Effective bioengineered niche for cardiac differentiation and/or beating induction of iPS and P19CL6 cells

摘要

Introduction: Various cues for cardiac differentiation of stem cells have been attracting great attentions. We are focusing on the cell culture substrates as biomechanical cues. In general, the differentiation profiles have been evaluated with cardiac gene expression, but they do not necessarily relate to the self-beating function. The purpose of this study is to study the suitable culture substrates for each step of the differentiation cascade (cardiac differentiation, beating induction, and beating duration) separately. To this end, in addition to iPS cells and P19CL6 cell, beating behaviors of neonatal cardiomyocytes (NCMs) were also analyzed (Figure 1). Materials and Methods: The iPS cells (iPS-MEF-Ng-178B-5) were provided by the RIKEN Bioresource Center.[Y1] Neonatal cardiomyocytes (NCMs)[y2] were isolation from neonatal SD rat hearts. P19CL6 cells were purchased from Riken Bio Resource Center. Soft hydrogel substrates were prepared based on the previous report. These elastic moduli measured by AFM were 9,20, and 180kPa. ECM proteins (collagen type Ⅰ(Col-Ⅰ), gelatin (Gel), and fibronectin (FN)) and other proteins were immobilized via N-sulfosuccinimidyl-6-(4-azido-2-nitrophenyl-amino) onto the gels and onto the tissue culture poly styrene (stiff surface). The cells were seeded on these substrates and treated with the conventional cardiac differentiation medium. The cardiac marker genes (GATA4, MEF2D, and MCN4) and the cell contractile proteins (a-MHC, b-MHC, TnC1, and TnT2) were quantified by qRT-PCR to evaluated cardiac differentiation and beating inductions. In addition, beating cells were visualized with calcium indicator dye Rhod-4 and counted under Eclipse TE-300 fluorescence microscope. Results and Discussion: It was found that each step of the cardiac differentiation was greatly improved by different substrates with the combination of modulus and protein. For example, Col-Ⅰ- and Gel-coated TCPS was very efficient to induce the cardiac marker gene expression but did not induce the beating well. In contrast, FN coated surface showed the opposite tendency. The duration period of the beating behavior of differentiated iPS was greatly elongated on the soft substrates irrespective of the coated materials. We also found a couple of matrices that are effective both in the cardiac differentiation and beating induction steps. Conclusions: By studying the cardiac differentiation of iPS cells and P19CL6 cells in a step-by-step manner, very effective processes for cardiac differentiation were established.