Neurogenesis-on-Chip: Electric field modulated transdifferentiation of human mesenchymal stem cell and mouse muscle precursor cell coculture

A number of bioengineering strategies, using biophysical stimulation, are being explored to guide the human mesenchymal stem cells (hMScs) into different lineages. In this context, we have limited understanding regarding the transdifferentiation of matured cells to another functional-cell type, when grown with stem cells, in a constrained cellular microenvironment under biophysical stimulation. While addressing such aspects, the present work reports the influence of the electric field (EF) stimulation on the phenotypic and functionality modulation of the coculture of mouse myoblasts (C2C12) with hMScs(hMSc:C2C12 = 1:10)in a custom designed polymethylmethacrylate (PMMA) based microfluidic devicewith in-built metal electrodes. The quantitative and qualitative analysis of the immunofluorescence study confirms that the cocultured cells in the conditioned medium with astrocytic feed, exhibit differentiation towards neural-committed cells under biophysicalstimulation in the range of the endogenous physiological electric field strength (8 ± 0.06 mV/mm). The control experiments using similar culture protocols revealed that while C2C12 monoculture exhibited myotube-like fused structures, and the hMScs exhibited the formation of neurosphere-like clusters with SOX2, nestin, βIII-tubulin expression. The electrophysiological study indicates the significant role of intercellular calcium signallingamongthe differentiated cells towards transdifferentiation. Furthermore, the depolarization induced calcium influx strongly support neural-like behaviour for the electric field stimulatedcells in coculture. The intriguing results are explained in terms of the paracrine signallingamongthe transdifferentiated hMScs and the C2C12 cells in the electric fieldstimulated cellular microenvironment in the PMMA microfluidic device. In summary, the present study establishes the potential for neurogenesis on-chip for the coculture of hMSc and C2C12 cells under tailored electric field stimulation, in vitro.