Open Access Highly Accessed Research article

Extended passaging increases the efficiency of neural differentiation from induced pluripotent stem cells

Karl R Koehler12, Philippe Tropel5, Jonathan W Theile13, Takako Kondo12, Theodore R Cummins13, Stéphane Viville45 and Eri Hashino123*

Author Affiliations

1 Stark Neurosciences Research Institute

2 Department of Otolaryngology

3 Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, USA

4 Service de Biologie de la Reproduction, Centre Hospitalier Universitaire, Strasbourg, F-67000 France

5 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de Santé et de Recherche Médicale (INSERM) U964/Centre National de Recherche Scientifique (CNRS) UMR 1704/Université de Strasbourg, 67404 Illkirch, France

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BMC Neuroscience 2011, 12:82  doi:10.1186/1471-2202-12-82

Published: 10 August 2011

Additional files

Additional File 1:

Supplementary information. Supplementary Figures 1-3 and Tables 1-3. Supplementary Figure 1 - iPSCs (GG3.3 and miPS-20) at various stages of neural differentiation. Representative micrographs of miPS-20 (A) and GG3.3 (B) iPSCs prior to differentiation, on day 5 of EB formation and on days 3 and 7 of neural induction. (C-E) Examples of aberrant cell types with endodermal (C) and mesodermal (D, E) morphologies that were prevalent during all early-passage iPSC Ni experiments. Scale bars represent 100 μm. Supplementary Figure 2 - The GG3.1 cell line is a competent iPSC line with no detectable transgene re-expression during neural differentiation. (A) Alkaline phosphatase staining of ESC and GG3.1 cells indicates pluripotent cells in undifferentiated cultures and a gradual loss of pluripotency during the EB stage. (B) Primers amplifying an untranslated region (UTR) of the Oct4, Sox2 and Klf4 genes were compared to exon expression in undifferentiated and neural induction days 3 and 7. All expression levels were normalized to undifferentiated expression levels. The identical pattern of expression indicates a lack of transgene re-expression. (C-D) The GG3.1 cell line displays similar expression levels of the Dlk1-Dio3 locus genes Gtl2 and Rian, which is an indirect measure of complete reprogramming. Equivalent expression of these genes was validated using 2 different primer sets; one novel and one published by Stadtfeld et al., 2010. Values are mean ± SD for 2-3 independent samples. Supplementary Figure 3 - Expression of neural lineage and subtype specific genes throughout Ni of early-passage GG3.1 iPSCs. (A-C) Representative micrographs showing the presence and abundance of HuC/D, Map2, neurofilament (NF) and Calretinin (Calr) positive cells at Ni day 7. Scale bars represent 150 μm. (D) The pluripotency marker Rex1 is downregulated during differentiation. (E) The anterior neurodevelopmental gene Otx2 is expressed by day 5 of EB. The neurotrophin receptor TrkB is expressed during the EB stage, but expression is elevated by day 7 of Ni. Calretinin is expressed by Ni day 3. (F) Markers of glutamatergic neurons, vesicular glutamate transporter 2 (VGLUT2) and the AMPA receptor subunit GluR2 are highly expressed by days 7 and 15 of Ni. Likewise, the GABAergic neuronal marker, glutamic acid decarboxylase 1 (GAD1) is unregulated by days 7 and 15. Values are mean ± SD for 2-3 independent samples. Supplementary Table 1 - Pluripotent stem cell lInes. Supplementary Table 2 - Primers. Supplementary Table 3 - Antibodies.

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