Embryonic Stem Cells/induced Pluripotent Stem Cells
Article first published online: 24 APR 2013
Copyright © 2013 AlphaMed Press
Volume 31, Issue 5, pages 941–952, May 2013
How to Cite
Shaltouki, A., Peng, J., Liu, Q., Rao, M. S. and Zeng, X. (2013), Efficient Generation of Astrocytes from Human Pluripotent Stem Cells in Defined Conditions. STEM CELLS, 31: 941–952. doi: 10.1002/stem.1334
Author contributions: A.S.: collection and/or assembly of data, data analysis and interpretation, and manuscript writing; J.P. and Q.L.: collection and/or assembly of data; M.S.R.: conception and design and manuscript writing; X.Z.: conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS January 22, 2013.
- Issue published online: 24 APR 2013
- Article first published online: 24 APR 2013
- Accepted manuscript online: 22 JAN 2013 08:54AM EST
- Manuscript Accepted: 24 DEC 2012
- Manuscript Received: 3 JUL 2012
- California Institute for Regenerative Medicine. Grant Numbers: TR-01856, CL1-00501
Additional Supporting Information may be found in the online version of this article.
|sc-12-0606_sm_SupplFigure1.pdf||165K||Supplemental Figure S1. NSC cultured on geltrex exhibit neuroepithelial characteristics. Immunocytochemistry showing human NSC (H14:passage14) express neuronal markers such as SOX1 and NESTIN (A&B), Sox2 (C&D), and PAX6 (E&F) after long term in vitro expansion. Scale bars in A-F, 100μM.|
|sc-12-0606_sm_SupplFigure2.tif||2801K||Supplemental Figure S2. (A) Efficiency of differentiation of hESC-derived NSC (H9) and iPSC-derived NSC (BC1) into astrocytes in defined medium is similar. To calculate the percentage GFAP positive Cells, four different fields per three independent experiments were counted blindly. The percent of GFAP positive cells was calculated as the ratio of cells showing GFAP positive staining compared to the total number of cells with Hoechst-stained nuclei. Total of 700 GFAP positive cells were counted per experiment. *P < 0.001, Student's t-test. (B) Cytoplasmic colocalization of S100β and GFAP in 4 weeks differentiated NSC (H14), and (C) (BC1). Scale bars in B and C, 100μM|
|sc-12-0606_sm_SupplFigure3.pdf||116K||Supplemental Figure S3. FACS analysis of ESC- and iPSC-derived NSC cultured in defined medium for 35 days. Flow cytometry was assessed using a Diva 6.1 software. Isotype IgG (red gragh) and secondary-only fluorophores (data not shown) were used as controls. For staining of intracellular GFAP(green graph), differentiated NSC were fixed with 2% paraformaldehyde for 15 min, washed and permeabilized with 0.2% Triton in blocking solution (10% goat serum, 1%BSA in Phosphate Buffer Saline) for 20 mins. Cells were incubated at room temperature with primary anti-GFAP antibodies for 1 h and subsequently stained with secondary antibodies for 30 min. (A) As shown in the historgram (left panel) 69% of live cells stained positive for GFAP in differentiated NSC (H14) after 35 days incubation using the defined medium, and this ratio was increased to 80% when iPSC-derived NSC (BC1) were differentiated in defined medium for 35 days (right panel)(B).|
|sc-12-0606_sm_SupplFigure4.pdf||181K||Supplemental Figure S4. Time course study of NSC (BC1) differentiation using defined medium into astrocytes as imaged by GFAP staining. (A) GFAP immunostaining of cells cultured in defined medium for two weeks. (B) Higher magnification of (A). (C&D) At 3-week time point, the number of cells immunopositive for GFAP staining began to increase and continued (E&F). By 5 weeks, majority of cells stained positive for GFAP and had differentiated into astrocytes (G&H). Similar results were obtained for NSCs derived from hESC (H9)(data not shown). Scale bars in A, C, E and G, 100μM. Scale bars in B, D, F and H, 50μM.|
|sc-12-0606_sm_SupplFigure5.pdf||144K||Supplemental Figure S5. Cells grown in defined medium can be cryopreserved at intermediate stages without losing their ability to further differentiate into astrocytes (A&B) Cells were cryopreserved at various time point (14 days of differentiation) in defined medium, and thawed in defined medium. (B) Higher magnification of (A). (C) NSC (H14) were differentiated in defined medium. The cells were then cryopreserved at different time points (10 days and 14 days) after differentiation. Viability of the cryopreserved astrocytes was calculated by Trypan blue-dye exclusion assay at 10, and 14 days after differentiation (solid black), and compared to the viability of NSC in Neurobasal Medium at similar time points. Viability was calculated as a ratio of live cells (which possess intact cell membranes and exclude Trypan blue) to the total number of cells. Values are the mean out of three separate experiments. Error bars represent the standard deviations. (D&E) Thawed cells were further differentiated in defined medium for additional 3 weeks (total of 5 weeks). (E) Higher magnification of (D). Scale bars in A, B, D and E, 100μM.|
|sc-12-0606_sm_SupplFigure6.tif||2915K||Supplemental Figure S6. Shown are graphs of several astrocyte markers and their normalized intensity at different time points throughout the differentiation process|
|sc-12-0606_sm_SupplFigure7.tif||2905K||Supplemental Figure S7. The hierarchical clustering of highly enriched genes from murine astrocytes with array data from human astrocytes revealed the gene expression similarities between mouse and human astrocytes. (A) Overview of the gene expression patterns of the top 40 enriched genes from murine astrocytes across NSC derived astrocytes at different stages of differentiation presented as a heat map. Each row represents a single gene and four columns represent Day 14, 21, 28 and 35 of differentiation (B) Clustering of Murine astrocyte-specific genes with two additional set of arrays, one from human astrocytes, and a second one from fetal derived APC, identified genes shared across the experimental samples between mouse and human astrocytes. Each row represents a single gene and five columns correspond to four independent dataset from fetal derived APC and one from human astrocytes. Upregulated astrocyte-associated genes are shown in red, whereas downregulated astrocyte-associated genes are shown in green.|
|sc-12-0606_sm_SupplFigure8.pdf||169K||Supplemental Figure S8. (A) Shown are graphs of several homeodomain transcriptional markers and their normalized intensity at different time points throughout the astrocyte differentiation process. (B) Immunocytochemistry analysis of 35 Days differentiated astrocytes with antibodies against NKX2-2, and (C) OTX2. Scale bars in B and C, 50μM.|
|sc-12-0606_sm_SupplFigure9.tif||2790K||Supplemental Figure S9. Differentiation of Human Fetal Glial Restricted Precursor cells (GRP) into astrocytes as a consequence of culture in defined medium. (A&D) GFAP and Tuj1 staining of GRP cultured in defined medium for one week. (D) Higher magnificaton of (A). (B&E) At 2-week time point, the number of cells immunopositive for GFAP began to increase and continued. (C&F) By 3 weeks, majority of GRP cells stained positive for GFAP and had differentiated into astrocytes. (E&F). Higher magnification of (B) and (C), respectively. Scale bars in A, B and C, 100μM. Scale bars in D, E and F, 50 μM.|
|sc-12-0606_sm_SupplFigure10.tif||2905K||Supplemental Figure S10. Immunohistochemical characterization of transplanted astrocytes. (A) Confocal images of immunostainned sections obtained from site of transplantation showed the absence of Thy1 staining in human astrocytes. (B) Staining of the same section with anti-HNP antibody confirmed the human identity of the cells. (C) Hoechst staining of the panel in (B). (D) Transplanted cells did not express neuronal marker as determined by co-immunostaining with Thy1 and human nuclear antigen antibodies. Scale bars in A-D, 20μM.|
|sc-12-0606_sm_SupplTable1.pdf||86K||Supplementary Table 1|
|sc-12-0606_sm_SupplTable2.pdf||91K||Supplementary Table 2|
|sc-12-0606_sm_SupplTable3.pdf||78K||Supplementary Table 3|
|sc-12-0606_sm_SupplTable4.pdf||57K||Supplementary Table 4|
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