Abstract
The generation of human induced neurons (hiNs) via exogenous delivery of neural transcription factors represents a novel technique to obtain disease and patient specific neurons. These cells have the potential to be used for disease modeling, diagnostics and drug screening, and also to be further developed for brain repair. In the present study, we utilized hiNs to develop an unbiased screening assay for small molecules that increase the conversion efficiency. Using this assay, we screened 307 compounds from five annotated libraries and identified six compounds that were very potent in potentiating the reprogramming process. When combined in an optimal combination and dose, these compounds increased the reprogramming efficiency of human fibroblasts more than 6-fold. Global gene expression and CellNet analysis at different timepoints during the reprogramming process revealed that neuron-specific genes and gene regulatory networks (GRNs) became progressively more activated while converting cells shut down fibroblast-specific GRNs. Further bioinformatics analysis revealed that the addition of the six compound resulted in the accelerated upregulation of a subset of neuronal genes, and also increased expression of genes associated with transcriptional activity and mediation of cellular stress response.
Highlights
Somatic cell reprogramming using defined transcription factors enables the generation of induced pluripotent stem cells[1,2,3], as well as allows for the direct conversion of somatic cells into terminally differentiated cells, including subtype-specific and functional neurons[4,5,6,7,8,9,10]
20 small molecules were selected as primary hits for increasing the neuronal purity above the set threshold (Fig. S1 and Table S1), and one additional compound was selected based on its ability to induce a mature neuronal morphology (EL38)
The use of small molecules to enhance efficiency of reprogramming, reduce the number of genes required for reprogramming as well as to gain further mechanistic insight into the reprogramming process has successfully been applied in somatic cell reprogramming to pluripotency[11,12,13,14,18,19,20,35,36]
Summary
Somatic cell reprogramming using defined transcription factors enables the generation of induced pluripotent stem (iPS) cells[1,2,3], as well as allows for the direct conversion of somatic cells into terminally differentiated cells, including subtype-specific and functional neurons[4,5,6,7,8,9,10]. A number of studies have shown that addition of small molecules during re-programming into pluripotency[11,12,13,14] or during direct cell fate conversion[15,16] increase the efficiency and/or survival[17], and in some cases allow for chemical replacement of individual reprogramming genes[18,19,20] or even completely replace the need for transgene expression[21]. We first performed CellNet analysis, which classifies cells using a large body of publicly available data (29, 30), which revealed initiation of neuron- specific gene regulatory networks (GRNs) as well as ablation of fibroblasts- specific GRNs, which occurred at similar rates among all groups of converting hiNs. Further bioinformatics analysis of this time course experiment enabled for a more detailed view on transcriptional changes and revealed that the addition of the six compounds resulted in the accelerated upregulation of a subset of neuronal genes, and increased expression of genes associated with transcriptional activity and mediation of cellular stress response early during the reprogramming process
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