Directed differentiation of functional astroglial subtypes from human pluripotent stem cells

Abstract

Regionally and functionally diverse types of astrocytes exist throughout the central nervous system and participate in nearly every aspect of normal and abnormal neural function. Therefore, human astrocyte subtypes are useful tools for understanding brain function, modulating disease processes and promoting neural regeneration. Here we describe a protocol for directed differentiation and maintenance of functional astroglia from human pluripotent stem cells in a chemically defined system. Human stem cells are first differentiated into neuroepithelial cells with or without exogenous patterning molecules (days 0-21). Regular dissociation of the neuroepithelial clusters in suspension, and in the presence of mitogens, permits generation of astroglial subtypes over a long-term expansion (days 21-90). Finally, the astroglial progenitors are either amplified for an extended time or differentiated into functional astrocytes on removal of mitogens and the addition of ciliary neurotrophic factor (days >90). This method generates robust populations of functionally diversified astrocytes with high efficiency.

Figure 1. Timeline of astrocyte generation

Cells are directed through developmental identities of human pluripotent stem cells (PSC), embryoid bodies (EB), neuroepithelial cells (NE), progenitors, and then immature astrocytes through 3 stages composed of a total of 13 steps. Cell identity may be confirmed by immunostaining after CNTF treatment of dissociated cells (quality control)at the recommended timepoints. For regional patterning, morphogens can be added from days 10–21.

Figure 2. Stages of astroglial differentiation

(a) PSCs should exhibit defined edges without (b) signs of differentiation. (c) Multilayered columnar cells are indicative of neuroepithelial origin, while (d) flattened or dark clusters are non neural. (e, g, i) Astroglial progenitors will display a stellate morphology when attached and eventually reform astrospheres in the presence of mitogens. (f, h) In contrast, non neural fibroblast-like cells will remain attached. Scale bars = 100 μm.

Figure 3. Confirmations of astroglial differentiation with astrocyte markers

The efficiency of astrocyte generation can be followed by the step wise expression of S100β and then GFAP (Hoechst nuclear stain = Ho). Eventually all cells express both markers and display a star shaped morphology. Scale bars = 50 μm.