Here we demonstrate that iPSC retain an epigenetic memory of their tissue of origin. Our data reveal several important principles that relate to the technical limitations inherent in the process of reprogramming, and which in practice influence the differentiation propensity of specific isolates of iPSC.
First, tissue source influences the efficiency and fidelity of reprogramming9,10,11
. From aged mice, blood cells were reprogrammed more closely to fESC than dermal fibroblasts, which yielded only incompletely reprogrammed cells. Neural progenitor-derived iPSC were most similar to fESC, consistent with evidence that such cells can be reprogrammed with fewer transcription factors38
. Whereas neural progenitors are not readily accessible, iPSC can be generated by direct reprogramming of human blood39
Second, analysis of DNA methylation reveals substantial differences between iPSC and embryo-derived ESC (ntESC and fESC). iPSC derived from non-hematopoietic cells (neural progenitors and fibroblasts) retain residual methylation at loci required for hematopoietic fate, which manifests as reduced blood-forming potential in vitro
. Residual methylation signatures link iPSC to their tissue of origin, and even discriminate between the myeloid and lymphoid origins of blood-derived iPSC. Prior studies reporting residual hypermethylation in iPSC37,22
did not establish a link between DMRs at specific loci, tissue of origin, and altered differentiation potential. While residual methylation is mostly repressive, we have shown for Wnt3 that residual gene body methylation in blood-derived iPSC is associated with enhanced blood potential. Interestingly, the poor blood potential of neural progenitor-derived iPSC, which lack this epigenetic mark and express lower levels of endogenous Wnt3, can be enhanced by supplementing differentiating cultures with exogenous Wnt3a cytokine, indicating that manipulating culture conditions can overcome epigenetic barriers.
Third, the differentiation propensity and methylation profile of iPSC can be reset. When blood-deficient neural progenitor-derived iPSC (NP-iPSC) are differentiated into blood and then reprogrammed to pluripotency, their blood-forming potential is markedly increased. Alternatively, treatment of NP-iPSC with chromatin-modifying compounds increases blood-forming potential and is associated with reduced methylation at hematopoietic loci. For some applications, epigenetic memory of the donor cell may be advantageous, as directed differentiation to specific tissue fates remains a challenge.
Fourth, nuclear transfer-derived ESC are more faithfully reprogrammed than most iPSC generated from adult somatic tissues. Like the immediate and rapid demethylation of the sperm pronucleus following fertilization, somatic nuclei are rapidly demethylated by nuclear transfer into ooplasm, prompting speculation that the egg harbors an active demethylase14
. In contrast, demethylation is a late phenomenon in factor-based reprogramming, and likely occurs passively13
. Studying how ooplasm erases methylation might identify biochemical functions that would enhance factor-based reprogramming. Failure to demethylate pluripotency genes is associated with intermediate or partial states of reprogramming12,29,40
, and knock-down of the maintenance methyltranferase DNMT1 or treatment with the demethylating agent 5-AZA can convert intermediate states to full pluripotency. Demethylation appears passage dependent13
, and reprogramming efficiency correlates with the rate of cell division and the passage number41
. In our experiments, we compared pluripotent stem cells of comparable low passage number (Supplemental Table 5
), but continued serial passage may homogenize the differentiation potential of pluripotent cell types.
The mRNA expression program of iPSC and fESC are strikingly similar42
. Minor differences in mRNA and microRNA expression have been reported40
, but removal of transgenes reduces the differences43
. The Dlk1-Dio3 locus, whose expression correlates with capacity to generate “all-iPSC” mice44
, is not differentially methylated and expressed in at least some iPSC lines that manifest epigenetic memory (our unpublished observations). Thus even the most stringently-defined iPSC might retain epigenetic memory. Importantly, differences between iPSC and fESC may not manifest until differentiation, when the specific loci that retain residual epigenetic marks are expressed, influencing cell fates. Methylation is but one molecular feature of “epigenetic memory” in iPSC. Faulty restoration of bivalent domains, which mark developmental loci with both active and repressive histone modifications 45
, and loss of pioneer factors, which in fESC and iPSC occupy enhancers of genes expressed only in differentiated cells46
, represent two other potential mechanisms.
Although ideal, generic iPSC may be functionally and molecularly indistinguishable from fESC, we have shown in practice that even rigorously selected iPSC can retain epigenetic marks characteristic of the donor cell that influence differentiation propensity. Epigenetic differences are unlikely to be essential features of iPSC, but rather reflect stochastic variations associated with the technical challenges of achieving complete reprogramming. Given that we lack reporter genes for selecting human iPSC, and cannot qualify their pluripotency by assaying embryo chimerism, the behavior of human cells will likely be influenced by epigenetic memory. Human ESC can also manifest variable differentiation potential47
. These data highlight our limited understanding of the epigenetic heterogeneity of pluripotent stem cells, and the need for improved methods to ensure reprogramming of somatic cells to a fully naïve, “ground state” of pluripotency48