Cardiomyoblast (H9c2) Differentiation on Tunable Extracellular Matrix Microenvironment

Abstract

Extracellular matrices (ECM) obtained from in vitro-cultured cells have been given much attention, but its application in cardiac tissue engineering is still limited. This study investigates cardiomyogenic potential of fibroblast-derived matrix (FDM) as a novel ECM platform over gelatin or fibronectin, in generating cardiac cell lineages derived from H9c2 cardiomyoblasts. As characterized through SEM and AFM, FDM exhibits unique surface texture and biomechanical property. Immunofluorescence also found fibronectin, collagen, and laminin in the FDM. Cells on FDM showed a more circular shape and slightly less proliferation in a growth medium. After being cultured in a differentiation medium for 7 days, H9c2 cells on FDM differentiated into cardiomyocytes, as identified by stronger positive markers, such as a-actinin and cTnT, along with more elevated gene expression of Myl2 and Tnnt compared to the cells on gelatin and fibronectin. The gap junction protein connexin 43 was also significantly upregulated for the cells differentiated on FDM. A successive work enabled matrix stiffness tunable; FDM crosslinked by 2wt% genipin increased the stiffness up to 8.5 kPa, 100 times harder than that of natural FDM. The gene expression of integrin subunit a5 was significantly more upregulated on FDM than on crosslinked FDM (X-FDM), whereas no difference was observed for b1 expression. Interestingly, X-FDM showed a much greater effect on the cardiomyoblast differentiation into cardiomyocytes over natural one. This study strongly indicates that FDM can be a favorable ECM microenvironment for cardiomyogenesis of H9c2 and that tunable mechanical compliance induced by crosslinking further provides a valuable insight into the role of matrix stiffness on cardiomyogenesis.

Three‐dimensional spherical gelatin bubble‐based scaffold improves the myotube formation of H9c2 myoblasts

“…A previous study reported that the myoblasts had different behaviors on laminin, fibronectin, collagen type I, and collagen type IV substrates (Duffy, Sun, & Feinberg, ). Similarly, cardiomyoblasts showed improved cardiomyogenic potentials on the cell‐derived matrices (Suhaeri et al,). However, muscle cells lose normal organizations and transform to random distributions during in vitro cultivation (Huang et al, ).…”

Highly efficient cardiac differentiation and maintenance by thrombin-coagulated fibrin hydrogels enriched with decellularized porcine heart extracellular matrix

“…An important aim in the design of the hydrogel system was to match the physical stiffness of native myocardium (10-50kPa range, [60][8]). To mechanically reinforce the intrinsically soft cardiac dECM, we chose the specific coagulation of fibrinogen by thrombin, as opposed to more generic agents such as transglutaminase [30][61] or genipin [39][62] We find the thrombin-coagulated fibrin gels to be softer than similar gels crosslinked by transglutaminase [30], but we could compensate for this by increasing the fibrinogen concentration correspondingly.…”

“…Fibroblast-derived matrix has been shown to enhance retinoid-based differentiation of H9c2 cells [39]. Here, we assess whether a similar favorable effect on the H9c2 cardiogenic differentiation can be obtained by direct coculture with Nor-10 fibroblasts [55].…”

“…Yet, upon exposure to retinoic acid, cardiac differentiation can be recovered [38]. The cardiac differentiation efficiency of H9c2 cells can be further enhanced by the presence of fibroblast-derived matrix in addition to the retinoic acid [39]. Our goal is to optimize cardiogenic differentiation of H9c2 by combining fibroblast support with appropriate mechanical and matrix cues in composites of coagulated fibrin and porcine cardiac dECM hydrogels.…”

Cardiomyocyte differentiation: Experience and observations from 2 laboratories

“…Yet a fourth variable, which has received some attention, is the surface on which the cells are cultured and differentiated. In this regard it has been reported that the degree of differentiation is influenced by interventions such as growing cells on an extracellular matrix (32) or growing cells on a surface with micro grooves. (33) The most significant limitation of this study is that it was done in a retrospective manner, in other words the research efforts of the 2 laboratories were not planned or coordinated in advance.…”