Induction and differentiation of human induced pluripotent stem cells into functional cardiomyocytes on a compartmented monolayer of gelatin nanofibers

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Induction and differentiation of human induced pluripotent stem cells into functional cardiomyocytes on a compartmented monolayer of gelatin nanofibers, Nanoscale, 2016, 8, 14530-14540


Culture dishes, flasks and multi-well plates are widely used for cell biology studies and cell-based assays but they are not ideal for culture and differentiation of pluripotent stem cells, including both embryonic (ESCs) and induced pluripotent stem cells (iPSCs). Indeed, cells cultured with these conventional culture supports have to adapt a two-dimensional (2D) surface with large exogenous contact and limited uptake and self-organization efficiency, while the pluripotent stem cells generally require more adapted cellular microenvironments to maintain their pluripotency or differentiation homogeneity. Previously, culture substrates coated with Matrigel, a gelatinous protein mixture derived from mouse tumor cells, or recombinant proteins such as laminin2 and vitronectin have been proposed for iPSC culture and differentiation. Other substrates such as oxygen plasma etched plates, porous materials and electrospun nanofibers are used, showing sufficient interest in textured surface morphology. However, these approaches do not recapitulate the three-dimensional (3D) organization of extracellular matrix proteins of stem cell niche. Without further improvement, conventional approaches will not be able to overcome risks of genetic instability and tumorigenicity. On the other hand, conventional approaches are not appropriate to support tissue formation because of the lack of required mechanic and/or physiological conditions. Previously, cardiac sheets were obtained by culture on and release from temperature-sensitive polymer layers. This approach is ingenious but it does not allow fabrication of robust and thick cardiac sheets due to limitation of nutrient diffusion. Alternatively, cardiac layers can be formed on topographic patterns and nanofibrous substrates, but their application potential has to be demonstrated. Most recently, we proposed a culture patch method for culture and differentiation of human iPSCs (hiPSCs) into motor neurons.



We put forward the culture patch method for hiPSC culture and differentiation towards cardiomyocytes. First, the crosslinked monolayer gelatin nanofibers allow minimizing the exogenous material contact of hiPSCs because of the large pore sizes of the patch and the use of a natural polymer. If necessary, the culture patch can be coated with vitronectin or other types of extracellular matrix proteins for more functional cell–nanofiber coupling. In all cases, the mechanical stress on cells should be reduced and cell–cell coupling should be enhanced with respect to the cells cultured on flat substrates. Accordingly, cells can be more easily self-organized, which explains the formation of single hiPSC colonies in each of the honeycomb compartments as well as the formation of homogeneous cardiac tissues after differentiation.


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Nanoscale, 2016, 8, 14530


Extensive efforts have been devoted to develop new substrates for culture and differentiation of human induced pluripotent stem cells (hiPSCs) toward cardiac cell-based assays. A more exciting prospect is the construction of cardiac tissue for robust drug screening and cardiac tissue repairing. Here, we developed a patch method by electrospinning and crosslinking of monolayer gelatin nanofibers on a honeycomb frame made of poly(ethylene glycol) diacrylate (PEGDA). The monolayer of the nanofibrous structure can support cells with minimal exogenous contact and a maximal efficiency of cell–medium exchange whereas a single hiPSC colony can be uniformly formed in each of the honeycomb compartments. By modulating the treatment time of the ROCK inhibitor Y-27632, the shape of the hiPSC colony could be controlled from a flat layer to a hemisphere. Afterwards, the induction and differentiation of hiPSCs were achieved on the same patch, leading to a uniform cardiac layer with homogeneous contraction. This cardiac layer could then be used for extracellular recording with a commercial multi-electrode array, showing representative field potential waveforms of matured cardiac tissues with appropriate drug responses.


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Induction and differentiation of human induced pluripotent stem cells into functional cardiomyocytes on a compartmented monolayer of gelatin nanofibers



Yadong Tang, Li Liu, Junjun Li, Leqian Yu, Li Wang, Jian Shi and Yong Chen



Nanoscale, 2016, 8, 14530-14540



DOI : 10.1039/C6NR04545F