deCLUTTER2+ pipeline to analyze calcium traces in a novel stem cell model for ventral midbrain patterned astrocytes.

Astrocytes are the most populous cell type of the human central nervous system and are essential for physiological brain function. Increasing evidence suggests multiple roles for astrocytes in Parkinson's disease (PD), nudging a shift in the research focus, which historically pivoted around the ventral midbrain dopaminergic neurons (vmDANs). Studying human astrocytes and other cell types in vivo remains challenging. However, in vitro reprogrammed human stem cell-based models provide a promising alternative. Here, we describe a novel protocol for astrocyte differentiation from human stem cell-derived vmDANs-generating progenitors. This protocol simulates the regionalization, gliogenic switch, radial migration, and final differentiation that occur in the developing human brain. We have characterized the morphological, molecular, and functional features of these ventral midbrain patterned astrocytes with a broad palette of techniques, and identifiesd novel candidate midbrain-astrocyte specific markers. In addition, we have developed a new pipeline for calcium imaging data analysis called deCLUTTER2+ (deconvolution of Ca2+ fluorescent patterns) that can be used to discover spontaneous or cue-dependent patterns of Ca2+ transients. Altogether, our protocol enables the characterization of the functional properties of human ventral midbrain patterned astrocytes under physiological conditions and in disease.