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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE269743 | GEO | 2024/10/15

REPOSITORIES: GEO

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Project description:Purpose: The goal of this study is to compare the NGS-derived from transcriptome profiling (RNA-seq) of human iPSC, human iPSC-derived astrocytes from control and Parkinson’s disease LRRK2 G2019S, and human commercial astrocytes to gain insight into the identity of human iPSC-derived astrocytes in vitro during the differentiation process. Total RNA was assayed for quantity and quality using Qubit® RNA HS Assay (Life Technologies) and RNA 6000 Nano Assay on a Bioanalyzer 2100. The RNASeq libraries were prepared from total RNA using the TruSeq®Stranded mRNA LT Sample Prep Kit (Illumina Inc., Rev.E, October 2013). Briefly, 500ng of total RNA was used as the input material and was enriched for the mRNA fraction using oligo-dT magnetic beads. The mRNA was fragmented in the presence of divalent metal cations and at high temperature (resulting RNA fragment size was 80-250nt, with the major peak at 130nt). The second strand cDNA synthesis was performed in the presence of dUTP instead of dTTP, this allowed to achieve the strand specificity. The blunt-ended double stranded cDNA was 3´adenylated and Illumina indexed adapters were ligated. The ligation product was enriched with 15 PCR cycles and the final library was validated on an Agilent 2100 Bioanalyzer with the DNA 7500 assay. The libraries were sequenced on HiSeq2000 (Illumina, Inc) in paired-end mode with a read length of 2x76bp using TruSeq SBS Kit v4. We generated over 30 million paired-end reads for each sample in a fraction of a sequencing v4 flow cell lane, following the manufacturer’s protocol. Image analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA 1.18.66.3) and followed by generation of fastq.gz sequence files by CASAVA. Results: We found that transcriptome of human iPSC-derived astrocytes is similar to human commercial astrocytes than human iPSC. Conclusion: These results suggest that iPSC-derived astrocytes resemble human commercial astrocytes validating the differentiating protocol used.

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

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