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.

We have developed a procedure to explant fibroblasts from the anterior cruciate ligament (ACL) and the medical collateral ligament (MCL) of the rabbit knee, and have optimized conditions for maintaining them in culture. Maximal growth for both ACL and MCL cells was obtained with Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum and 250 microM ascorbate. ACL and MCL fibroblasts displayed intrinsic differences in their responses to changes in culture parameters. Specifically, they displayed different growth responses when plated at different densities and responded to RPMI 1640 medium in very different ways. There were also biochemical differences between the cell types. Both cell types produced similar amounts of collagen in culture, but the ratio of type I to type III, the major collagen subtypes produced by these cells, were different. ACL fibroblasts produced 86.7% type I and 13.3% type III, and MCL fibroblasts produced 71.1% type I and 28.9% type III. In addition, total protein produced by ACL fibroblasts was higher than that produced by MCL cells. This confirms the suggestions of previous researchers that such differences might exist.

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Human Mesenchymal cells (hMSCs) are promising in regenerative medicine for their multi-lineage differentiation capability. It has been demonstrated that lineage specification is governed by both chemical and mechanical cues. Among all the different mechanical cues known to control hMSCs fate, substrate stiffness is the most well-studied. It has been shown that the naive mesenchymal stem cells when cultured on soft gel, they commit towards adipogenic lineage while when cultured on stiff gel they become osteogenic. Soft substrates also cause less cell spreading, less traction, less focal adhesion assembly and stress fibre formation. Furthermore, chromatin condensation increases when cells are cultured on soft substrates. As the nucleus has been postulated to be mechanosensor and mechanotransducer, in this paper we asked the question how mechanosensing and mechanoresponse process will be influenced if we change the chromatin condensation by using an external chemical stimulus. To address this question, we treated hMSCs cultured on soft polyacrylamide (PA) gels with a histone deacetylase inhibitor (HDACi) called Valproic Acid (VA) which decondense the chromatin by hyperacetylation of histone proteins. We found that the treatment with VA overrides the effect of soft substrates on hMSCs morphology, cellular traction, nuclear localization of mechnosensory protein YAP, and differentiation. VA treated cells behaved as if they are on stiff substrates in all aspects tested here. Furthermore, we have shown that VA controls hMSCs differentiation via activation of ERK/MAPK pathway by increasing the p-ERK expression which inhibits adipogenic differentiation potential of mesenchymal stem cells. Collectively, these findings for the first time demonstrate that inhibiting histone acetylation can override the mechanoresponse of hMSCs. This work will help us to fundamentally understand the mechanosignalling process and to control the hMSCs differentiation in tissue engineering and regenerative medicine.

Human mesenchymal stem cells (hMSCs) are multipotent cells that can differentiate into adipocytes, chondrocytes and osteoblasts. Due to their differentiation potential, hMSCs are among the most frequently used cells for therapeutic applications in tissue engineering and regenerative medicine. However, the number of cells obtained through isolation alone is insufficient for hMSC-based therapies and basic research, necessitating their in-vitro expansion. Conventionally, this is often carried out on rigid surfaces such as tissue culture petriplates (TCPs). However, during in-vitro expansion, hMSCs lose their proliferative ability and multilineage differentiation potential, making them unsuitable for clinical use. Although multiple approaches have been tried to maintain hMSC stemness over prolonged expansion, finding a suitable culture system to achieve this remains an unmet need. Recently, few research groups including ours have shown that hMSCs maintain their stemness over long passages when cultured on soft substrate. In addition, it has been shown that hMSCs cultured on soft substrates have more condensed chromatin and lower levels of histone acetylation compared to those cultured on stiff substrates. It has also been shown that condensing/decondensing chromatin by deacetylation/acetylation can delay/hasten replicative senescence in hMSCs during long-term expansion on TCPs. However, how chromatin condensation/decondensation influences nuclear morphology and DNA damage - which are strongly related to the onset of senescence and cancer - is still not known. To answer this question, here we cultured hMSCs for long duration (P4-P11) in presence of epigenetic modifiers histone acetyltransferase inhibitor (HATi) which promotes chromatin condensation by preventing histone acetylation and histone deacetylase inhibitor (HDACi) which promotes chromatin decondensation and investigated their effect on various nuclear markers related to senescence and cancer. We have found that consistent acetylation causes severe nuclear abnormalities whereas chromatin condensation by deacetylation helps in safeguarding nucleus from damages caused by in-vitro expansion.