Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency

Abstract

Summary Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered reactivation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies.

Resetting the epigenome for heart regeneration.

“…However, inhibition of H3K27 methylases (PRC1/2) limits pluripotency in some studies [119,121], but enhances pluripotency in others [122], suggesting context-dependent functions for H3K27 methylation during reprogramming. It was recently demonstrated that H3K27 methylation is only required at a core set of lineage-specific developmentally regulated genes [123] and loss of H3K27 methylation at these sites greatly diminishes reprogramming efficiency [123].…”

Bioinformatic and Genomic Analyses of Cellular Reprogramming and Direct Lineage Conversion

“…A recently generated secondary system for human reprogramming has been reported where differentiation of iPS colonies into fibroblasts created lines with clonal integrations of inducible reprogramming factors and constitutive hTERT to overcome senescence and the ability to expand clones. As with other systems, this secondary system generates human iPS cells that are generally very similar to ES cells in their gene expression profiles and histone modifications [15].…”

“…Generally, there is agreement that iPS reprogramming generates cells that are very similar to ES cells [15,31,55,61,74]; however, genomic-scale bioinformatics has been able to identify subtle differences between ES and iPS cells. An early meta-analysis of expressional profiling identified a subset of genes and miRs that can differentiate human iPS and ES cells [18].…”

“…The improving feasibility of reliable single-cell expressional profiling also opens the possibilities of assessing the gene network changes that occur during more rapid forms of cellular reprogramming, such as reprogramming by nuclear transfer and cell fusion [100]. Finally, it is exciting that human reprogramming methodologies and systems for integrative analysis are starting to catch up to the progress made in mice [15]. While human reprogramming is required for regenerative purposes, studying the interspecies differences between mice and human can illuminate the feasibility of using mouse reprogramming systems for both drug screening and disease modelling.…”

“…To this end, further analysis of the mechanisms by which these handfuls of factors can reprogram a cell will be indispensable to understand reprogramming, as has been performed in both iPS and iN reprogramming [8,11,47,52,95]. To achieve the precise timing and high efficiency that are so critical for these types of integrative analyses, secondary reprogramming systems, such as those that have been generated for iPS cells, have served as a profitable system for bioinformatic analysis [15,35,40,50,61,74,90,96]. While these secondary systems abrogate a great deal of the cellular heterogeneity found during reprogramming, they cannot overcome the cell-to-cell variability that is inherent to this system.…”