Assessment and Evaluation of Contemporary Approaches for Astrocyte Differentiation from hiPSCs: A Modeling Paradigm for Alzheimer's Disease

Background: Astrocytes have recently gained attention as key players in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease. Numerous differentiation protocols have been developed to study human astrocytes in vitro. However, the properties of the resulting glia are inconsistent, making it difficult to select an appropriate method for a given research question. Therefore, we compared three approaches for the generation of iPSC-derived astrocytes. We performed a detailed analysis using a widely used long serum-free (LSFP) and short serum-free (SSFP) protocol, as well as a TUSP protocol using serum for a limited time of differentiation. Results: We used RNA sequencing and immunochemistry to characterize the cultures. Astrocytes generated by the LSFP and SSFP methods differed significantly in their characteristics from those generated by the TUSP method using serum. The TUSP astrocytes had a less neuronal pattern, showed a higher degree of extracellular matrix formation, and were more mature. The short-term presence of FBS in the medium facilitated the induction of astroglia characteristics but did not result in reactive astrocytes. Data from cell-type deconvolution analysis applied to bulk transcriptomes from the cultures assessed their similarity to primary and fetal human astrocytes. Conclusions: Overall, our analyses highlight the need to consider the advantages and disadvantages of a given differentiation protocol for solving specific research tasks or drug discovery studies with iPSC-derived astrocytes. Overall design: The differentiation of iPSC-derived NPCs into astrocytes was performed using three different protocols: LSFP method (1), SSFP method (2) and TUSP method (3). For each method of astrocyte differentiation, cell pellet samples were taken after NPC expansion (AE), astrocyte induction (AI) and maturation (AM). At the end of the maturation phase of the TUSP method of astrocyte differentiation and treatment, cells were treated in triplicate with 500 nM AB42 or 150 uM H2O2. All collected cellular pellets were analysed with RNASeq.