Manipulating cell fate through reprogramming: approaches and applications

Abstract

Cellular plasticity progressively declines with development and differentiation, yet these processes can be experimentally reversed by reprogramming somatic cells to induced pluripotent stem cells (iPSCs) using defined transcription factors. Advances in reprogramming technology over the past 15 years have enabled researchers to study diseases with patient-specific iPSCs, gain fundamental insights into how cell identity is maintained, recapitulate early stages of embryogenesis using various embryo models, and reverse aspects of aging in cultured cells and animals. Here, we review and compare currently available reprogramming approaches, including transcription factor-based methods and small molecule-based approaches, to derive pluripotent cells characteristic of early embryos. Additionally, we discuss our current understanding of mechanisms that resist reprogramming and their role in cell identity maintenance. Finally, we review recent efforts to rejuvenate cells and tissues with reprogramming factors, as well as the application of iPSCs in deriving novel embryo models to study pre-implantation development.

Keywords: Cell fate; Epigenetics; Induced pluripotent stem cells; Reprogramming; Small molecules; Transcription factors.

Fig. 1.

Different strategies to produce iPSCs and applications of iPSC technology. Diverse somatic cell types can be reliably reprogrammed into iPSCs using either transcription factors, small molecules or a combination of both. Reprogramming technology has been leveraged for diverse applications, including disease modeling and studies aimed at identifying fundamental mechanisms that maintain cell identity or facilitate cellular plasticity. More recently, modified reprogramming strategies have been employed in vivo to study tissue regeneration and aging, as well as in vitro to assemble versatile models of early embryogenesis. iPSC, induced pluripotent stem cell; Oct4, octamer-binding transcription factor 4; Klf4, Krüppel-like factor 4; Sox2, sex-determining region Y-box 2; c-Myc, cellular myelocytomatosis oncogene.

Fig. 2.

Comparison of chemical- versus transcription factor-based reprogramming approaches. (A) Small molecule-based reprogramming occurs in a stepwise manner involving distinct intermediate stages that include an epithelial and extra-embryonic endoderm (XEN)-like state. XEN cells are cultured stem cells representative of the primitive endoderm of blastocyst embryos. (B) Transcription factor (TF)-based reprogramming using fibroblasts also passes through characteristic intermediate stages, including an early mesenchymal-to-epithelial transition (MET) and the late activation of pluripotency genes. (C) During TF-induced reprogramming, the somatic program is first extinguished by OKSM-mediated sequestration of somatic TFs, leading to somatic enhancer decommissioning, as well as a gain of repressive H3K27me3 at associated promoters via PRC2 (left panel). Pluripotency networks are subsequently activated via Tet2-dependent active demethylation of pluripotency-associated regulatory elements, leading to their activation (right panel). The dotted pink lines represent RNA being transcribed. PRC2, polycomb repressive complex 2; Tet2, ten-eleven translocation methylcytosine dioxygenase 2; H3K27me3, histone H3 lysine-27-trimethylation; O, octamer-binding transcription factor 4; K, Krüppel-like factor 4; S, sex-determining region Y-box 2; M, cellular myelocytomatosis oncogene. (D) Factors that resist reprogramming are often shared with mechanisms that maintain cell identity. For example (1), nucleosome assembly via the histone chaperone CAF-1, and histone methylation at histone H3K9 and H3K36 residues, maintain somatic gene expression programs and/or repress the acquisition of pluripotency-associated programs. Moreover (2), Nudt21 controls the protein levels of fate-instructive chromatin regulators by modulating the use of alternative polyA sites of associated RNAs. (3) Lysine SUMOylation modifies the activity or stability of regulatory factors by attaching SUMO peptides via the conjugating enzyme Ubc9. (4) Chromatin remodeling via the NuRD/Mbd3 histone deacetylase complex reportedly prevents deterministic reprogramming of somatic cells towards iPSCs by keeping pluripotency genes silenced. Mbd3, methyl-CpG binding domain protein 3; NuRD, nucleosome remodeling and deacetylation; CAF-1, chromatin assembly factor 1; H3K9me, histone H3 lysine-9 methylation; H3K36me, histone H3 lysine-36 methylation; Nudt21, nudix hydrolase 21; SUMO, small ubiquitin-like modifier; Ubc9, ubiquitin-conjugating enzyme 9.

Fig. 3.

Applications of reprogramming technology in rejuvenation research and embryo modeling. (A) Partial reprogramming involves short pulses of OKSM expression in cultured cells or mice (left panel). This approach is reportedly insufficient to generate iPSCs in vitro or teratomas in vivo but sufficient to reverse some hallmarks of aging, consistent with a rejuvenation-like phenotype. Rejuvenation-like phenotypes after OKSM pulses have been observed at the molecular, tissue and organism levels (right panel), including, for example, the restoration of H3K9 and H4K20 methylation towards a youthful state, improved tissue architecture and, in some models, an extension of lifespan. H4K20me, histone H4 lysine-20 methylation; H3K9me, histone H3 lysine-9 methylation. (B) OKSM expression in fibroblast cultures reportedly gives rise to both extra-embryonic-like (blue) and embryonic-like (red) cell types that can self-assemble into 3D blastocyst-like models. Trophoblast stem cells (TSCs) are cultured stem cells representative of trophectoderm precursors of the blastocyst embryo, which give rise to differentiated cells of the placenta. TSCs can either be derived directly from fibroblast cultures undergoing reprogramming (dotted line) or indirectly from naive iPSCs using cytokines (solid line). iPSC, induced pluripotent stem cell; O, octamer-binding transcription factor 4; K, Krüppel-like factor 4; S, sex-determining region Y-box 2; M, cellular myelocytomatosis oncogene.