Specification of transplantable astroglial subtypes from human pluripotent stem cells - PubMed (original) (raw)

Specification of transplantable astroglial subtypes from human pluripotent stem cells

Robert Krencik et al. Nat Biotechnol. 2011.

Abstract

Human pluripotent stem cells (hPSCs) have been differentiated efficiently to neuronal cell types. However, directed differentiation of hPSCs to astrocytes and astroglial subtypes remains elusive. In this study, hPSCs were directed to nearly uniform populations of immature astrocytes (>90% S100β(+) and GFAP(+)) in large quantities. The immature human astrocytes exhibit similar gene expression patterns as primary astrocytes, display functional properties such as glutamate uptake and promotion of synaptogenesis, and become mature astrocytes by forming connections with blood vessels after transplantation into the mouse brain. Furthermore, hPSC-derived neuroepithelia, patterned to rostral-caudal and dorsal-ventral identities with the same morphogens used for neuronal subtype specification, generate immature astrocytes that express distinct homeodomain transcription factors and display phenotypic differences of different astroglial subtypes. These human astroglial progenitors and immature astrocytes will be useful for studying astrocytes in brain development and function, understanding the roles of astrocytes in disease processes and developing novel treatments for neurological disorders.

PubMed Disclaimer

Figures

Figure 1. Differentiation of astroglia from hPSCs

(a) Illustration of the astroglial differentiation process. hPSCs were first differentiated to early neuroepithelial cells (NE) in the absence of exogenous growth factors for 10 days, followed by patterning with morphogens between days 10 and 21. The neural/astroglial progenitors were expanded in the presence of EGF and FGF2, during which progenitors were differentiated for 7 days with CNTF every 30 days for characterization with cell type-specific markers. (b) At day-180, immature astrocytes display a stellate morphology, express S100β in both the cytoplasm and nuclei, and express GFAP in a filamentous pattern throughout the cytoplasm. Nuclei are indicated by Hoechst (Ho) staining. (c) Temporal course comparison of S100β (120 days, p = 0.0055) and GFAP (120 days, p = 0.001; 180 days, p = 0.0066) expression of RA- and FGF8-specified astroglia (from 3 separate passages of the H9 hESC line) among total cells. (d) hESC-GFAP+ cells express Aldh1L1, but not NG2, as compared to mouse primary astrocytes. (e) Western blotting analysis confirms the expression of GFAP and GLT-1 in day-180 astroglia. (f) Subsets of astroglia express A2B5, whereas the majority of immature astrocytes express CD44 by 90 days. (g) NFIA is not expressed in early NE at day-11, but it begins to be expressed in a small number of progenitors with concomitant down regulation of PAX6 at day-30 (arrows). By day-180, all cells express NFIA. (h) Incorporation of BrdU by RA- and FGF8-specified astroglial progenitors at 60 days (n = 6, p = 0.0043) demonstrates differential proliferation of subtypes. By 180 days, BrdU uptake is not different between groups, and is completely absent in cells after removal of growth factors and addition of CNTF. Scale bars = 50 μM. Data are represented as mean +/− SEM.

Figure 2. Astroglial subtypes express region-specific proteins

(a) Differential treatment with patterning molecules (RA, FGF8, or SHH) from days 10–21 generates cells with distinct expression of homeodomain transcription factors, which is maintained as cells differentiate from neural progenitors (NP) to immature astrocytes. (b) At day-60, FGF8-, but not RA-, specified S100β+ astroglia express Otx2 in nuclei. (c) RA-, but not FGF8-, specified S100β+ astroglia express Hoxb4. GFAP+ immature astrocytes continue to express (b) Otx2 and (c) Hoxb4 at day-180. (d) Quantification of regional marker expression of day-120 GFAP+ immature astrocytes (FGF8-specified; Otx2 = 92.1% ± 2.5, Hoxb4 = 0. GFAP+ RA-specified; Otx2 = 3.2% ± 1.3, Hoxb4 = 97.6% ± 2.1). (e) Quantification of day-30 ventralized astroglial progenitors (−SHH; Nkx2.1 = 0. +SHH; Nkx2.1 = 82.6% ± 7.0). (f) Day-60 S100β+ astroglia differentiated from SHH-ventralized neural progenitors express Nkx2.1. Scale bar = 50 μM.

Figure 3. Functional characteristics of hPSC-derived immature astrocytes

(a) Immature astrocytes were analyzed by whole cell patch clamping. (i–iii) Voltage steps (clamped at −70 mV and stepped from −50 to +50 mV at 10 mV increments for 500 ms) induced outward currents in red fluorescent labeled 4-month astroglia, which significantly decreased in the presence of neurons for 2 weeks (n = 10 for both groups). Action potentials could not be elicited (inset in i). (bi-ii) The inward current response by AMPA was blocked with CNQX and AP5, and the L-glutamate response was partially reduced. (biii) L-glutamate induced inward current was reduced by glutamate transporter inhibitors DHK and SOS. (c) Kinetics of cellular uptake of L-glutamate (starting at 50 uM) was measured in the absence or presence of PDC and Na+ and normalized to μg of protein (n = 3 for each group). (d) Immature astrocytes propagate calcium waves to adjacent cells upon mechanical stimulation. Calcium wave propagation was measured for anterior and posterior immature astrocytes (20 seconds; RA = 59.2 μm ± 3.2, FGF8 = 76.0 μm ± 3.1, p = 0.0196. 30 seconds; RA = 62.4 μm ± 3.8, FGF8 = 91.4 μm ± 7.8, p = 0.0288, n = 3 for all groups), both of which were inhibited by 2-APB (20 second p values; RA = 0.0144, FGF8 = 0.0104. 30 second p values; RA = 0.0147, FGF8 = 0.0197). (e) Co-culturing of hESC-derived neurons and immature astrocytes for 3 weeks results in an increased presence of Synapin 1+ puncta (Syn, n = 3, p = 0.0119). Scale bar = 50 μm.

Figure 4. hPSC-derived astroglia retain their identity in vivo

(a) Illustration of intraventricular transplantation of hESC-derived astroglia and the resulting position of grafted cells. (b) One hundred days post transplantation, both RA-specified (n = 3) and FGF8-specified (n = 4) human cells (hNuc+ = red) are present in ventricular areas (outlined with dashed lines), and express GFAP. Arrows indicate the human cells magnified in the insets. (c) Grafted, RA-specified human astrocytes (hNuc+ = blue) in the corpus callosum (outlined with dashed lines) express Hoxb4 (red, 65/65). In contrast, all of the FGF8-specified hNuc+/GFAP+ cells express Otx2 (red, 52/52). (d) Illustration of hippocampal transplantation. (e) Human astrocytes (red) survive and express GFAP but (f) not βIII-tubulin 6 weeks after transplantation to the adult hippocampus (n = 4 for each group). (g) Six months following transplantation of day-21 hESC-derived neural progenitors, human astrocytes (white arrow, hNuc+/GFAP+ shown in upper inset) extend processes onto endogenous blood vessels (outlined with dashes) in a form of end feet (yellow arrow, shown in the lower inset on a single plane). (h) Mouse and human astrocytes exhibit distinct phenotypes, including process length and blood vessel association. Scale bar = 50 μm.

Figure 5. Hypothesis of astroglial subtype specification

The regional identity (anterior-posterior, dorsal-ventral) of astrocytes is determined when early neuroepithelial cells (NE, neural stem cells) are patterned to regional progenitors by morphogens such as RA and SHH. The regionalized progenitors first give rise to subclasses of neurons and then, during gliogenesis, generate regional-specific astrocytes with potential functionally distinct characteristics.

References

1. Barres BA. The mystery and magic of glia: a perspective on their roles in health and disease. Neuron. 2008;60:430–440. - PubMed
1. Kettenmann H, Verkhratsky A. Neuroglia: the 150 years after. Trends Neurosci. 2008;31:653–659. - PubMed
1. Zhang SC. Defining glial cells during CNS development. Nat. Rev. Neurosci. 2001;2:840–843. - PubMed
1. Rowitch DH, Kriegstein AR. Developmental genetics of vertebrate glial-cell specification. Nature. 2010;468:214–222. - PubMed
1. Ullian EM, Sapperstein SK, Christopherson KS, Barres BA. Control of synapse number by glia. Science. 2001;291:657–661. - PubMed

Specification of transplantable astroglial subtypes from human pluripotent stem cells - PubMed (original) (raw)