Direct cell reprogramming: approaches, mechanisms and progress

Abstract

The reprogramming of somatic cells with defined factors, which converts cells from one lineage into cells of another, has greatly reshaped our traditional views on cell identity and cell fate determination. Direct reprogramming (also known as transdifferentiation) refers to cell fate conversion without transitioning through an intermediary pluripotent state. Given that the number of cell types that can be generated by direct reprogramming is rapidly increasing, it has become a promising strategy to produce functional cells for therapeutic purposes. This Review discusses the evolution of direct reprogramming from a transcription factor-based method to a small-molecule-driven approach, the recent progress in enhancing reprogrammed cell maturation, and the challenges associated with in vivo direct reprogramming for translational applications. It also describes our current understanding of the molecular mechanisms underlying direct reprogramming, including the role of transcription factors, epigenetic modifications, non-coding RNAs, and the function of metabolic reprogramming, and highlights novel insights gained from single-cell omics studies.

Fig. 1 |. Principles of indirect and direct reprogramming.

Direct reprogramming (also known as transdifferentiation) refers to a change in cell fate that, unlike in indirect reprogramming, does not involve a pluripotent intermediate state (usually the production of induced pluripotent stem cells). Due to the self-renewal capacity of the intermediate pluripotent stem cells, indirect reprogramming can produce target cells in a large scale and is suitable for ex vivo cell production. On the other hand, by not requiring this intermediate step, direct reprogramming is a faster and more efficient process and, in principle, as it can occur both ex vivo and in situ (in the target tissue), it is more suitable for in vivo tissue repair. Moreover, direct reprogramming could retain epigenetic hallmarks of the cell of origin, for example, ageing hallmarks, in the reprogrammed cell compared with indirect reprogramming, making the cells obtained through direct reprogramming more suitable for modelling ageing-related disease.

Fig. 2 |. Direct reprogramming across germ layers.

Direct reprogramming can induce cell fate conversions between cell lineages that are derived from the same embryonic germ layer but can also cross the germ layer barrier. That is, cells derived from one germ layer can be converted to cell types originating from another germ layer. Fibroblasts originating from the mesoderm have been used as starting cells in most direct reprogramming experiments owing to their availability and high plasticity. Other cell types, such as macroglial cells from the ectoderm and α-cells from the endoderm, have also been used for successful direct reprogramming. The combinations of reprogramming factors used for each cell type conversion are shown; pioneer factors that are crucial for successful direct reprogramming are highlighted. Small molecules and microRNAs are also used for direct reprogramming (not shown).

Fig. 3 |. Functions of reprogramming factors during direct reprogramming.

a | At the initial stages of fate conversion, unlike other transcription factors, pioneer factors can access closed chromatin and bind to regions that are in an open conformation in the target cell type to allow cell type-specific gene expression. b | Reprogramming factors recruit other factors and work cooperatively to activate or inhibit target gene expression. c | Reprogramming factors could refine the binding profile of other reprogramming factors during direct reprogramming. The expression of a single reprogramming factor may induce the expression of lineage genes non-specific to the target cell type. The co-expression of other reprogramming factors limited such non-specific binding, thus refining the induced gene programme in the end-product cells.

Fig. 4 |. Histone modifications that regulate gene expression during direct reprogramming.

a | The types and functions of single histone modifications during direct reprogramming. H3K4me3, a histone modification that is associated with active transcription, serves as a hallmark for successful activation of the transcriptional programme that is characteristic of the desired cell type. H3K27me3, a repressive histone modification, can be used as a marker of successful silencing of the starting cell transcriptional programme. H3K9me3 is a histone modification that is associated with heterochromatin, which is refractory to transcription activation and constitutes a major barrier for successful reprogramming. H2AK119Ub is a repressive mark that has been identified at cardiac-specific loci in fibroblasts and the removal of this epigenetic mark enhances cardiac reprogramming. b | The types and functions of different combinations of histone modification during direct reprogramming. The co-enrichment of H3K4me3 and H3K27ac marks the promoters of expressed genes and genes that become activated during reprogramming. The simultaneous presence of H3K4me and H3K27ac marks the enhancers of active genes. The coexistence of H3K4me, H3K27ac and H3K9me3 (trivalent chromatin) promotes the binding of Ascl1 to neuron-specific genes during the conversion of fibroblasts to induced neurons and is an indicator of the efficiency of Ascl1-driven induced neuron reprogramming. c | Histone variants play a part in reprogramming. The histone H3 variant H3.3 has a dual role during direct reprogramming: it is important for the maintenance of the gene expression programme of the starting cell type at early stages of reprogramming and is required for the establishment of the gene expression programme of the desired cell lineage in the late stages of reprogramming. TF, transcription factor.

Fig. 5 |. Metabolic switch during direct reprogramming.

The switch from glycolysis to oxidative phosphorylation (OxPhos) is important for direct reprogramming both in vitro and in vivo. Treating mouse embryonic fibroblasts with oligomycin A, an inhibitor of OxPhos, completely abolished the reprogramming of these fibroblasts to induced neurons (iNs) following overexpression of Ascl1 and Neurog2. Reactive oxygen species (ROS) are a by-product of the glycolysis-to-OxPhos switch, and aberrantly high levels of ROS could impede cell fate conversion. The overexpression of Bcl-2, an anti-apoptotic protein, or treatment with anti-oxidant compounds, such as vitamin E or α-tocotrienol, drastically increased the efficiency of reprogramming to iNs both in vitro and in vivo. Exposing the cardiac fibroblast to an anti-oxidant (selenium) led a 5–15-fold increase in reprogramming efficiency when mouse cardiac fibroblasts were induced to convert to induced cardiomyocytes (iCMs) in vitro via the forced expression of microRNAs. TCA, tricarboxylic acid.

Fig. 6 |. Single-cell omics in direct reprogramming.

Computational approaches that produce information on reprogramming trajectories based on single-cell RNA-seq (scRNA-seq) data facilitate the identification of alternative routes in both direct neuronal and cardiac reprogramming. Two examples of the type of information that can be obtained are shown. a | Clustering analysis based on scRNA-seq at the late stages of Ascl1-mediated (circle) or BAM-mediated (triangle) neuronal reprogramming showed three distinct cell clusters with specific lineage gene expression of neuron (red), fibroblast (blue) and myocyte (green) fate, suggesting the existence of an alternative cell fate at the late stage of neuronal reprogramming. The plot is modified based on the data from REF.. b | Trajectory analysis revealed two separate paths in human cardiac reprogramming. When cells engage in a ‘reprogramming path’, they gradually acquire a cardiomyocyte cell fate; however, cells can also follow a ‘refractory route’ and revert towards a fibroblast cell fate. Differential gene expression analysis identified genes involved in different pathways or cellular processes that are activated or suppressed while cells follow either route. The plot is modified based on the data in REF.. c | The alternative reprogramming outcomes of direct reprogramming revealed by scRNA-seq analysis. In neuronal reprogramming mediated by Ascl1 only, most of the cells gained a transcription programme similar to myocytes. The addition of Brn2 and Myt1l suppressed the aberrant myogenic programme. In cardiac reprogramming mediated by Gata4, Mef2c and Tbx5 (GMT), most of the cells successfully gained a cardiac programme as expected. A small population of cells gained transcription signatures of vasculature and blood vessel development. scRNA-seq analysis of human cardiac reprogramming also revealed the existence of a refractory route where the cells reverted to a fibroblast fate. ECM, extracellular matrix; iCM, induced cardiomyocyte; iN, induced neuron; LLE, locally linear embedding; tSNE, t-distributed stochastic neighbour embedding. Part a adapted from REF., Springer Nature Limited; part b adapted with permission from REF., Elsevier.