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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE255748 | GEO | 2024/04/08

REPOSITORIES: GEO

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Project description:It remains controversial whether the routes from differentiated cells to iPSCs are related to the reverse order of normal developmental processes or independent of them. Here, we generated iPSCs from mouse astrocytes by three (Oct3/4, Klf4 and Sox2 (OKS)), two (OK), or four (OKS plus c-Myc) factors. Sox1, a neural stem cell (NSC)-specific transcription factor, is transiently upregulated during reprogramming and Sox1-positive cells become iPSCs. The upregulation of Sox1 is essential for OK-induced reprogramming. Genome-wide analysis revealed that the gene expression profile of Sox1-expressing intermediate-state cells resembles that of NSCs. Furthermore, the intermediate-state cells are able to generate neurospheres, which can differentiate into both neurons and glial cells. Remarkably, during MEF reprogramming, neither Sox1 upregulation nor an increase in neurogenic potential occurs. Thus, astrocytes are reprogrammed through an NSC-like state, suggesting that reprogramming partially follows the retrograde pathway of normal developmental processes. To investigate the gene expression profile of intermediate-state cells during astrocyte reprogramming, we performed genome-wide gene expression analysis in five samples; starting astrocytes, intermediate-state cells expressing Sox1-GFP, NSCs, iPSCs established from astrocytes, and iPSCs established from MEFs (iPS-MEF-Ng-20D-17) that had previously been reported (Okita, K. et al. Nature 448: 313-317 (2007)). Two (NSCs, iPSCs from astrocytes and MEFs) or three (astrocytes, intermediate-state cells) biological replicates were prepared for microarray samples. Total RNA was extracted with an RNeasy kit (Qiagen). cDNA synthesis and transcriptional amplification were performed using 50-100 ng of total RNA with the GeneChip WT PLUS Reagent Kit (Affymetrix). Fragmented and biotin-labeled cDNA targets were hybridized to GeneChip Mouse Gene 1.0 ST arrays (Affymetrix) according to the manufacturerâ??s protocol. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner.

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

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