The induced pluripotent stem cell (iPSC) technology has transformed in vitro research and holds great promise to advance regenerative medicine. iPSCs have the capacity for an almost unlimited expansion, are amenable to genetic engineering, and can be differentiated into most somatic cell types. iPSCs have been widely applied to model human development and diseases, perform drug screening, and develop cell therapies. In this review, we outline key developments in the iPSC field and highlight the immense versatility of the iPSC technology for in vitro modeling and therapeutic applications. We begin by discussing the pivotal discoveries that revealed the potential of a somatic cell nucleus for reprogramming and led to successful generation of iPSCs. We consider the molecular mechanisms and dynamics of somatic cell reprogramming as well as the numerous methods available to induce pluripotency. Subsequently, we discuss various iPSC-based cellular models, from mono-cultures of a single cell type to complex three-dimensional organoids, and how these models can be applied to elucidate the mechanisms of human development and diseases. We use examples of neurological disorders, coronavirus disease 2019 (COVID-19), and cancer to highlight the diversity of disease-specific phenotypes that can be modeled using iPSC-derived cells. We also consider how iPSC-derived cellular models can be used in high-throughput drug screening and drug toxicity studies. Finally, we discuss the process of developing autologous and allogeneic iPSC-based cell therapies and their potential to alleviate human diseases. 

Induced pluripotent stem cells (iPSCs): molecular mechanisms of induction and applications

Chromatin structure and dynamics: one nucleosome at a time.

Histone H3 lysine 9 methylation (H3K9me) epigenetically silences gene expression by forming heterochromatin. Proteins called HP1, which contain specialized reader domains, bind to H3K9me and recruit factors that regulate epigenetic silencing. Though these interactions have been identified in vitro, we do not understand how HP1 proteins specifically and selectively bind to heterochromatin-associated factors within the nucleus. Using fission yeast as a model system, we measured the single-molecule dynamics associated with two archetypal HP1 paralogs, Swi6 and Chp2, and inferred how they form complexes with their interacting partners: Epe1, a putative H3K9 demethylase; Clr3, a histone deacetylase; and Mit1, a chromatin remodeler. Through a series of genetic perturbations that affect H3K9 methylation and HP1-mediated recruitment, we were able to track altered diffusive properties associated with each HP1 protein and its binding partner. Our findings show that the HP1-interacting proteins we investigated only co-localize with Swi6 and Chp2 at sites of H3K9me. When H3K9me is absent, Epe1 and Swi6 exhibit diffusive states consistent with off-chromatin interactions. Our results suggest that histone modifications like H3K9 methylation are not simply inert binding platforms but rather, they can shift the balance of HP1 complex assembly toward a predominantly chromatin-bound state. By inferring protein-protein interactions based on the altered mobilities of proteins in living cells, we propose that H3K9 methylation can stimulate the assembly of diverse HP1-associated complexes on chromatin. SIGNIFICANCE STATEMENT During differentiation, epigenetic silencing is essential for preserving cellular identity. Establishing and maintaining epigenetic silencing depends on histone H3 lysine 9 methylation, which HP1 proteins recognize and bind with low micromolar affinity and millisecond-scale kinetics. HP1 proteins also recruit diverse histone modifiers to maintain gene silencing. HP1 protein biochemistry has revealed what can happen, but the state-of-the-art in this field includes little about what does happen in the complex and crowded environment of the nucleus. Using single-molecule tracking of HP1 proteins and their binding partners, we identified the rules that govern their complex formation in the native chromatin context, and we found that chromatin— previously thought to be an inert platform—enhances complex formation between HP1 and its binding partners.

/pdf/h3k9-methylation-enhances-hp1-associated-epigenetic-2ecmxfuh.pdf

H3K9 methylation enhances HP1-associated epigenetic silencing complex assembly and suppresses off-chromatin binding

Abstract The discovery of mouse embryonic stem cells in 1981 transformed research in mammalian developmental biology and functional genomics. The subsequent generation of human pluripotent stem cells (PSCs) and the development of molecular reprogramming have opened unheralded avenues for drug discovery and cell replacement therapy. Here, I review the history of PSCs from the perspective that long‐term self‐renewal is a product of the in vitro signaling environment, rather than an intrinsic feature of embryos. I discuss the relationship between pluripotent states captured in vitro to stages of epiblast in the embryo and suggest key considerations for evaluation of PSCs. A remaining fundamental challenge is to determine whether naïve pluripotency can be propagated from the broad range of mammals by exploiting common principles in gene regulatory architecture. 

Propagating pluripotency – The conundrum of self‐renewal

Induced pluripotent stem cells (iPSCs) exhibit inconsistent differentiation potential, negatively impacting their downstream applications. Here, we potentiated their reprogramming by adding ten-eleven translocation 1 (TET1), a DNA demethylase, to produce TET1-iPSCs (T-iPSCs). By comparing the differentiation efficiencies of 46 in-house-generated iPSC clones, we identified an extraembryonic gene signature linked to differentiation defects. The extraembryonic signature was upregulated when all three TET genes were knocked out in human embryonic stem cells. T-iPSCs with enhanced epithelialized morphology, a trait attributable to TET1 activity during reprogramming, exhibited uniform TET1 expression and lacked the identified extraembryonic signature. These T-iPSCs differentiated into ventral midbrain dopaminergic neurons with unprecedented fidelity and efficiency. Our data collectively revealed a deviation from the genuine embryonic gene profile in conventional human iPSCs, which was amendable by including TET1 in the reprogramming cocktail. We recommend the proposed T-iPSC production pipeline as a de facto standard for human iPSC reprogramming.

TET1 potentiates human iPS cell reprogramming by rectifying extraembryonic gene noises

Enhancers are genomic DNA sequences that bind transcription factors, chromatin regulators and non-coding transcripts to modulate the expression of target genes. They have been found to act from different locations relative to a gene and to modulate the activity of promoters up to ∼1 Mb away. Here we report the first 3D genome structures of single mouse ES cells as they are induced to exit pluripotency, transition through a formative stage and undergo neuroectodermal differentiation. In order to directly study how interactions between enhancers and promoters are reconfigured genome wide we have determined 3D structures of haploid cells using single cell Hi-C, where we can unambiguously map the contacts to particular chromosomes. We find that there is a remarkable reorganisation of 3D genome structure where inter-chromosomal intermingling increases dramatically in the formative state. This intermingling is associated with the formation of a large number of multiway hubs that bring together enhancers and promoters with similar chromatin states from typically 5-8 distant chromosomal sites that are often separated by many Mb from each other. Genes important for pluripotency exit establish contacts with emerging enhancers within multiway chromatin hubs as cells enter the formative state, consistent with these structural changes playing an important role in establishing new cell identities. Furthermore, we find that different multiway chromatin hubs, and thus different enhancer-promoter interactions, are formed in different individual cells. Our results suggest that studying genome structure in single cells will be important to identify key changes in enhancer-promoter interactions that occur as cells undergo developmental transitions.

/pdf/enhancer-promoter-interactions-are-reconfigured-through-the-1kyp3pn0.pdf

Enhancer-promoter interactions are reconfigured through the formation of long-range multiway chromatin hubs as mouse ES cells exit pluripotency

Abstract To understand how the nucleosome remodeling and deacetylase (NuRD) complex regulates enhancers and enhancer–promoter interactions, we have developed an approach to segment and extract key biophysical parameters from live-cell three-dimensional single-molecule trajectories. Unexpectedly, this has revealed that NuRD binds to chromatin for minutes, decompacts chromatin structure and increases enhancer dynamics. We also uncovered a rare fast-diffusing state of enhancers and found that NuRD restricts the time spent in this state. Hi-C and Cut&amp;Run experiments revealed that NuRD modulates enhancer–promoter interactions in active chromatin, allowing them to contact each other over longer distances. Furthermore, NuRD leads to a marked redistribution of CTCF and, in particular, cohesin. We propose that NuRD promotes a decondensed chromatin environment, where enhancers and promoters can contact each other over longer distances, and where the resetting of enhancer–promoter interactions brought about by the fast decondensed chromatin motions is reduced, leading to more stable, long-lived enhancer–promoter relationships. 

Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD.

Enhancers bind transcription factors, chromatin regulators, and non-coding transcripts to modulate the expression of target genes. Here, we report 3D genome structures of single mouse ES cells as they are induced to exit pluripotency and transition through a formative stage prior to undergoing neuroectodermal differentiation. We find that there is a remarkable reorganization of 3D genome structure where inter-chromosomal intermingling increases dramatically in the formative state. This intermingling is associated with the formation of a large number of multiway hubs that bring together enhancers and promoters with similar chromatin states from typically 5-8 distant chromosomal sites that are often separated by many Mb from each other. In the formative state, genes important for pluripotency exit establish contacts with emerging enhancers within these multiway hubs, suggesting that the structural changes we have observed may play an important role in modulating transcription and establishing new cell identities. 

Enhancer-promoter interactions are reconfigured through the formation of long-range multiway hubs as mouse ES cells exit pluripotency.

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Enhancer-promoter interactions are reconfigured through the formation of long-range multiway chromatin hubs as mouse ES cells exit pluripotency

Live-cell three-dimensional single-molecule tracking reveals modulation of enhancer dynamics by NuRD.

Enhancer-promoter interactions are reconfigured through the formation of long-range multiway hubs as mouse ES cells exit pluripotency.