The P-type ATPases family consists of ion and lipid transporters. Their unique diversity in function and expression is critical for normal development. In this study we investigated human pluripotent stem cells (hPSC) and different neural progenitor states to characterize the expression of the plasma membrane calcium ATPases (PMCAs) during human neural development and in mature mesencephalic dopaminergic (mesDA) neurons. Our RNA sequencing data identified a dynamic change in ATPase expression correlating with the differentiation time of the neural progenitors, which was independent of the neuronal progenitor type. Expression of ATP2B1 and ATP2B4 were the most abundantly expressed, in accordance with their main role in Ca2+ regulation and we observed all of the PMCAs to have a subcellular punctate localization. Interestingly in hPSCs ATP2B1 and ATP2B3 were highly expressed in a cell cycle specific manner and ATP2B2 and ATP2B4 were highly expressed in a hPSC sub-population. In neural rosettes a strong apical PMCA expression was identified in the luminal region. Lastly, we confirmed all PMCAs to be expressed in mesDA neurons, however at varying levels. Our results reveal that PMCA expression dynamically changes during stem cell differentiation and highlights the diverging needs of cell populations to regulate and properly integrate Ca2+ changes, which can ultimately correspond to changes in specific stem cell transcription states.

Gene editing in human embryonic stem cells (hESCs) has been significantly enhanced by the discovery and development of CRISPR Cas9, a programmable nuclease system that can introduce targeted double-stranded breaks. The system relies on the optimal selection of a sgRNA sequence with low off-targets and high efficiency. We designed an improved monomeric red fluorescent protein reporter, GEmCherry2, for assessing CRISPR Cas9 activity and for optimizing sgRNA. By incorporating an out-of-frame sequence to the N-terminal of the red fluorescent protein mCherry, we created a visual tool for assessing the indel frequency after cutting with CRISPR Cas9. When a sgRNA-Cas9 construct is co-transfected with a corresponding GEmCherry2 construct, single nucleotide indels can move the GEmCherry2 sequence back in-frame and allow quantification and comparison of the efficiency of different sgRNA target sites by measuring red fluorescence. With this GEmCherry2 assay, we compared four target sites in the safe harbor AAVS1 locus and found significant differences in target site activity. We verified the activity using TIDE, which ranked our target sites in a similar order as the GEmCherry2 system. We also identified an AAV short inverted terminal repeat sequence within the Cas9 construct that, upon removal significantly improved transient transfection and expression in hESCs. Moreover, using GEmCherry2, we designed a sgRNA to target SORCS2 in hESCs and successfully introduced indels into the coding sequence of SORCS2.