Epigenetic memory in induced pluripotent stem cells.

Pluripotent stem cells and their characterizationa, Experimental schema. fESC, ntESC, F-iPSC, and B-iPSC were derived from B6/CBA F1 mice by reprogramming and/or cell culture, characterized for pluripotency by criteria applied to human cells, followed by differentiation analysis for osteogenic or hematopoietic lineages. b, Expression of pluripotency markers NANOG and OCT4 by immunohistochemistry. 4,6-Diamidino-2-phenylindole (DAPI) staining for total cell content. Feeder fibroblasts serve as internal negative controls. c, Teratoma analysis: tumor histology from indicated cell lines shows highly cystic structures consisting of differentiated elements of all three embryonic germ layers. Scale bar, 200μm.

Differentiation of cell linesa, Hematopoietic colony number per 100,000 EB cells differentiated from indicated cell lines. b, Alizarin Red staining of osteogenic cultures of B-iPSC (left) and F-iPSC (right). Top: 3 cm culture dish; Bottom: stained colonies; scale bar, 500 μm. c, Quantification of elemental calcium by inductively coupled plasma – atomic emission spectroscopy49 in 5×105 cells after osteogenic differentiation of indicated cell lines. d, Q-PCR of osteogenic genes, Bglap, Sp7, and Runx2 in indicated cell lines after osteogenic differentiation. Gene expression was normalized to Actin. n=number of independent clones tested. Error bars=s.d.

Analysis of methylation in stem cell linesa, Cluster dendrogram using probes from DMRs that distinguish B-iPSC and F-iPSC. Cell clones are described in Supplemental Table 5. b, Enrichment of DMRs for hematopoiesis and fibroblast-related transcription factors in B-iPSC and F-iPSC, relative to chance (blue histogram; 100,000 random permutations). Left panel: 20 of 74 hematopoiesis-related transcription factors overlap DMRs hypermethylated in F-IPSC (p=0.0034); Right panel: 115 of 764 fibroblast-specific genes overlap DMRs hypermethylated in B-iPSC (p=10−5).

Stringently-defined pluripotent stem cells and their characterizationa, Experimental schema. Four horizontal lines indicate integrated proviruses carrying dox-inducible reprogramming factors in some experiments. Characteristics of individual clones in all subsequent panels can be found in Supplemental Table 5. b, Hematopoietic colony number per 100,000 EB cells differentiated from indicated cell lines. n=number of independent clones tested. Error bars=s.d., added for clones repeated three or more times. c, Cluster dendrogram using probes from DMRs that distinguish Bl-iPSC and NP-iPSC.