An Efficient Platform for Astrocyte Differentiation from Human Induced Pluripotent Stem Cells

Abstract

SummaryGrowing evidence implicates the importance of glia, particularly astrocytes, in neurological and psychiatric diseases. Here, we describe a rapid and robust method for the differentiation of highly pure populations of replicative astrocytes from human induced pluripotent stem cells (hiPSCs), via a neural progenitor cell (NPC) intermediate. We evaluated this protocol across 42 NPC lines (derived from 30 individuals). Transcriptomic analysis demonstrated that hiPSC-astrocytes from four individuals are highly similar to primary human fetal astrocytes and characteristic of a non-reactive state. hiPSC-astrocytes respond to inflammatory stimulants, display phagocytic capacity, and enhance microglial phagocytosis. hiPSC-astrocytes also possess spontaneous calcium transient activity. Our protocol is a reproducible, straightforward (single medium), and rapid (<30 days) method to generate populations of hiPSC-astrocytes that can be used for neuron-astrocyte and microglia-astrocyte co-cultures for the study of neuropsychiatric disorders.

Viral infections and multiple sclerosis

“…Several models of demyelination are known [20,21]. In vitro models based on glial cell lines include the use of oligodendrocyte [22,23], astrocyte [24,25] and microglial [26,27] cell lines [28] and progenitor-derived glial cells [29][30][31][32][33]. In vivo models include the most extensively studied animal model for MS, experimental autoimmune encephalomyelitis (EAE), obtained by immunization with myelin proteins with adjuvants, or passive transfer of autoreactive T cells with or without pathogenic auto-antibodies in susceptible animals [34][35][36][37].Various clinical types of EAE can be induced in rodents and primates (marmosets and rhesus macaques), ranging from acute monophasic [38] to a chronic course, either relapsing/remitting [39] or chronic progressing [40].…”

CD49f is a novel marker to purify functional human iPSC-derived astrocytes

“…Hierarchical clustering of RNA-Seq data showed strong similarities between CD49f + astrocytes and hiPSC-astrocytes generated from an alternative differentiation protocol 21 as well as primary human astrocytes, versus clear differences from other cell types such as neurons, oligodendrocytes, microglia, and endothelial cells ( Figure 2D). Despite the patterning during the first few days of differentiation with caudalizing and ventralizing agents retinoic acid and sonic hedgehog, it is interesting to note that the transcriptomic profile of our samples is very similar to the one of forebrain astrocytes generated by the Brennand lab 21 , raising the questions of whether astrocyte heterogeneity stems from regional differences 39 , and whether these differences can be recapitulated in a dish using iPSC-derived cells. It is not surprising that iPSC-astrocytes cluster closer to fetal than adult primary cells, as this is true for all iPSC-derived CNS cells 40 -again highlighting the importance of an astrocyte-specific cell surface marker that can be used to isolate astrocytes from organoids at this age.…”

“…The advent of human embryonic stem cell and induced pluripotent stem cell (iPSC) technology has enabled large-scale generation of human astrocytes and other CNS cells that retain the genetic information of the patient, as powerful human in vitro models of disease (reviewed in 14 ) . Several protocols to generate astrocytes have been developed by independent groups, using either a specific gradient of patterning agents to mimic embryonic development [15][16][17][18][19][20][21] or overexpression of critical transcription factors 22,23 . Usually, iPSC differentiation into astrocytes in monolayer cultures does not require a purification step; in recent years, however, more complex 3D cultures of CNS organoids have been established that contain neural progenitor cells, neurons, oligodendrocyte lineage cells and microglia, in addition to astrocytes 22,[24][25][26][27][28][29][30][31] .…”

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

“…Monolayer generation methods of astrocyte‐like cell types are less common due in part to early iterations reliance on factors that can induce reactive astrogliosis such as TNF‐alpha and/or the addition of various types and concentrations of serum, a xenotropic reagent. While more refined versions of the monolayer approach minimizing the use of serum are entering the mainstream, the use of xenotropic reagents in any capacity will be problematic for maintaining consistency and repeatability (Lundin et al, ; Roybon et al, ; TCW et al, ). Because this method is less common it will not be further detailed.…”

“…While more refined versions of the monolayer approach minimizing the use of serum are entering the mainstream, the use of xenotropic reagents in any capacity will be problematic for maintaining consistency and repeatability (Lundin et al, 2018;Roybon et al, 2013;TCW et al, 2017). Because this method is less common it will not be further detailed.…”