Asparagine Synthetase Marks a Distinct Dependency Threshold for Cardiomyocyte Dedifferentiation.

BACKGROUND: Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition. METHODS: Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte-targeted delivery of reprogramming factors (Oct4, Sox2, Klf4, and Myc) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD. RESULTS: RNA sequencing and integrated analysis identified Asparagine Synthetase (Asns) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although Asns overexpression in Oct4, Sox2, Klf4, and Myc cardiomyocytes augmented hallmarks of CMDD, Asns deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in Oct4, Sox2, Klf4, and Myc cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway. CONCLUSIONS: We discovered a novel gene Asns as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an Asns/mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration.

The homeobox transcription factor DUXBL controls exit from totipotency.

In mice, exit from the totipotent two-cell (2C) stage embryo requires silencing of the 2C-associated transcriptional program. However, the molecular mechanisms involved in this process remain poorly understood. Here we demonstrate that the 2C-specific transcription factor double homeobox protein (DUX) mediates an essential negative feedback loop by inducing the expression of DUXBL to promote this silencing. We show that DUXBL gains accessibility to DUX-bound regions specifically upon DUX expression. Furthermore, we determine that DUXBL interacts with TRIM24 and TRIM33, members of the TRIM superfamily involved in gene silencing, and colocalizes with them in nuclear foci upon DUX expression. Importantly, DUXBL overexpression impairs 2C-associated transcription, whereas Duxbl inactivation in mouse embryonic stem cells increases DUX-dependent induction of the 2C-transcriptional program. Consequently, DUXBL deficiency in embryos results in sustained expression of 2C-associated transcripts leading to early developmental arrest. Our study identifies DUXBL as an essential regulator of totipotency exit enabling the first divergence of cell fates.

Inducing Pluripotency in Somatic Cells: Historical Perspective and Recent Advances.

Inducing pluripotency in somatic cells is mediated by the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc. The resulting induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine by virtue of their ability to differentiate into different types of functional cells. Specifically, iPSCs derived directly from patients offer a powerful platform for creating in vitro disease models. This facilitates elucidation of pathological mechanisms underlying human diseases and development of new therapeutic agents mitigating disease phenotypes. Furthermore, genetically and phenotypically corrected patient-derived iPSCs by gene-editing technology or the supply of specific pharmaceutical agents can be used for preclinical and clinical trials to investigate their therapeutic potential. Despite great advances in developing reprogramming methods, the efficiency of iPSC generation remains still low and varies between donor cell types, hampering the potential application of iPSC technology. This paper reviews histological timeline showing important discoveries that have led to iPSC generation and discusses recent advances in iPSC technology by highlighting donor cell types employed for iPSC generation.

Antagonism among DUX family members evolved from an ancestral toxic single homeodomain protein.

Double homeobox (DUX) genes are unique to eutherian mammals, expressed transiently during zygotic genome activation (ZGA) and involved in facioscapulohumeral muscular dystrophy (FSHD) and cancer when misexpressed. We evaluate the 3 human DUX genes and the ancestral single homeobox gene sDUX from the non-eutherian mammal, platypus, and find that DUX4 cytotoxicity is not shared with DUXA or DUXB, but surprisingly is shared with platypus sDUX, which binds DNA as a homodimer and activates numerous ZGA genes and long terminal repeat (LTR) elements. DUXA, although transcriptionally inactive, has DNA binding overlap with DUX4, and DUXA-VP64 activates DUX4 targets and is cytotoxic. DUXA competition antagonizes the activity of DUX4 on its target genes, including in FSHD patient cells. Since DUXA is a DUX4 target gene, this competition potentiates feedback inhibition, constraining the window of DUX4 activity. The DUX gene family therefore comprises antagonistic members of opposing function, with implications for their roles in ZGA, FSHD, and cancer.

Antagonism among DUX family members evolved from an ancestral toxic single homeodomain protein.

Double homeobox (DUX) genes are unique to eutherian mammals and normally expressed transiently during zygotic genome activation. The canonical member, DUX4, is involved in facioscapulohumeral muscular dystrophy (FSHD) and cancer, when misexpressed in other contexts. We evaluate the 3 human DUX genes and the ancestral single homeobox gene sDUX from the non-eutherian mammal, platypus, and find that DUX4 activities are not shared with DUXA or DUXB, which lack transcriptional activation potential, but surprisingly are shared with platypus sDUX. In human myoblasts, platypus sDUX drives cytotoxicity, inhibits myogenesis, and induces DUX4 target genes, particularly those associated with zygotic genome activation (ZGA), by binding DNA as a homodimer in a way that overlaps the DUX4 homeodomain crystal structure. DUXA lacks transcriptional activity but has DNA-binding and chromatin accessibility overlap with DUX4 and sDUX, including on ZGA genes and LTR elements, and can actually be converted into a DUX4-like cytotoxic factor by fusion to a synthetic transactivation domain. DUXA competition antagonizes the activity of DUX4 on its target genes, including in FSHD patient cells. Since DUXA is an early DUX4 target gene, this activity potentiates feedback inhibition, constraining the window of DUX4 activity. The DUX gene family therefore comprises cross-regulating members of opposing function, with implications for their roles in ZGA, FSHD, and cancer. HIGHLIGHTS: Platypus sDUX is toxic and inhibits myogenic differentiation.DUXA targets overlap substantially with those of DUX4.DUXA fused to a synthetic transactivation domain acquires DUX4-like toxicity.DUXA behaves as a competitive inhibitor of DUX4.

Generation of Induced Pluripotent Stem Cells from Lymphoblastoid Cell Lines by Electroporation of Episomal Vectors.

Background and Objectives: Lymphoblastoid cell lines (LCLs) deposited from disease-affected individuals could be a valuable donor cell source for generating disease-specific induced pluripotent stem cells (iPSCs). However, generation of iPSCs from the LCLs is still challenging, as yet no effective gene delivery strategy has been developed. Methods and Results: Here, we reveal an effective gene delivery method specifically for LCLs. We found that LCLs appear to be refractory toward retroviral and lentiviral transduction. Consequently, lentiviral and retroviral transduction of OCT4, SOX2, KFL4 and c-MYC into LCLs does not elicit iPSC colony formation. Interestingly, however we found that transfection of oriP/EBNA-1-based episomal vectors by electroporation is an efficient gene delivery system into LCLs, enabling iPSC generation from LCLs. These iPSCs expressed pluripotency makers (OCT4, NANOG, SSEA4, SALL4) and could form embryoid bodies. Conclusions: Our data show that electroporation is an effective gene delivery method with which LCLs can be efficiently reprogrammed into iPSCs.

Evaluating the Feasibility and Acceptability of an Artificial-Intelligence-Enabled and Speech-Based Distress Screening Mobile App for Adolescents and Young Adults Diagnosed with Cancer: A Study Protocol.

Adolescents and young adults (AYAs) diagnosed with cancer are an age-defined population, with studies reporting up to 45% of the population experiencing psychological distress. Although it is essential to screen and monitor for psychological distress throughout AYAs' cancer journeys, many cancer centers fail to effectively implement distress screening protocols largely due to busy clinical workflow and survey fatigue. Recent advances in mobile technology and speech science have enabled flexible and engaging methods to monitor psychological distress. However, patient-centered research focusing on these methods' feasibility and acceptability remains lacking. Therefore, in this project, we aim to evaluate the feasibility and acceptability of an artificial intelligence (AI)-enabled and speech-based mobile application to monitor psychological distress among AYAs diagnosed with cancer. We use a single-arm prospective cohort design with a stratified sampling strategy. We aim to recruit 60 AYAs diagnosed with cancer and to monitor their psychological distress using an AI-enabled speech-based distress monitoring tool over a 6 month period. The primary feasibility endpoint of this study is defined by the number of participants completing four out of six monthly distress assessments, and the acceptability endpoint is defined both quantitatively using the acceptability of intervention measure and qualitatively using semi-structured interviews.

Reversible reprogramming of cardiomyocytes to a fetal state drives heart regeneration in mice.

Cardiomyocyte (CM) replacement is very slow in adult mammalian hearts, preventing regeneration of damaged myocardium. By contrast, fetal hearts display considerable regenerative potential owing to the presence of less mature CMs that still have the ability to proliferate. In this study, we demonstrate that heart-specific expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) induces adult CMs to dedifferentiate, conferring regenerative capacity to adult hearts. Transient, CM-specific expression of OSKM extends the regenerative window for postnatal mouse hearts and induces a gene expression program in adult CMs that resembles that of fetal CMs. Extended expression of OSKM in CMs leads to cellular reprogramming and heart tumor formation. Short-term OSKM expression before and during myocardial infarction ameliorates myocardial damage and improves cardiac function, demonstrating that temporally controlled dedifferentiation and reprogramming enable cell cycle reentry of mammalian CMs and facilitate heart regeneration.

CHD4 ensures stem cell lineage fidelity during skeletal muscle regeneration.

Regeneration of skeletal muscle requires resident stem cells called satellite cells. Here, we report that the chromatin remodeler CHD4, a member of the nucleosome remodeling and deacetylase (NuRD) repressive complex, is essential for the expansion and regenerative functions of satellite cells. We show that conditional deletion of the Chd4 gene in satellite cells results in failure to regenerate muscle after injury. This defect is principally associated with increased stem cell plasticity and lineage infidelity during the expansion of satellite cells, caused by de-repression of non-muscle-cell lineage genes in the absence of Chd4. Thus, CHD4 ensures that a transcriptional program that safeguards satellite cell identity during muscle regeneration is maintained. Given the therapeutic potential of muscle stem cells in diverse neuromuscular pathologies, CHD4 constitutes an attractive target for satellite cell-based therapies.

Biological importance of OCT transcription factors in reprogramming and development.

Ectopic expression of Oct4, Sox2, Klf4 and c-Myc can reprogram somatic cells into induced pluripotent stem cells (iPSCs). Attempts to identify genes or chemicals that can functionally replace each of these four reprogramming factors have revealed that exogenous Oct4 is not necessary for reprogramming under certain conditions or in the presence of alternative factors that can regulate endogenous Oct4 expression. For example, polycistronic expression of Sox2, Klf4 and c-Myc can elicit reprogramming by activating endogenous Oct4 expression indirectly. Experiments in which the reprogramming competence of all other Oct family members tested and also in different species have led to the decisive conclusion that Oct proteins display different reprogramming competences and species-dependent reprogramming activity despite their profound sequence conservation. We discuss the roles of the structural components of Oct proteins in reprogramming and how donor cell epigenomes endow Oct proteins with different reprogramming competences.

Donor cell memory confers a metastable state of directly converted cells.

Generation of induced oligodendrocyte progenitor cells (iOPCs) from somatic fibroblasts is a strategy for cell-based therapy of myelin diseases. However, iOPC generation is inefficient, and the resulting iOPCs exhibit limited expansion and differentiation competence. Here we overcome these limitations by transducing an optimized transcription factor combination into a permissive donor phenotype, the pericyte. Pericyte-derived iOPCs (PC-iOPCs) are stably expandable and functionally myelinogenic with high differentiation competence. Unexpectedly, however, we found that PC-iOPCs are metastable so that they can produce myelination-competent oligodendrocytes or revert to their original identity in a context-dependent fashion. Phenotypic reversion of PC-iOPCs is tightly linked to memory of their original transcriptome and epigenome. Phenotypic reversion can be disconnected from this donor cell memory effect, and in vivo myelination can eventually be achieved by transplantation of O4+ pre-oligodendrocytes. Our data show that donor cell source and memory can contribute to the fate and stability of directly converted cells.

Directed Evolution of an Enhanced POU Reprogramming Factor for Cell Fate Engineering.

Transcription factor-driven cell fate engineering in pluripotency induction, transdifferentiation, and forward reprogramming requires efficiency, speed, and maturity for widespread adoption and clinical translation. Here, we used Oct4, Sox2, Klf4, and c-Myc driven pluripotency reprogramming to evaluate methods for enhancing and tailoring cell fate transitions, through directed evolution with iterative screening of pooled mutant libraries and phenotypic selection. We identified an artificially evolved and enhanced POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of reprogramming speed and efficiency. In contrast to Oct4, not only can ePOU induce pluripotency with Sox2 alone, but it can also do so in the absence of Sox2 in a three-factor ePOU/Klf4/c-Myc cocktail. Biochemical assays combined with genome-wide analyses showed that ePOU possesses a new preference to dimerize on palindromic DNA elements. Yet, the moderate capacity of Oct4 to function as a pioneer factor, its preference to bind octamer DNA and its capability to dimerize with Sox2 and Sox17 proteins remain unchanged in ePOU. Compared with Oct4, ePOU is thermodynamically stabilized and persists longer in reprogramming cells. In consequence, ePOU: 1) differentially activates several genes hitherto not implicated in reprogramming, 2) reveals an unappreciated role of thyrotropin-releasing hormone signaling, and 3) binds a distinct class of retrotransposons. Collectively, these features enable ePOU to accelerate the establishment of the pluripotency network. This demonstrates that the phenotypic selection of novel factor variants from mammalian cells with desired properties is key to advancing cell fate conversions with artificially evolved biomolecules.

Permissive epigenomes endow reprogramming competence to transcriptional regulators.

Identifying molecular and cellular processes that regulate reprogramming competence of transcription factors broadens our understanding of reprogramming mechanisms. In the present study, by a chemical screen targeting major epigenetic pathways in human reprogramming, we discovered that inhibiting specific epigenetic roadblocks including disruptor of telomeric silencing 1-like (DOT1L)-mediated H3K79/K27 methylation, but also other epigenetic pathways, catalyzed by lysine-specific histone demethylase 1A, DNA methyltransferases and histone deacetylases, allows induced pluripotent stem cell generation with almost all OCT factors. We found that simultaneous inhibition of these pathways not only dramatically enhances reprogramming competence of most OCT factors, but in fact enables dismantling of species-dependent reprogramming competence of OCT6, NR5A1, NR5A2, TET1 and GATA3. Harnessing these induced permissive epigenetic states, we performed an additional screen with 98 candidate genes. Thereby, we identified 25 transcriptional regulators (OTX2, SIX3, and so on) that can functionally replace OCT4 in inducing pluripotency. Our findings provide a conceptual framework for understanding how transcription factors elicit reprogramming in dependency of the donor cell epigenome that differs across species.

Reprogramming competence of OCT factors is determined by transactivation domains.

OCT4 (also known as POU5F1) plays an essential role in reprogramming. It is the only member of the POU (Pit-Oct-Unc) family of transcription factors that can induce pluripotency despite sharing high structural similarities to all other members. Here, we discover that OCT6 (also known as POU3F1) can elicit reprogramming specifically in human cells. OCT6-based reprogramming does not alter the mesenchymal-epithelial transition but is attenuated through the delayed activation of the pluripotency network in comparison with OCT4-based reprogramming. Creating a series of reciprocal domain-swapped chimeras and mutants across all OCT factors, we clearly delineate essential elements of OCT4/OCT6-dependent reprogramming and, conversely, identify the features that prevent induction of pluripotency by other OCT factors. With this strategy, we further discover various chimeric proteins that are superior to OCT4 in reprogramming. Our findings clarify how reprogramming competences of OCT factors are conferred through their structural components.

Generation of a human iPSC line (MPIi007-A) from a patient with Metachromatic leukodystrophy.

Here we have generated a human induced pluripotent stem cells (hiPSC) line (MPIi007-A) from skin fibroblasts of a 4-year-old male Metachromatic leukodystrophy (MLD) patient with a heterozygous 1178C > G (Thr393Ser) mutation in arylsulfatase A (ARSA) gene via retroviral expression of OCT4, SOX2, KLF4 and c-MYC. The MPIi007-A iPSC line displayed typical embryonic stem cell-like morphology, carried the ARSA gene mutation, expressed several pluripotent stem cell makers, retained normal karyotype (46, XY) and were capable of forming teratomas containing three germ layers. The MPIi007-A line can be used for the characterization of MLD-associated pathomechanisms and developing new therapeutic options.

ATAC-seq footprinting unravels kinetics of transcription factor binding during zygotic genome activation.

While footprinting analysis of ATAC-seq data can theoretically enable investigation of transcription factor (TF) binding, the lack of a computational tool able to conduct different levels of footprinting analysis has so-far hindered the widespread application of this method. Here we present TOBIAS, a comprehensive, accurate, and fast footprinting framework enabling genome-wide investigation of TF binding dynamics for hundreds of TFs simultaneously. We validate TOBIAS using paired ATAC-seq and ChIP-seq data, and find that TOBIAS outperforms existing methods for bias correction and footprinting. As a proof-of-concept, we illustrate how TOBIAS can unveil complex TF dynamics during zygotic genome activation in both humans and mice, and propose how zygotic Dux activates cascades of TFs, binds to repeat elements and induces expression of novel genetic elements.

Attenuated Epigenetic Suppression of Muscle Stem Cell Necroptosis Is Required for Efficient Regeneration of Dystrophic Muscles.

Somatic stem cells expand massively during tissue regeneration, which might require control of cell fitness, allowing elimination of non-competitive, potentially harmful cells. How or if such cells are removed to restore organ function is not fully understood. Here, we show that a substantial fraction of muscle stem cells (MuSCs) undergo necroptosis because of epigenetic rewiring during chronic skeletal muscle regeneration, which is required for efficient regeneration of dystrophic muscles. Inhibition of necroptosis strongly enhances suppression of MuSC expansion in a non-cell-autonomous manner. Prevention of necroptosis in MuSCs of healthy muscles is mediated by the chromatin remodeler CHD4, which directly represses the necroptotic effector Ripk3, while CHD4-dependent Ripk3 repression is dramatically attenuated in dystrophic muscles. Loss of Ripk3 repression by inactivation of Chd4 causes massive necroptosis of MuSCs, abolishing regeneration. Our study demonstrates how programmed cell death in MuSCs is tightly controlled to achieve optimal tissue regeneration.

Keeping Fibrotic Responses in Contractile Tissues at Bay: The Plot t(Hic1)ens.

Two recent papers in Cell Stem Cell by Soliman et al. (2020) and Scott et al. (2019) explore the contributions of mesenchymal progenitor cells to fibrosis in heart and skeletal muscle. They find tissue-dependent roles for a subpopulation of mesenchymal progenitors expressing Hic1 with varied differentiation capacities and pathophysiologic contributions.

Research and therapy with induced pluripotent stem cells (iPSCs): social, legal, and ethical considerations.

Induced pluripotent stem cells (iPSCs) can self-renew indefinitely in culture and differentiate into all specialized cell types including gametes. iPSCs do not exist naturally and are instead generated ("induced" or "reprogrammed") in culture from somatic cells through ectopic co-expression of defined pluripotency factors. Since they can be generated from any healthy person or patient, iPSCs are considered as a valuable resource for regenerative medicine to replace diseased or damaged tissues. In addition, reprogramming technology has provided a powerful tool to study mechanisms of cell fate decisions and to model human diseases, thereby substantially potentiating the possibility to (i) discover new drugs in screening formats and (ii) treat life-threatening diseases through cell therapy-based strategies. However, various legal and ethical barriers arise when aiming to exploit the full potential of iPSCs to minimize abuse or unauthorized utilization. In this review, we discuss bioethical, legal, and societal concerns associated with research and therapy using iPSCs. Furthermore, we present key questions and suggestions for stem cell scientists, legal authorities, and social activists investigating and working in this field.

Connect-four: genomic analyses of regenerating stem cells identifies zygotic Dux factors as tumor initiators.

How, if and in which cell types embryonic gene expression programs are elicited to induce tumor formation remains poorly understood. Through genomic analyses of regenerating, p53 deficient muscle stem cells we identified various oncogenomic amplifications, including but not limited to, the zygotic transcription factor Duxbl/DUXB to initiate tumorigenic transformation.