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.

BioTalentum Ltd Godollo Hungary

Department of Biochemistry Faculty of Science Masaryk University Brno Czech Republic

Department of Pharmacology and Toxicology Faculty of Medicine in Pilsen Charles University Prague Czech Republic

Department of Physiology and Animal Health Institute of Physiology and Animal Nutrition Hungarian University of Agriculture and Life Sciences Godollo Hungary

Laboratory of Neurobiology and Pathological Physiology Institute of Animal Physiology and Genetics The Czech Academy of Science Veveří 97 60200 Brno Czech Republic

Psychiatric Clinic University Hospital and Faculty of Medicine in Pilsen Charles University Pilsen Czech Republic

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