Directed Differentiation of Human Pluripotent Stem Cells into Radial Glia and Astrocytes Bypasses Neurogenesis

Abstract

Derivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome, impeding their use in biomedical research. Here, we developed a highly efficient chemically defined astrocyte differentiation strategy that overcomes current limitations. This approach largely bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and in vitro experiments. hPSCs were first differentiated into radial glial cells (RGCs) exhibiting in vivo-like radial glia signatures. Activation of NOTCH and JAK/STAT pathways in bona fide RGCs resulted in direct astrogliogenesis confirmed by expression of various glial markers (NFIA, NFIB, SOX9, CD44, S100B, GFAP). Transcriptomic and genome-wide epigenetic analyses confirmed RGC-to-astrocyte differentiation and absence of neurogenesis. The morphological and functional identity of hPSC-derived astrocytes was confirmed by using an array of methods (e.g. electron microscopy, calcium imaging, co-culture with neurons, grafting into mouse brains). Lastly, the scalable protocol was adapted to a robotic platform and used to model Alexander disease. In conclusion, our findings uncover remarkable plasticity in neural lineage progression that can be exploited to manufacture large numbers of human hPSC-derived astrocytes for drug development and regenerative medicine.

Gain of 1q confers an MDM4-driven growth advantage to undifferentiated and differentiating hESC while altering their differentiation capacity

“…We then queried the data for the expression of different sets of markers for embryonic and extra-embryonic lineages that appear in early human development. Table S1 shows the lists of gene sets we curated from published data [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44] . We found NE 1q showed high levels of expression of markers of non-central nervous system (non-CNS) ectodermal lineages such as non-neural ectoderm and of cranial placode (Fig.…”

Gain of 1q confers an MDM4-driven growth advantage to undifferentiated and differentiating hESC while altering their differentiation capacity

“…We then queried the data for the expression of different sets of markers for embryonic and extra-embryonic lineages that appear in early human development. Table S1 shows the lists of gene sets we curated from published data [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44] . We found NE 1q showed high levels of expression of markers of non-central nervous system (non-CNS) ectodermal lineages such as non-neural ectoderm and of cranial placode (Figure 2E and Figure 2F, Figure S3).…”

Small-molecule screen reveals pathways that regulate C4 secretion in stem cell-derived astrocytes

“…Human induced pluripotent stem cells (hiPSCs) offer the unique ability to generate large numbers of patient-specific differentiated cells. However, many standard protocols for differentiating astrocytes from pluripotent stem cells require an extensive culture time (up to 6 months) ( Dezonne et al., 2017 ; Krencik et al., 2011 ; Palm et al., 2015 ; Sloan et al., 2017 ) and/or shorter protocols that rely on the isolation of intermediate cells, such as neural and oligodendrocyte progenitor cells ( Barbar et al., 2020 ; Jiang et al., 2013 ), or repeated cell passages ( Byun et al., 2020 ; Jovanovic et al., 2021 ; Leng et al., 2021 ; Lundin et al., 2018 ; Peteri et al., 2021 ; Santos et al., 2017 ; Soubannier et al., 2020 ; Tcw et al., 2017 ). To successfully develop valuable preclinical models for high-throughput screening of astrocytes, it would be preferable to have differentiation protocols that can rapidly and reproducibly generate large numbers of those cells.…”