Table of Contents
ISRN Cell Biology
Volume 2012, Article ID 124878, 12 pages
http://dx.doi.org/10.5402/2012/124878
Research Article

Reprogramming of Human Huntington Fibroblasts Using mRNA

1Fraunhofer Institute for Cell Therapy and Immunology, Perlickstraβe 1, 04103 Leipzig, Germany
2Institute of Virology, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany
3Interdisciplinary Centre for Bioinformatics, University of Leipzig, Härtelstraβe 16-18, 04107 Leipzig, Germany
4Department Stereotactic and Functional Neurosurgery, University Hospital of Freiburg, Breisacher Straβe 67, 79106 Freiburg, Germany
5Institute of Human Genetics, Newcastle University, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK

Received 10 August 2011; Accepted 7 September 2011

Academic Editors: A. Kretsovali, L. Shevde, and A. Tavares

Copyright © 2012 Antje Arnold et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

Supplemental Figure 1: mRNA-iPS cells from foreskin fibroblasts (A) Morphology of mRNA-iPS cells with surrounding donor cells (passage 0) and on donor feeder cells (passages 1, 2, 3 and 5). About 80% of the colonies were ALP-positive at passages 1 and 2. (B) Immunofluorescence staining in mRNA-iPS (foreskin, 11 years, factors: OSK) for Oct4, Sox2, Nanog, SSEA-3, SSEA4 and Tra-1-60. All cells were also stained with DAPI and images were merged with lineage markers. (C) Karyotyping of fibroblasts and fibroblast-derived mRNA-iPS cells (foreskin, 11 years, factors: OSK).

Supplemental Figure 2: In vitro differentiation of mRNA-iPS cells derived from foreskin fibroblasts (A) mRNA-iPS cells (factors: OSK) derived from fibroblasts (foreskin, 11 years). Spontaneous differentiation into embryoid bodies (EBs) was followed for some days and EBs stained positive for various differentiation markers. (B) mRNA-iPS cells (factors: OSK) derived from fibroblasts (foreskin, 11 years) were differentiated in monolayers toward neuronal (GFAP and tubulin III), chondrocyte (collagen II and aggrecan), hepatocyte (AFP and cytokeratin 18), keratinocyte (ß-integrin and cytokeratin 14) lineages. All cells were also stained with DAPI and images were merged with lineage markers. Immunofluorescence staining was performed after two weeks in differentiation medium (see Experimental Procedures). In addition, the cells were differentiated towards osteoblasts (Alizarin Red-positive) and adipocytes (Oil Red O-positive).

Supplemental Figure 3: Analysis of viPS colonies derived from adult fibroblasts Virus-derived iPS cells (factors: ONSTKM, donor 56 years, Huntington patient). (A) Virus-derived iPS colonies stained for pluripotency markers (Oct4, Sox2, Tra1-60, Nanog, SSEA3 and SSEA4) and DNA. (B) As negative controls, human adult Huntington disease fibroblasts were stained with all pluripotency markers used (Oct4, Sox2, Tra1-60, Nanog, SSEA3 and SSEA4) and DNA.

Supplemental Figure 4: In vitro differentiation of viral iPS cells derived from Huntington fibroblasts (A) Viral iPS cells differentiated in monolayers toward neuronal (GFAP and tubulin III), chondrocyte (collagen II and aggrecan), hepatocyte (AFP and cytokeratin 18), keratinocyte (ß-integrin and cytokeratin 14) lineages. All cells were also stained with DAPI and images were merged with lineage markers. Immunofluorescence staining after two weeks in differentiation medium (see Experimental Procedures). (B) Karyotyping of donor fibroblasts and virus-derived iPS cells.

Supplemental Figure 5: Characterization of iPS cells (A) Up-regulation in expression of key pluripotency genes in mRNA-iPS cells vs. donor fibroblasts (adult male, 56, Huntington patient). The table shows the list of genes obtained in three-class comparisons that are similarly expressed in the iPS cells and hESCs but differentially expressed between iPS cells and their donors. The given fold changes (FC) refer to differential expression with respect to iPS cells. (B) The promoter regions of Nanog, Oct4 and Rex1 were analyzed for the methylation state in human (foreskin, 11 years) mRNA-iPS cells (factors: OSK).

Supplemental Table 1: Primer sequences

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