Histone deacetylase inhibitor overrides the effect of soft hydrogel on the mechanoresponse of human mesenchymal stem cells

Human Mesenchymal stem 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 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 reduce the chromatin condensation in hMSCs cultured on soft substrates by using an external reagent. 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 (yes-associated protein), and differentiation. VA treated cells behaved as if they are on stiff substrates in all aspects tested here. In contrast, when we increase chromatin condensation using histone acetyl transferase inhibitor (HATi), adipogenic differentiation on soft gel increases. Furthermore, we have shown that VA controls hMSCs differentiation via activation of ERK/MAPK pathway by increasing the p-ERK expression. Collectively, these findings demonstrate for the first time that inhibiting histone deacetylase 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. SignificanceTo harness the clinical potential of Human mesenchymal stem cells (hMSCs), it is important to understand the working mechanism of various signals in deciding cell fate. It has been shown in the recent past that the substrate rigidity influence hMSCs differentiation. Though the mechanism is not fully understood, the mechanosignals are known to alter the cell fate via reorganizing the chromatin packing. In this work, we ask if altering chromatin packing can change the cell fate as dictated by the mechanical properties of the substrates. We have shown that almost all the known effector of mechanosignals get modified when we tweak the chromatin packing via histone deacetylase inhibitor (HDACi). This study is helpful for developing a better understanding of the mechano-signalling process. It gives us a tool to override the effect of the substrates, particularly when the mechanical properties of biomaterials are not conducive for the desired differentiation outcome. Additionally, as HDACi are being used in the therapy of cancer and different neurodegenerative diseases, our understanding of their crosstalk with tissue stiffness in controlling cell fate will help to understand any unknown and undesirable side effects of using HDACi as therapeutic agent.