Comparison of two protocols for the generation of iPSC-derived human astrocytes.

BACKGROUND: Astrocytes have recently gained attention as key contributors to the pathogenesis of neurodegenerative disorders including Parkinson's disease. To investigate human astrocytes in vitro, numerous differentiation protocols have been developed. However, the properties of the resulting glia are inconsistent, which complicates the selection of an appropriate method for a given research question. Thus, we compared two approaches for the generation of iPSC-derived astrocytes. We phenotyped glia that were obtained employing a widely used long, serum-free ("LSF") method against an in-house established short, serum-containing ("SSC") protocol which allows for the generation of astrocytes and midbrain neurons from the same precursor cells. RESULTS: We employed high-content confocal imaging and RNA sequencing to characterize the cultures. The astrocytes generated with the LSF or SSC protocols differed considerably in their properties: while the former cells were more labor-intense in their generation (5 vs 2 months), they were also more mature. This notion was strengthened by data resulting from cell type deconvolution analysis that was applied to bulk transcriptomes from the cultures to assess their similarity with human postmortem astrocytes. CONCLUSIONS: Overall, our analyses highlight the need to consider the advantages and disadvantages of a given differentiation protocol, when designing functional or drug discovery studies involving iPSC-derived astrocytes.

Vitamin K-dependent carboxylation regulates Ca2+ flux and adaptation to metabolic stress in ß cells.

Vitamin K is a micronutrient necessary for γ-carboxylation of glutamic acids. This post-translational modification occurs in the endoplasmic reticulum (ER) and affects secreted proteins. Recent clinical studies implicate vitamin K in the pathophysiology of diabetes, but the underlying molecular mechanism remains unknown. Here, we show that mouse ß cells lacking γ-carboxylation fail to adapt their insulin secretion in the context of age-related insulin resistance or diet-induced ß cell stress. In human islets, γ-carboxylase expression positively correlates with improved insulin secretion in response to glucose. We identify endoplasmic reticulum Gla protein (ERGP) as a γ-carboxylated ER-resident Ca2+-binding protein expressed in ß cells. Mechanistically, γ-carboxylation of ERGP protects cells against Ca2+ overfilling by diminishing STIM1 and Orai1 interaction and restraining store-operated Ca2+ entry. These results reveal a critical role of vitamin K-dependent carboxylation in regulation of Ca2+ flux in ß cells and in their capacity to adapt to metabolic stress.