteristic of a senescence state, in other words, an irreversible cell division arrest which makes the cells unsuitable for clinical use. This issue has been addressed and is still looking for a solution . In this part, we study two hydrogels: a system with PEG20000 having an average shear elastic modulus of around 600 Pa and a matrix with PEG6000 around twice as stiff, having an average shear elastic modulus of around 1400 Pa (Figure ).…”

“…According to the International Society for Cellular Therapy's standard criteria, in cell‐based therapy, only CD73, CD90, and CD105‐positive hMSCs are administered to patients in clinics . Culturing hMSCs on soft substrates (typically, E ≈ 10 to 1 kPa) has been reported to be a better alternative to TCPS, keeping hMSCs' immunophenotyping intact . We intend to study these functions on soft substrates with topography.…”

“…It is known that stiff substrates favor osteogenic differentiation with a less considerable adipogenic differentiation potential . Cells favor adipogenic differentiation on soft substrates with a round morphology . Normally, the hard surfaces would then be expected to produce osteoblasts and the adipogenic differentiation potential would vanish.…”

“…Especially after several number of passages, usually after around the twelfth passage (P12) and multiple differentiation steps, they tend to go in a phase called “senescence” where the intrinsic function (stemness) of stem cells fades and cannot be recovered. This represents a major issue in tissue engineering where a culture system that maintains the stemness of the hMSCs is needed to efficiently expand them in vitro . The impact of patterned surfaces on cellular behavior has also been investigated in recent years.…”