Supplementary figures (Zhang)

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1 Supplementary figures (Zhang) Supplementary figure 1. Characterization of hesc-derived astroglia. (a) Treatment of astroglia with LIF and CNTF increases the proportion of GFAP+ cells, whereas the percentage is decreased by FBS. (b) Morphological change induced by FBS treatment. (c) Known astroglia-related genes are upregulated (fold change) in both RA- and FGF8-patterned day-210 astroglia compared to day-17 neuroepithelial cells as determined by qrt-pcr, reported in arbitrary units. Scale bars = 50 um.

2 Supplementary figure 2. Differential phenotypes in region-specific astroglial subtypes. (a) Region specific markers can distinguish between RA- or FGF8-patterned hesc-derived GFAP- and GFAP+ astroglia (day-120) and between astroglia progenitors patterned with or without SHH (day-30). (b) Expression of selective developmental and functional genes was compared by qrt-pcr between RA-, FGF8-, or non-specified (control) human astroglia at day-210. (c) Astrocytes prepared from E13.5 mouse cortex and ventral midbrain express the anterior marker Otx2, whereas those from the spinal cord express the posterior marker Hoxb4. (d) In situ staining of P1 mouse tissue demonstrated that a subset of GFAP+ cells express Hoxb4 in the spinal cord, Otx2 in areas of the cortex, and Nkx2.1 in the ventral striatum (white arrows). Yellow arrows indicate GFAP+ astrocytes with weak or no expression of regional markers. Scale bars = 50 um.

3 Supplementary figure 3. Astroglial cell differentiation of various pluripotent stem cell lines. (a) Line H7 exhibits the same protein expression profile including expression of S100β, then GFAP. (b) Induced pluripotent stem cells line ipsc(imr90)-4 can efficiently generate a nearly pure population of astroglia by 180 days. Additionally, ipsc(imr90)-4 derived astroglia (c) express CD44, and (d) can be specified to a posterior identity by the addition of RA. Scale bars = 50 um.

4 Supplementary figure 4. Long range calcium wave propagation is dependent upon extracellular ATP signaling. Calcium waves persist in anterior hesc-astroglia in the presence of the gap junction blocker carbenoxolone, but are reduced in the presence of apyrase.

5 Antibody Vendor Type Dilution A2B5 Chemicon Monoclonal 1:100 Aldh1L1 Antibodies Incorporated Monoclonal 1:50 BrdU Accurate Chemicals Polyclonal 1:500 CD44 BD Biosciences Monoclonal 1:1000 GFAP Dako Polyclonal 1:5000 GFAP Chemicon Monoclonal 1:5000 GLT-1 Gift from Dr. Jeff Rothstein Polyclonal 1:25000 hnuc Chemicon Monoclonal 1:400 Hoxb4 DSHB Monoclonal 1:100 Ki67 Zymed Polyclonal 1:200 NFIA Gift from Dr. Ben Deneen Polyclonal 1:2000 NG2 Chemicon Polyclonal 1:40 Nkx2.1 Chemicon Monoclonal 1:500 Otx2 R and D Systems Polyclonal 1:2000 PAX6 DSHB Monoclonal 1:5000 S100B Abcam Polyclonal 1:2000 Synapsin I EMD Biosciences Polyclonal 1:1000 βiii-tubulin Covance Polyclonal 1:1000 Supplementary Table 1. List of antibodies used for immunochemistry.

6 Gene ID Accession number Forward Reverse CHL1 NM_ ATGGAGCCGCTTTTACTTGGA GGCAACTTGGACTTTTGACTGT AQP4 NM_ GTGGGGTAAGTGTGGACCTTT GCCAGAAATTCCGCTGTGAC NFIA NM_ ACAGGTGGGGTTCCTCAATC GTGGGACGCTGCAACTTTT NFIX NM_ ATGTACTCCCCGTACTGCCTC ACATCCGCTTTTCATGCTTCTT GLT-1 NM_ ATCTTGGCTCAGAGGAACCCA CAGGATGACACCAAACACCG GLAST NM_ ATCCTTGGATTTACCCTCCGA CGCCATTCCTGTGACAAGAC HOXA3 NM_ TCTTTGGTCTAACTCACCTCCC GCCGGTAAGGTCCGTGTAG HOXB3 NM_ CCAGTGCCACTAGCAACAG CGTTTGCCTCGACTCTTTCATC PAX3 NM_ GGAACTGGAGCGTGCTTTTG GGCGGTTGCTAAACCAGAC SP8 NM_ CTAGGGGAAGAACCGAGGTTG GCCTTTGCCGAAGGAAGAAGA DLX1 NM_ AAAGATCCGTAAACCCAGGACG CAACGCACTACCCTCCAGA SPARCL1 NM_ GCACCTGACAACACTGCAATC TTTCAGCCTTATGGTGGGAATC NTRK2 NM_ TGTTCAGCACATCAAGCGACA GCTCAGGACAGAGGTTATAGCAT CD74 NM_ CTGGAAGGTCTTTGAGAGCTG GTCAGTGGGCTTTTGCTCC SLIT1 NM_ GCCTGGAACTCAATGGCAAC CTGGTTTCGGTTCAGTCGCA CXCL12 NM_ ATGCCCATGCCGATTCTTCG GCCGGGCTACAATCTGAAGG SLC38A1 NM_ TGACAGTGCCCGAGGATGATA TGGCTGTTTGTGAGACTTCTTC SLC26A7 NM_ AGAAGGCGACTGCCCATTTT ACTGCCAACATTATCCCAGACA Supplementary Table 2. Primers used for qrt-pcr. Supplementary movie 1. Representative example of calcium wave propagation in FGF8-specified astroglia. 1 frame = 2 seconds. Frame rate = 5 frames / second. Supplementary movie 2. Z series of human astrocyte image in Fig. 4g stained for GFAP (green), human nuclei (red), and total nuclei (blue).