Epigenetic memory in induced pluripotent stem cells

Abstract

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.

Figure 1. Pluripotent stem cells and their characterization

a, 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.

Figure 2. Differentiation of cell lines

a, 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 spectroscopy 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.

Figure 3. Analysis of methylation in stem cell lines

a, 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).

Figure 4. Stringently-defined pluripotent stem cells and their characterization

a, 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.