GSE75568
BAF Complex Enhances Reprogramming of Adult Human Fibroblasts
Chromatin remodeling molecules of the BAF complex, Brg1 and Baf155, as well as other chromatin remodeling modeling molecules have been described to enhance Oct4, Sox2, Klf4 and c-Myc mediated reprogramming of mouse somatic cells into induced pluripotent stem cells (iPSCs). Brg1 maintains pluripotency of mouse embryonic stem cells (mESCs) by modulating the expression of pluripotency genes including Oct4 in synergism with LIF/STAT signaling. Interestingly, unlike mESCs, BAF complex in human embryonic stem cells (hESCs) is composed of both BAF155 and BAF170, where BAF170 plays a role in maintaining hESCs pluripotency. In this study, we describe that BRG1 and BAF155 enhance reprogramming of adult human fibroblasts. Overexpression of BAF155 does not affect pluripotency of hiPSCs as tested by global gene expression profiling as well as in vivo and in vitro assays. Additionally, these findings show that BRG1 and BAF155 expression can enhance reprogramming even in the absence of LIF/STAT signaling. Overall design: RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300 ng of total RNA per sample were used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12hrs of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer's instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates. The bead intensities were mapped to the corresponding gene information using BeadStudio 3.2 (Illumina) and background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model (Irizarry et al., 2003). Variance stabilization was performed using the log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in MATLAB. Hierarchical clusters of genes and samples were performed with the one minus the sample correlation metric and the Unweighted Pair-Group Method using Average (UPGMA) linkage method. 19 samples were analyzed: H1, H1 human Embryonic Stem Cells (ESCs), 3 replicates H9, H9 human Embryonic Stem Cells (ESCs), 3 replicates H4FiPS1, Human 4 factors (Oct4, Sox2, Klf4 and Myc) induced pluripotent stem (iPS) cells, clone 1, 3 replicates H4FiPS2, Human 4 factors (Oct4, Sox2, Klf4 and Myc) induced pluripotent stem (iPS) cells, clone 2, 3 replicates H6FiPS2, Human 6 factors (Oct4, Sox2, Klf4, Brg1 and Baf155) induced pluripotent stem (iPS) cells, clone 2, 3 replicates H6FiPS3, Human 6 factors (Oct4, Sox2, Klf4, Brg1 and Baf155) induced pluripotent stem (iPS) cells, clone 3, 3 replicates Fibro, F134 Human Fibroblast, 1 replicate