Direct lineage conversions: unnatural but useful?

Abstract

Classic experiments such as somatic cell nuclear transfer into oocytes and cell fusion demonstrated that differentiated cells are not irreversibly committed to their fate. More recent work has built on these conclusions and discovered defined factors that directly induce one specific cell type from another, which may be as distantly related as cells from different germ layers. This suggests the possibility that any specific cell type may be directly converted into any other if the appropriate reprogramming factors are known. Direct lineage conversion could provide important new sources of human cells for modeling disease processes or for cellular-replacement therapies. For future applications, it will be critical to carefully determine the fidelity of reprogramming and to develop methods for robustly and efficiently generating human cell types of interest.

Figure 1. Epigenetic models of development and reprogramming

During development, cells are gradually restricted in their developmental potential (left). It is believed that this irreversible restriction is accompanied and caused by the progressive acquisition of epigenetic modifications (symbolized as accumulating black stripes in bars) that help to stabilize cell fate decisions and to restrict the adoption of inappropriate fates. The pluripotent state is characterized by a highly “open” chromatin configuration, which is thought to permit differentiation to a variety of cell types. In one model (Model A) reprogramming to a pluripotent state (by nuclear transfer, cell fusion, or by defined transcription factors) could occur by the stepwise erasure of epigenetic marks associated with differentiation (red arrows), which would allow cells to regain the open chromatin state and, by default, pluripotency. Alternatively, (Model B) the pluripotent state can be thought of as a defined and actively regulated epigenetic state rather than an epigenetically erased space. This model would suggest that the reprogramming factors actively establish the pluripotent chromatin state (red arrow), and that reprogramming represents an acquisition of pluripotent characteristics (red bars) rather than a loss of epigenetic lineage-restriction (green bars). This model suggests that inducing pluripotency should not be considered fundamentally different than inducing other defined cell types, and that it should be possible to convert one differentiated cell type into another (blue bars) with the right combination of factors.

Figure 2. Various modes of induced cell fate changes

a) Dedifferentiation: reversion to a less differentiated state. Typical examples include iPS cell reprogramming and loss of Pax5 in mature B cells b) Transdetermination: conversion between two closely related progenitor cells that share a direct common progenitor. Also, most gain-of-function experiments interrogating lineage-determining factors during embryonic development fall into this category. c) Transdifferentiation: direct fate switch between two distinct cell types. Examples include lineage conversion of mature hematopoietic cell types, exocrine to endocrine pancreatic cells, or the conversion of fibroblasts into cardiac cells, skeletal myocytes, neurons, or hepatocytes. d) Directly induced differentiation: direct conversion studies suggest that it might be possible to directly induce a more differentiated cell type without passing through the corresponding intermediate progenitor state. For example, MyoD1 expression in human ES cells rapidly generates multinucleated myotubes . Similarly, forced expression of iN cell reprogramming factors in pluripotent human cells rapidly generates neurons ^,

Figure 3. Potential mechanism of the stepwise activation of silent genes by reprogramming factors

a) How can a transcription factor or a combination of transcription factors modify gene expression at epigenetically silenced loci? The various repressive marks are symbolized with red objects of different shapes (left) on DNA (blue line) or nucleosomes (blue cylinders). Green objects represent active chromatin marks (right). Dark green circles represent lineage reprogramming factors (TFs) that promote gene transcription in a permissive chromatin state. b) The repressed chromatin state is likely to be more dynamic than classically assumed. Epigenetic marks may stochastically fluctuate between active and repressive states with the majority of marks being repressed at any given time point. During cell division there is also a potential window for epigenetic plasticity as unmodified histones are incorporated into duplicated strands of DNA. c) The stochastic loss of repressive modifications or the sliding/displacement of nucleosomes may allow transcription factors to weakly bind DNA and access their cognate binding sites. This interaction may interfere with the stochastic fluctuation, and can potentially recruit additional coactivators and/or histone modifying enzymes to this regulatory region, thus stabilizing transcription factor binding and eventually leading to transcriptional activation. d) The newly activated genes may code for endogenous reprogramming factors or other transcription factors that could promote activation of the novel transcriptional program via a positive feedback loop. This would activate a self-maintaining transcriptional program and the exogenous reprogramming factors would no longer required to maintain the new cell lineage identity.