Fast Generation of Functional Subtype Astrocytes from Human Pluripotent Stem Cells

Abstract

SummaryDifferentiation of astrocytes from human pluripotent stem cells (hPSCs) is a tedious and variable process. This hampers the study of hPSC-generated astrocytes in disease processes and drug development. By using CRISPR/Cas9-mediated inducible expression of NFIA or NFIA plus SOX9 in hPSCs, we developed a method to efficiently generate astrocytes in 4–7 weeks. The astrocytic identity of the induced cells was verified by their characteristic molecular and functional properties as well as after transplantation. Furthermore, we developed a strategy to generate region-specific astrocyte subtypes by combining differentiation of regional progenitors and transgenic induction of astrocytes. This simple and efficient method offers a new opportunity to study the fundamental biology of human astrocytes and their roles in disease processes.

An update on human astrocytes and their role in development and disease

“…The most conventional approach, 3D generation and maintenance, has become the standard method on which the majority of subsequent protocols have been based, including significant portions of direct conversion methods (Juopperi et al, 2012;Krencik & Zhang, 2011;Mormone, D'Sousa, Alexeeva, Bederson, & Germano, 2014). Finally, direct conversion through TF expression significantly decreases the induction protocols length but stands as a relatively new and unexplored approach (Caiazzo et al, 2014;Canals et al, 2018;Li, Tao, et al, 2018;Li, Tian, et al, 2018;Tchieu et al, 2019).…”

“…These mutations cause the formation of intracellular aggregates called Rosenthal fibers that are thought to cause astrocyte toxicity through toxic gain of function (Prust et al, 2011). This is sufficient to induce neurodegeneration in patients affected by AxD (Li, Tao, et al, 2018;Li, Tian, et al, 2018). Many mouse models have been developed to recapitulate this disorder (Hagemann et al, 2005;Messing et al, 1998), however none of them show a complete array of all the main characteristic of the disease.…”

Modeling genetic epilepsies in a dish

“…Various protocols have been developed to derive neural cells from hPSCs that are most relevant to epilepsy, including cortical-like excitatory neurons, 17,18 cortical-like inhibitory neurons (γ-aminobutyric acid [GABA]-expressing interneurons), [19][20][21] and hippocampallike neurons. 22,112 Recent developments in optimizing the protocols of neural differentiation also make it possible to generate astrocytes, [113][114][115] midbrain dopamine neurons 116,117 and spinal motor neurons 118,119 from hPSCs. Most of these protocols involve the application of small molecules to hPSC monolayers or the forced expression of specific transcription factors.…”

Plasminogen binding inhibitors demonstrate unwanted activities on GABA A and glycine receptors in human iPSC derived neurons

“…Evaluation of calcium signaling is performed to functionally validate and assess the astrocyte biology of hPSC derived astrocytic models. Functional calcium signaling is shown in several hPSC derived models in response to mechanical (15,16,230,242,244), electrical (245), ATP (20,183,230) and glutamate simulation (246). Spontaneous calcium waves are also observed in hPSC derived models (183) together with calcium leakage from the ER, identified by inhibiting the major ER release channels RYR, IP3R and SERCA (245).…”

“…The astrocyte specific marker in rodent, GFAP, is for example also expressed by various other cell types in humans of which also the GFAPδ isotype mark specific progenitors not observed in mice (14). Human astrocyte heterogeneity is partly due to regional identity which has been mimicked in vitro (15,16), whereas functional diversity seemingly is dependent on neuronal input (17)(18)(19).…”