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How master regulators such as Oct4 can shape 3D genome architecture during cell reprogramming remains poorly understood. In this issue of Cell Stem Cell, Wang et al. (2021) demonstrate that Oct4 undergoes phase separation to reconfigure TAD architecture for cell fate control.

Direct reprogramming has revolutionized the fields of stem cell biology and regenerative medicine. However, the common mechanisms governing how reprogramming cells undergo transcriptome and epigenome remodeling (i.e., regulatome remodeling) have not been investigated. Here, by characterizing early changes in the regulatome of three different types of direct reprogramming, we identify lineage-specific features as well as common regulatory transcription factors. Of particular interest, we discover that the neuronal factor Ascl1 possesses cross-lineage potential; together with Mef2c, it drives efficient cardiac reprogramming toward a mature and induced cardiomyocyte phenotype. Through ChIP-seq and RNA-seq, we find that MEF2C drives the shift in ASCL1 binding away from neuronal genes toward cardiac genes, guiding their co-operative epigenetic and transcription activities. Together, these findings demonstrate the existence of common regulators of different direct reprogramming and argue against the premise that transcription factors possess only lineage-specific capabilities for altering cell fate - the basic premise used to develop direct reprogramming approaches.

Organ constructs are organ-like structures grown in vitro or in vivo that harbor the components, architecture, and function of in vivo organs, in part or in toto. The convergence of stem cell biology, bioengineering, and gene editing tools have substantially broadened our ability to generate various types of organ constructs for regenerative medicine as well as to address pressing biomedical questions. In this Review, we highlight prevailing approaches for generating organ constructs, from organoids to chimeric organ engineering. We also discuss design principles of different approaches, their utility and limitations, and propose strategies to resolve existing hurdles.

Summary Transcription factor (TF)-mediated reprogramming to pluripotency is a slow and inefficient process, because most pluripotency TFs fail to access relevant target sites in a refractory chromatin environment. It is still unclear how TFs actually orchestrate the opening of repressive chromatin during the long latency period of reprogramming. Here, we show that the orphan nuclear receptor Esrrb plays a pioneering role in recruiting the core pluripotency factors Oct4, Sox2, and Nanog to inactive enhancers in closed chromatin during the reprogramming of epiblast stem cells. Esrrb binds to silenced enhancers containing stable nucleosomes and hypermethylated DNA, which are inaccessible to the core factors. Esrrb binding is accompanied by local loss of DNA methylation, LIF-dependent engagement of p300, and nucleosome displacement, leading to the recruitment of core factors within approximately 2 days. These results suggest that TFs can drive rapid remodeling of the local chromatin structure, highlighting the remarkable plasticity of stable epigenetic information.

Topological-associated domains (TADs) are thought to be relatively stable across cell types, although some TAD reorganization has been observed during cellular differentiation. However, little is known about the mechanisms through which TAD reorganization affects cell fate or how master transcription factors affect TAD structures during cell fate transitions. Here, we show extensive TAD reorganization during somatic cell reprogramming, which is correlated with gene transcription and changes in cellular identity. Manipulating TAD reorganization promotes reprogramming, and the dynamics of concentrated chromatin loops in OCT4 phase separated condensates contribute to TAD reorganization. Disrupting OCT4 phase separation attenuates TAD reorganization and reprogramming, which can be rescued by fusing an intrinsically disordered region (IDR) to OCT4. We developed an approach termed TAD reorganization-based multiomics analysis (TADMAN), which identified reprogramming regulators. Together, these findings elucidate a role and mechanism of TAD reorganization, regulated by OCT4 phase separation, in cellular reprogramming.

Although cellular reprogramming enables the generation of new cell types for disease modeling and regenerative therapies, reprogramming remains a rare cellular event. By examining reprogramming of fibroblasts into motor neurons and multiple other somatic lineages, we find that epigenetic barriers to conversion can be overcome by endowing cells with the ability to mitigate an inherent antagonism between transcription and DNA replication. We show that transcription factor overexpression induces unusually high rates of transcription and that sustaining hypertranscription and transgene expression in hyperproliferative cells early in reprogramming is critical for successful lineage conversion. However, hypertranscription impedes DNA replication and cell proliferation, processes that facilitate reprogramming. We identify a chemical and genetic cocktail that dramatically increases the number of cells capable of simultaneous hypertranscription and hyperproliferation by activating topoisomerases. Further, we show that hypertranscribing, hyperproliferating cells reprogram at 100-fold higher, near-deterministic rates. Therefore, relaxing biophysical constraints overcomes molecular barriers to cellular reprogramming.

Cell fate conversion generally requires reprogramming effectors to both introduce fate programs of the target cell type and erase the identity of starting cell population. Here, we reveal insights into the activity of microRNAs, miR-9/9* and miR-124 (miR-9/9*-124) as reprogramming agents that orchestrate direct conversion of human fibroblasts into motor neurons, by first eradicating fibroblast identity and promoting uniform transition to a neuronal state in sequence. We identify KLF-family transcription factors as direct target genes for miR-9/9*-124 and show their repression is critical for erasing fibroblast fate. Subsequent gain of neuronal identity requires upregulation of a small nuclear RNA, RN7SK, which induces accessibilities of chromatin regions and neuronal gene activation to push cells to a neuronal state. Our study defines deterministic components in the microRNA-mediated reprogramming cascade.

Cell-fate conversion generally requires reprogramming effectors to both introduce fate programs of the target cell type and erase the identity of starting cell population. Here, we reveal insights into the activity of microRNAs miR-9/9∗ and miR-124 (miR-9/9∗-124) as reprogramming agents that orchestrate direct conversion of human fibroblasts into motor neurons by first eradicating fibroblast identity and promoting uniform transition to a neuronal state in sequence. We identify KLF-family transcription factors as direct target genes for miR-9/9∗-124 and show their repression is critical for erasing fibroblast fate. Subsequent gain of neuronal identity requires upregulation of a small nuclear RNA, RN7SK, which induces accessibilities of chromatin regions and neuronal gene activation to push cells to a neuronal state. Our study defines deterministic components in the microRNA-mediated reprogramming cascade.

Human pluripotent stem cells harbor the capacity to differentiate into cells from the three embryonic germ layers, and this ability grants them a central role in modeling human disorders and in the field of regenerative medicine. Here, we review pluripotency in human cells with respect to four different aspects: (1) embryonic development, (2) transcriptomes of pluripotent cell stages, (3) genes and pathways that reprogram somatic cells into pluripotent stem cells, and finally (4) the recent identification of the human pluripotent stem cell essentialome. These four aspects of pluripotency collectively culminate in a broader understanding of what makes a cell pluripotent.

Summary Following fertilization, totipotent cells undergo asymmetric cell divisions, resulting in three distinct cell types in the late pre-implantation blastocyst: epiblast (Epi), primitive endoderm (PrE), and trophectoderm (TE). Here, we aim to understand whether these three cell types can be induced from fibroblasts by one combination of transcription factors. By utilizing a sophisticated fluorescent knockin reporter system, we identified a combination of five transcription factors, Gata3, Eomes, Tfap2c, Myc, and Esrrb, that can reprogram fibroblasts into induced pluripotent stem cells (iPSCs), induced trophoblast stem cells (iTSCs), and induced extraembryonic endoderm stem cells (iXENs), concomitantly. In-depth transcriptomic, chromatin, and epigenetic analyses provide insights into the molecular mechanisms that underlie the reprogramming process toward the three cell types. Mechanistically, we show that the interplay between Esrrb and Eomes during the reprogramming process determines cell fate, where high levels of Esrrb induce a XEN-like state that drives pluripotency and high levels of Eomes drive trophectodermal fate., Graphical Abstract, Highlights • Gata3, Eomes, Tfap2c, Myc, and Esrrb convert fibroblasts into iPSCs, iTSCs, and iXENs • Esrrb, but not other pluripotency genes, can shift the TSC fate into pluripotency • Esrrb induces pluripotency by the activation of a unique XEN-like state • The interplay between Eomes and Esrrb determines cell fate decision, Benchetrit and colleagues identified a combination of factors that can produce the three in vitro equivalent cell types of the blastocyst, iPSCs, iTSCs, and iXENs, from fibroblasts. They found that Esrrb was most potent in inducing pluripotency by the activation of a XEN-like state and Eomes most potent in promoting trophectoderm fate.

Summary A suitable source of progenitor cells is required to attenuate disease or affect cure. We present an “interrupted reprogramming” strategy to generate “induced progenitor-like (iPL) cells” using carefully timed expression of induced pluripotent stem cell reprogramming factors (Oct4, Sox2, Klf4, and c-Myc; OSKM) from non-proliferative Club cells. Interrupted reprogramming allowed controlled expansion yet preservation of lineage commitment. Under clonogenic conditions, iPL cells expanded and functioned as a bronchiolar progenitor-like population to generate mature Club cells, mucin-producing goblet cells, and cystic fibrosis transmembrane conductance regulator (CFTR)-expressing ciliated epithelium. In vivo, iPL cells can repopulate CFTR-deficient epithelium. This interrupted reprogramming process could be metronomically applied to achieve controlled progenitor-like proliferation. By carefully controlling the duration of expression of OSKM, iPL cells do not become pluripotent, and they maintain their memory of origin and retain their ability to efficiently return to their original phenotype. A generic technique to produce highly specified populations may have significant implications for regenerative medicine., Highlights • This approach harnesses residual epigenetic memory in the early reprogramming process • Interrupted reprogramming allows controlled expansion without traversing pluripotency • Lineage-committed induced progenitor-like cells can be obtained from mature cells • High-level engraftment of CFTR wild-type cells was possible in airway of CFTR-KO mice, In this article Waddell, Nagy, and colleagues present an “interrupted reprogramming” strategy to produce highly specified functional “induced progenitor-like cells” from mature quiescent cells. They propose that careful control of the duration of transient expression of iPSC reprogramming factors (OSKM) allows controlled expansion yet preservation of parental lineage without traversing the pluripotent state.

The ability to reprogram somatic cells into induced pluripotent stem cells (iPSCs) using defined factors provides new tools for biomedical research. However, some iPSC clones display tumorigenic and immunogenic potential, thus raising concerns about their utility and safety in the clinical setting. Furthermore, variability in iPSC differentiation potential has also been described. Here we discuss whether these therapeutic obstacles are specific to transcription-factor-mediated reprogramming or inherent to every cellular reprogramming method. Finally, we address whether a better understanding of the mechanism underlying the reprogramming process might improve the fidelity of reprogramming and, therefore, the iPSC quality.

Pluripotent genomes are folded in a topological hierarchy that reorganizes during differentiation. The extent to which chromatin architecture is reconfigured during somatic cell reprogramming is poorly understood. Here we integrate fine-resolution architecture maps with epigenetic marks and gene expression in embryonic stem cells (ESCs), neural progenitor cells (NPCs), and NPC-derived induced pluripotent stem cells (iPSCs). We find that most pluripotency genes reconnect to target enhancers during reprogramming. Unexpectedly, some NPC interactions around pluripotency genes persist in our iPSC clone. Pluripotency genes engaged in both "fully-reprogrammed" and "persistent-NPC" interactions exhibit over/undershooting of target expression levels in iPSCs. Additionally, we identify a subset of "poorly reprogrammed" interactions that do not reconnect in iPSCs and display only partially recovered, ESC-specific CTCF occupancy. 2i/LIF can abrogate persistent-NPC interactions, recover poorly reprogrammed interactions, reinstate CTCF occupancy, and restore expression levels. Our results demonstrate that iPSC genomes can exhibit imperfectly rewired 3D-folding linked to inaccurately reprogrammed gene expression.

Cellular reprogramming using chemically defined conditions, without genetic manipulation, is a promising approach for generating clinically relevant cell types for regenerative medicine and drug discovery. However, small-molecule approaches for inducing lineage-specific stem cells from somatic cells across lineage boundaries have been challenging. Here, we report highly efficient reprogramming of mouse fibroblasts into induced neural stem cell-like cells (ciNSLCs) using a cocktail of nine components (M9). The resulting ciNSLCs closely resemble primary neural stem cells molecularly and functionally. Transcriptome analysis revealed that M9 induces a gradual and specific conversion of fibroblasts toward a neural fate. During reprogramming specific transcription factors such as Elk1 and Gli2 that are downstream of M9-induced signaling pathways bind and activate endogenous master neural genes to specify neural identity. Our study provides an effective chemical approach for generating neural stem cells from mouse fibroblasts and reveals mechanistic insights into underlying reprogramming processes.

Summary The gastrointestinal (GI) epithelium is a highly regenerative tissue with the potential to provide a renewable source of insulin + cells after undergoing cellular reprogramming. Here, we show that cells of the antral stomach have a previously unappreciated propensity for conversion into functional insulin-secreting cells. Native antral endocrine cells share a surprising degree of transcriptional similarity with pancreatic β cells, and expression of β cell reprogramming factors in vivo converts antral cells efficiently into insulin + cells with close molecular and functional similarity to β cells. Induced GI insulin + cells can suppress hyperglycemia in a diabetic mouse model for at least 6 months and regenerate rapidly after ablation. Reprogramming of antral stomach cells assembled into bioengineered mini-organs in vitro yielded transplantable units that also suppressed hyperglycemia in diabetic mice, highlighting the potential for development of engineered stomach tissues as a renewable source of functional β cells for glycemic control.

For many years, stem cell metabolism was viewed as a byproduct of cell fate status rather than an active regulatory mechanism; however, there is now a growing appreciation that metabolic pathways influence epigenetic changes associated with lineage commitment, specification, and self-renewal. Here we review how metabolites generated during glycolytic and oxidative processes are utilized in enzymatic reactions leading to epigenetic modifications and transcriptional regulation. We discuss how "metabolic reprogramming" contributes to global epigenetic changes in the context of naive and primed pluripotent states, somatic reprogramming, and hematopoietic and skeletal muscle tissue stem cells, and we discuss the implications for regenerative medicine.

SummaryWe have recently demonstrated that reactive glial cells can be directly reprogrammed into functional neurons by a single neural transcription factor, NeuroD1. Here we report that a combination of small molecules can also reprogram human astrocytes in culture into fully functional neurons. We demonstrate that sequential exposure of human astrocytes to a cocktail of nine small molecules that inhibit glial but activate neuronal signaling pathways can successfully reprogram astrocytes into neurons in 8-10 days. This chemical reprogramming is mediated through epigenetic regulation and involves transcriptional activation of NEUROD1 and NEUROGENIN2. The human astrocyte-converted neurons can survive for >5 months in culture and form functional synaptic networks with synchronous burst activities. The chemically reprogrammed human neurons can also survive for >1 month in the mouse brain in vivo and integrate into local circuits. Our study opens a new avenue using chemical compounds to reprogram reactive glial cells into functional neurons.

SummaryAging is a major risk factor for many human diseases, and in vitro generation of human neurons is an attractive approach for modeling aging-related brain disorders. However, modeling aging in differentiated human neurons has proved challenging. We generated neurons from human donors across a broad range of ages, either by iPSC-based reprogramming and differentiation or by direct conversion into induced neurons (iNs). While iPSCs and derived neurons did not retain aging-associated gene signatures, iNs displayed age-specific transcriptional profiles and revealed age-associated decreases in the nuclear transport receptor RanBP17. We detected an age-dependent loss of nucleocytoplasmic compartmentalization (NCC) in donor fibroblasts and corresponding iNs and found that reduced RanBP17 impaired NCC in young cells, while iPSC rejuvenation restored NCC in aged cells. These results show that iNs retain important aging-related signatures, thus allowing modeling of the aging process in vitro, and they identify impaired NCC as an important factor in human aging.

Epigenetic and epitranscriptomic networks have important functions in maintaining the pluripotency of embryonic stem cells (ESCs) and somatic cell reprogramming. However, the mechanisms integrating the actions of these distinct networks are only partially understood. Here we show that the chromatin-associated zinc finger protein 217 (ZFP217) coordinates epigenetic and epitranscriptomic regulation. ZFP217 interacts with several epigenetic regulators, activates the transcription of key pluripotency genes, and modulates N6-methyladenosine (m(6)A) deposition on their transcripts by sequestering the enzyme m(6)A methyltransferase-like 3 (METTL3). Consistently, Zfp217 depletion compromises ESC self-renewal and somatic cell reprogramming, globally increases m(6)A RNA levels, and enhances m(6)A modification of the Nanog, Sox2, Klf4, and c-Myc mRNAs, promoting their degradation. ZFP217 binds its own target gene mRNAs, which are also METTL3 associated, and is enriched at promoters of m(6)A-modified transcripts. Collectively, these findings shed light on how a transcription factor can tightly couple gene transcription to m(6)A RNA modification to ensure ESC identity.

The National Heart, Lung, and Blood Institute's Next Generation Genetic Association Studies Consortium has used induced pluripotent stem cell technology to study the effects of common genetic variants in vitro. This issue of Cell Stem Cell and affiliated journals include several manuscripts describing the results of the consortium's efforts.

Although cellular reprogramming enables the generation of new cell types for disease modeling and regenerative therapies, reprogramming remains a rare cellular event. By examining reprogramming of fibroblasts into motor neurons and multiple other somatic lineages, we find that epigenetic barriers to conversion can be overcome by endowing cells with the ability to mitigate an inherent antagonism between transcription and DNA replication. We show that transcription factor overexpression induces unusually high rates of transcription and that sustaining hypertranscription and transgene expression in hyperproliferative cells early in reprogramming is critical for successful lineage conversion. However, hypertranscription impedes DNA replication and cell proliferation, processes that facilitate reprogramming. We identify a chemical and genetic cocktail that dramatically increases the number of cells capable of simultaneous hypertranscription and hyperproliferation by activating topoisomerases. Further, we show that hypertranscribing, hyperproliferating cells reprogram at 100-fold higher, near-deterministic rates. Therefore, relaxing biophysical constraints overcomes molecular barriers to cellular reprogramming.

Successful cloning of monkeys, the first non-human primate species, by somatic cell nuclear transfer (SCNT) attracted worldwide attention earlier this year. Remarkably, it has taken more than 20 years since the cloning of Dolly the sheep in 1997 to achieve this feat. This success was largely due to recent understanding of epigenetic barriers that impede SCNT-mediated reprogramming and the establishment of key methods to overcome these barriers, which also allowed efficient derivation of human pluripotent stem cells for cell therapy. Here, we summarize recent advances in SCNT technology and its potential applications for both reproductive and therapeutic cloning.

The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency.

Tissue regeneration requires dynamic cellular adaptation to the wound environment. It is currently unclear how this is orchestrated at the cellular level and how cell fate is affected by severe tissue damage. Here we dissect cell fate transitions during colonic regeneration in a mouse dextran sulfate sodium (DSS) colitis model, and we demonstrate that the epithelium is transiently reprogrammed into a primitive state. This is characterized by de novo expression of fetal markers as well as suppression of markers for adult stem and differentiated cells. The fate change is orchestrated by remodeling the extracellular matrix (ECM), increased FAK/Src signaling, and ultimately YAP/TAZ activation. In a defined cell culture system recapitulating the extracellular matrix remodeling observed in vivo, we show that a collagen 3D matrix supplemented with Wnt ligands is sufficient to sustain endogenous YAP/TAZ and induce conversion of cell fate. This provides a simple model for tissue regeneration, implicating cellular reprogramming as an essential element. The mechanism that governs tissue regeneration following severe damage to the colonic epithelium remains poorly understood. Jensen and colleagues show that the colonic epithelium undergoes a profound reprogramming into a more primitive state with fetal-like properties. Moreover, they demonstrate that YAP and TAZ operate as essential mechano-sensors during tissue reprogramming.

What exactly happens to chromatin as cells undergo an identity change? In this issue of Cell Stem Cell, Li et al. (2017) and Knaupp et al. (2017) offer new insights by mapping in exquisite detail chromatin accessibility dynamics as mouse embryonic fibroblasts are reprogrammed into induced pluripotent stem cells.

Direct cellular reprogramming provides a powerful platform to study cell plasticity and dissect mechanisms underlying cell fate determination. Here, we report a single-cell transcriptomic study of human cardiac (hiCM) reprogramming that utilizes an analysis pipeline incorporating current data normalization methods, multiple trajectory prediction algorithms, and a cell fate index calculation we developed to measure reprogramming progression. These analyses revealed hiCM reprogramming-specific features and a decision point at which cells either embark on reprogramming or regress toward their original fibroblast state. In combination with functional screening, we found that immune-response-associated DNA methylation is required for hiCM induction and validated several downstream targets of reprogramming factors as necessary for productive hiCM reprograming. Collectively, this single-cell transcriptomics study provides detailed datasets that reveal molecular features underlying hiCM determination and rigorous analytical pipelines for predicting cell fate conversion.

SummaryRecently, direct reprogramming between divergent lineages has been achieved by the introduction of regulatory transcription factors. This approach may provide alternative cell resources for drug discovery and regenerative medicine, but applications could be limited by the genetic manipulation involved. Here, we show that mouse fibroblasts can be directly converted into neuronal cells using only a cocktail of small molecules, with a yield of up to >90% being TUJ1-positive after 16 days of induction. After a further maturation stage, these chemically induced neurons (CiNs) possessed neuron-specific expression patterns, generated action potentials, and formed functional synapses. Mechanistically, we found that a BET family bromodomain inhibitor, I-BET151, disrupted the fibroblast-specific program, while the neurogenesis inducer ISX9 was necessary to activate neuron-specific genes. Overall, our findings provide a “proof of principle” for chemically induced direct reprogramming of somatic cell fates across germ layers without genetic manipulation, through disruption of cell-specific programs and induction of an alternative fate.