Efficient derivation of functional astrocytes from human induced pluripotent stem cells (hiPSCs)
Astrocytes are specialized glial cell types of the central nervous system (CNS) with remarkably high abundance, morphological and functional diversity. Astrocytes maintain neural metabolic support, synapse regulation, blood-brain barrier integrity and immunological homeostasis through intricate interactions with other cells, including neurons, microglia, pericytes and lymphocytes. Due to their extensive intercellular crosstalks, astrocytes are also implicated in the pathogenesis of CNS disorders, such as ALS (amyotrophic lateral sclerosis), Parkinson's disease and Alzheimer's disease. Despite the critical importance of astrocytes in neurodegeneration and neuroinflammation are recognized, the lack of suitable in vitro systems limits their availability for modeling human brain pathologies. Here, we report the time-efficient, reproducible generation of astrocytes from human induced pluripotent stem cells (hiPSCs). Our hiPSC-derived astrocytes expressed characteristic classical markers of mature astrocytes, such as GFAP, S100b, ALDH1L1 and AQP4. Furthermore, hiPSC-derived astrocytes displayed spontaneous calcium transients and responded to inflammatory stimuli by the secretion of type A1 and type A2 astrocyte-related cytokines. Overall design: Two control human iPSC lines (BIOT.009 and BIOT.021) were used in this study, derived from healthy female donors and characterized earlier. Neural progenitor cells (NPC) were generated from the iPSCs by dual inhibition of the SMAD signalling pathway using LDN193189 and SB431542 [18, 19], then subsequently used for astrocyte differentiation. The hiPSC-derived neural progenitor cells (NPCs) were propagated for at least 10 days in a completed neural maintenance medium. Cells were passaged with Accutase upon reaching confluence, and they were plated into poly-ornithine-laminin (POL)-coated plates with a seeding density of 50.000-150.000 cells/cm2. NPCs were seeded into matrigel-coated plates on day 14. To initiate the generation of astroglial progenitor cells (APCs), the culture medium was changed to medium into astrocyte growth medium the next day. The medium was changed every other day until the 21st day of differentiation. At the 21st day of astrocyte induction, APCs were seeded into matrigel-coated plates in an astrocyte induction medium with a density of 25.000-50.000 cells/cm2. The next day the medium was switched to astrocyte maturation medium, which was changed every other day until day 42. Total RNA from cellular pellet samples was isolated using TRI Reagent (Merck, USA) with RapidOutput DNA removal kit (ThermoFisher Scientific, USA). The quantity and quality of RNA extracts were analyzed by using a Qubit 4 fluorometer (ThermoFisher Scientific, USA) and Fragment Analyzer (Agilent, USA). Complementary DNA (cDNA) libraries were synthesized using the NEBNext® Poly(A) mRNA Magnetic Isolation Module and NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (NEB, UK). RNA sequencing was performed on the Illumina NovaSeq 6000 platform with the NovaSeq S4 300 kit generating 150 bp pair-end reads (Illumina, USA). The sequencing quality of each read was assessed by FastQC, erroneous reads were filtered out using Trimmed Galore.  Trimmed sequence reads were mapped to the human genome sequence hg38 by using the R package module Rbowtie2 and Rsamtooks. Differentially expressed genes (DEGs) with |log2 fold change| > 1 and q < 0.01 (FDR-adjusted P-values) were selected in DeSeq2.