Kele, M. | Search bioRxiv

bioRxiv2023

Defined human PSC culture conditions robustly maintain human PSC pluripotency through Ca2+ signaling.

Human pluripotent stem cells (hPSCs) have significant potential for disease modeling and cell therapies. However, their clinical applicability is limited due to the need for undefined conditions for PSC cultivation, which increase the risk of immunogenicity, result in batch-to-batch variability and finite scalability. These limitations may be circumvented by xeno-free, defined culture conditions. However, biological processes that preserve robust, homogenous PSCs in defined conditions remain to be characterized. Here, we compared gene expression data from over 100 hPSC cell lines cultivated in undefined and defined culture conditions. Defined culture conditions significantly reduced inter-PSC line variability, highlighting the importance of standardization to minimize PSC biases. This variability is concurrent with decreased germ layer differentiation and increased expression of Ca2+-binding proteins. The significance of tightly controlled Ca2+ signaling in hPSC pluripotency in defined culture conditions was also confirmed. A deeper understanding of these processes may aid in standardizing defined hPSC culture conditions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=174 HEIGHT=200 SRC="FIGDIR/small/552440v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@1e5729aorg.highwire.dtl.DTLVardef@c6fa28org.highwire.dtl.DTLVardef@1515b91org.highwire.dtl.DTLVardef@52c6b6_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefEidhof et al. compared gene expression array data obtained from more than 100 hPSC cell lines to study the biological differences between PSCs cultivated under defined and undefined culture conditions. HighlightsO_LIDefined culture conditions reduce inter-hPSC line variability. C_LIO_LIDefined conditions decrease the expression of germ layer differentiation markers. C_LIO_LICa2+ signaling associated genes capture specific hPSC states in defined conditions. C_LIO_LIHigh Ca2+ activity drives pluripotency, low Ca2+ activity drives differentiation. C_LI

cell biology↗

bioRxiv2019

Presynaptic dysfunction in CASK-related neurodevelopmental disorders

CASK-related disorders are a genetically defined group of neurodevelopmental syndromes. There is limited information about the effects of CASK mutations in human neurons. Therefore, we sought to delineate CASK mutation consequences and neuronal level effects using induced pluripotent stem cell-derived neurons from two mutation carriers; one male diagnosed with ASD and a female with MICPCH. We show a reduction of the CASK protein in maturing neurons from the mutation carriers, which leads to significant downregulation of gene sets involved in presynaptic development and CASK protein interactors. Furthermore, CASK-deficient neurons showed decreased inhibitory presynapse size as indicated by VGAT staining, which may alter the excitatory-inhibitory (E/I) balance in developing neural circuitries. Using in vivo magnetic resonance spectroscopy quantification of GABA in the male mutation carrier, we further highlight the possibility to validate in vitro cellular data in brain. Our data shows that future pharmacological and clinical studies on targeting presynapses and E/I imbalance could lead to specific treatments for CASK-related disorders. HighlightsModelling of CASK-related disorders using iPSC-derived human neuronal cells CASK mutations cause dysregulation of its protein interactor partners Reduced CASK levels primarily affect inhibitory presynapse development In vitro GABAergic phenotype predicts in vivo neurotransmitter levels

genetics↗