Protein Kinase-A Inhibition Is Sufficient to Support Human Neural Stem Cells Self-Renewal

Abstract

Human pluripotent stem cell-derived neural stem cells offer unprecedented opportunities for producing specific types of neurons for several biomedical applications. However, to achieve it, protocols of production and amplification of human neural stem cells need to be standardized, cost effective, and safe. This means that small molecules should progressively replace the use of media containing cocktails of protein-based growth factors. Here we have conducted a phenotypical screening to identify pathways involved in the regulation of hNSC self-renewal. We analyzed 80 small molecules acting as kinase inhibitors and identified compounds of the 5-isoquinolinesulfonamide family, described as protein kinase A (PKA) and protein kinase G inhibitors, as candidates to support hNSC self-renewal. Investigating the mode of action of these compounds, we found that modulation of PKA activity was central in controlling the choice between self-renewal or terminal neuronal differentiation of hNSC. We finally demonstrated that the pharmacological inhibition of PKA using the small molecule HA1004 was sufficient to support the full derivation, propagation, and long-term maintenance of stable hNSC in absence of any other extrinsic signals. Our results indicated that tuning of PKA activity is a core mechanism regulating hNSC self-renewal and differentiation and delineate the minimal culture media requirement to maintain undifferentiated hNSC in vitro. STEM CELLS 2015;33:3666-3672 SIGNIFICANCE STATEMENTPluripotent stem cells-derived neural stem cells offer unprecedented opportunities for producing human neurons for several biomedical applications. However, defining the minimal requirement to maintain neural stem cells self-renewal in vitro, using small molecules rather than protein based cocktails, is a pre-requisite to fully achieve the potential of these cells. Here, we have used high throughput screening to find small molecules able to replace the prototypical EGF/FGF-2 based cocktail uses to support neural stem cells self-renewal. This screen identified Protein Kinase A inhibitors and allowed us to delineate the minimal culture media requirement to maintain undifferentiated hNSC in vitro.

Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3β

“…All the results reveal that PKA phosphorylates Ctnnb1 and Gsk3β and plays a key role during NSC differentiation. Interestingly, inhibition of PKA is sufficient to maintain undifferentiated human NSC in vitro , which is consistent with our results. PKA activity may play a core role in controlling self‐renewal and differentiation of NSC.…”

Human Pluripotent Stem Cell-derived Cortical Neurons for High Throughput Medication Screening in Autism: A Proof of Concept Study in SHANK3 Haploinsufficiency Syndrome

“…Reprogramming of somatic cells into iPSC being potentially responsible for induction of epigenetic and imprinting alterations that could bias the study of gene regulation ( Pick et al, 2009 , Urbach et al, 2010 , Lister et al, 2011 ), the primary drug screening was conducted using neurons differentiated from the control hESC line SA001. The 202 compounds were screened in 4 biological replicates at one concentration determined for each compound based on data from the literature or from our own previous findings ( Boissart et al, 2013 , Georges et al, 2015 ). This primary drug screening identified 29 primary hits that increased SHANK3 mRNA content by at least 30% as compared to DMSO-treated cells and with a deviation from the DMSO mean of more than 2 σ ( Fig.…”

Rescuing compounds for Lesch-Nyhan disease identified using stem cell–based phenotypic screening

“…15) that are differentiated into neurons to study the effect of HGPRT deficiency. We previously showed that such neurons may be instrumental to identify potentially therapeutic pharmacological compounds using automated high-throughput drug screening coupled with image-based high-content analysis (16)(17)(18). In the current study, we show that LND cells demonstrate pathological changes during key steps of the differentiation of human pluripotent stem cellderived (PSC-derived) neural stem cells (NSC) into cortical neurons.…”