On-chip neural induction boosts neural stem cell commitment: toward a pipeline for iPSC-based therapies

The clinical translation of induced pluripotent stem cells (iPSCs) holds great potential for personalized therapeutics. However, the current workflow to generate iPSCs is expensive, time-consuming, and requires standardization. We present a simplified microfluidic approach for reprogramming fibroblasts into iPSCs and their subsequent differentiation into neural stem cells (NSCs). Our method exploits microphysiological technology, providing a 100-fold reduction in reagents for reprogramming and a 9-fold reduction in number of input cells. The iPSCs generated from microfluidic reprogramming of fibroblasts show upregulation of pluripotency markers and downregulation of fibroblast markers, on par with those reprogrammed in the standardized well-conditions. The NSCs differentiated in microfluidic chips show upregulation of neuroectodermal markers (ZIC1, PAX6, SOX1), highlighting their propensity for nervous system development. We then compare the cells obtained on conventional well plates and microfluidic chips for reprogramming and neural induction by bulk RNA sequencing. Pathway enrichment analysis of NSCs generated in the chip showed neural stem cell development enrichment and boosted commitment to the neural stem cell lineage in the initial phases of neural induction, attributed to the confined environment in a microfluidic chip. This method provides a cost-effective pipeline to reprogram and differentiate iPSCs which can be made compliant with the current good manufacturing practices for therapeutics. Overall design: To perform the reprogramming of somatic cells intro induced pluripotent stem cells on a microfluidic chip and compare it to a conventional well plate format. Further, we performed the differentiation of iPSCs into ectodermal lineage using dual-SMAD inhibition protocol on the microfluidic chip and conventional well plates to study the differences between the choice of culture format.