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The genome is intricately folded into chromatin compartments, topologically associating domains (TADs) and loops unique to each cell type. How this higher-order genome organization regulates cell fate transition remains elusive. Here we show how a single non-neural progenitor transcription factor, PTF1A, reorchestrates the 3D genome during fibroblast transdifferentiation into neural stem cells (NSCs). Multiomics analyses integrating Hi-C data, PTF1A and CTCF DNA-binding profiles, H3K27ac modification, and gene expression, demonstrate that PTF1A binds to subTAD boundaries subsequently associated with elevated CTCF binding and enhanced boundary insulation, and reorganizes chromatin loops, leading to gene expression changes that drive transdifferentiation into NSCs. Moreover, PTF1A activates enhancers and super-enhancers near low-insulation boundaries and modulates H3K27ac deposition, promoting cell fate transitions. Together, our data implicate an involvement of 3D genome in transcriptional and cell fate alterations, and highlight an essential role for PTF1A in gene expression control and multiscale 3D genome remodeling during cell reprogramming., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The mechanisms by which oncogenic mutations and anatomical locations work together to influence the immune environment within tumors are not well understood. In this issue of Immunity, Ross et al. show that H3.3K27M diffuse midline gliomas (DMGs) are enriched with disease-associated myeloid cells (DAMs). Myeloid-targeted strategies reprogram DAMs to a homeostatic state, reduce myeloid infiltration into tumors, and prolong survival., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Direct lineage reprogramming into dopaminergic (DA) neurons holds great promise for the more effective production of DA neurons, offering potential therapeutic benefits for conditions such as Parkinson's disease. However, the reprogramming pathway for fully reprogrammed DA neurons remains largely unclear, resulting in immature and dead-end states with low efficiency. In this study, using single-cell RNA sequencing, the trajectory of reprogramming DA neurons at multiple time points, identifying a continuous pathway for their reprogramming is analyzed. It is identified that intermediate cell populations are crucial for resetting host cell fate during early DA neuronal reprogramming. Further, longitudinal dissection uncovered two distinct trajectories: one leading to successful reprogramming and the other to a dead end. Notably, Arid4b, a histone modifier, as a crucial regulator at this branch point, essential for the successful trajectory and acquisition of mature dopaminergic neuronal identity is identified. Consistently, overexpressing Arid4b in the DA neuronal reprogramming process increases the yield of iDA neurons and effectively reverses the disease phenotypes observed in the PD mouse brain. Thus, gaining insights into the cellular trajectory holds significant importance for devising regenerative medicine strategies, particularly in the context of addressing neurodegenerative disorders like Parkinson's disease., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Dynamic alterations in cellular phenotypes during cancer progression are attributed to a phenomenon known as 'lineage plasticity'. This process is associated with therapeutic resistance and involves concurrent shifts in metabolic states that facilitate adaptation to various stressors inherent in malignant growth. Certain metabolites also serve as synthetic reservoirs for chromatin modification, thus linking metabolic states with epigenetic regulation. There remains a critical need to understand the mechanisms that converge on lineage plasticity and metabolic reprogramming to prevent the emergence of lethal disease. This review attempts to offer an overview of our current understanding of the interplay between metabolic reprogramming and lineage plasticity in the context of cancer, highlighting the intersecting drivers of cancer hallmarks, with an emphasis on solid tumors., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cartilage rarely heals spontaneously once damaged. Osteoarthritis (OA) is the most common degenerative joint disease among the elderly; however, effective treatment for OA is currently lacking. Autologous chondrocyte implantation (ACI), an innovative regenerative technology involving the implantation of healthy chondrocytes, may restore damaged lesions. Chondrocytes for ACI may potentially be induced from differentiated somatic cells using retrovirus (RV)-mediated transduction of three reprogramming factors (SOX9, KLF4, and c-MYC). However, the efficiency of the current induction system needs to be improved and the safety issues arising from the genomic integration of the vector DNA have to be addressed. To solve these problems, we used an RNA vector, termed the replication-defective and persistent Sendai virus vector (SeVdp), to express reprogramming factors for chondrocyte induction. Our results showed that the SeVdp-based vector induced chondrocytes more efficiently than the RV vector, probably because of robust and rapid expression of the transgenes, without any apparent integration of the SeVdp vector. The induced chondrocytes formed cartilage-like tissues when injected subcutaneously into mice. Thus, the SeVdp-based system for inducing chondrocytes may act as a foundation for developing safer and more effective treatments for damaged cartilage., (© 2024. The Author(s).)

External constraints, such as development, disease, and environment, can induce changes in epigenomic patterns that may profoundly impact the health trajectory of fetuses and neonates into adulthood, influencing conditions like obesity. Epigenetic modifications encompass processes including DNA methylation, covalent histone modifications, and RNA-mediated regulation. Beyond forward cellular differentiation (cell programming), terminally differentiated cells are reverted to a pluripotent or even totipotent state, that is, cellular reprogramming. Epigenetic modulators facilitate or erase histone and DNA modifications both in vivo and in vitro during programming and reprogramming. Noticeably, obesity is a complex metabolic disorder driven by both genetic and environmental factors. Increasing evidence suggests that epigenetic modifications play a critical role in the regulation of gene expression involved in adipogenesis, energy homeostasis, and metabolic pathways. Hence, we discuss the mechanisms by which epigenetic interventions influence obesity, focusing on DNA methylation, histone modifications, and non-coding RNAs. We also analyze the methodologies that have been pivotal in uncovering these epigenetic regulations, i.e., Large-scale screening has been instrumental in identifying genes and pathways susceptible to epigenetic control, particularly in the context of adipogenesis and metabolic homeostasis; Single-cell RNA sequencing (scRNA-seq) provides a high-resolution view of gene expression patterns at the individual cell level, revealing the heterogeneity and dynamics of epigenetic regulation during cellular differentiation and reprogramming; Chromatin immunoprecipitation (ChIP) assays, focused on candidate genes, have been crucial for characterizing histone modifications and transcription factor binding at specific genomic loci, thereby elucidating the epigenetic mechanisms that govern cellular programming; Somatic cell nuclear transfer (SCNT) and cell fusion techniques have been employed to study the epigenetic reprogramming accompanying cloning and the generation of hybrid cells with pluripotent characteristics, etc. These approaches have been instrumental in identifying specific epigenetic marks and pathways implicated in obesity, providing a foundation for developing targeted therapeutic interventions. Understanding the dynamic interplay between epigenetic regulation and cellular programming is crucial for advancing mechanism and clinical management of obesity., (© 2024. The Author(s).)

Overexpression of cardiac reprogramming factors, including GATA4, HAND2, TBX5, and MEF2C (GHT/M), can directly reprogram cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs). Adeno-associated virus (AAV) vectors are widely used clinically, and vectors targeting cardiomyocytes (CMs) have been extensively studied. However, safe and efficient AAV vectors targeting CFs for in vivo cardiac reprogramming remain elusive. Therefore, we screened multiple AAV capsids and promoters to develop efficient and safe CF-targeting AAV vectors for in vivo cardiac reprogramming. AAV-DJ capsids containing periostin promoter (AAV-DJ-Postn) strongly and specifically expressed transgenes in resident CFs in mice after myocardial infarction (MI). Lineage tracing revealed that AAV-DJ-Postn vectors expressing GHT/M reprogrammed CFs into iCMs, which was further increased 2-fold using activated MEF2C via the fusion of the powerful MYOD transactivation domain (M-TAD) with GHT (AAV-DJ-Postn-GHT/M-TAD). AAV-DJ-Postn-GHT/M-TAD injection improved cardiac function and reduced fibrosis after MI. Overall, we developed new AAV vectors that target CFs for cardiac reprogramming., Competing Interests: Declaration of interests M. Ieda holds a patent related to this work, U.S. Patent 9,517,250, entitled ‘‘Methods for Generating Cardiomyocytes,” issued on October 19, 2012., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ectopic expression of lineage-specific transcription factors (TFs) of another cell type can induce cell fate reprogramming. However, the heterogeneity of reprogramming cells has been a challenge for data interpretation and model evaluation. Here, we present a protocol to characterize cells expressing defined factors during direct cell reprogramming using a factor-indexing approach based on single-nuclei multiome sequencing (FI-snMultiome-seq). We describe the steps for barcoding TFs, converting human fibroblasts to pancreatic ductal-like cells using defined TFs, and preparing library for FI-snMultiome-seq analysis. For complete details on the use and execution of this protocol, please refer to Fei et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: iPSC reprogramming technology exhibits significant promise in the realms of clinical therapeutics, disease modeling, pharmaceutical drug discovery, and various other applications. However, the extensive utilization of this technology has encountered impediments in the form of inefficiency, prolonged procedures, and ambiguous biological processes. Consequently, in order to improve this technology, it is of great significance to delve into the underlying mechanisms involved in iPSC reprogramming. The BET protein BRD4 plays a crucial role in the late stage of reprogramming; however, its precise function in the early stage remains unclear., Results: Our study aims to investigate BRD4's role in the early stages of iPSC reprogramming. Our investigation reveals that early inhibition of BRD4 substantially enhances iPSC reprogramming, whereas its implementation during the middle-late stage impedes the process. During the reprogramming, ribosome DNA expression initially increases before decreasing and then gradually recovers. Early inhibition of BRD4 improved the decline and restoration of rDNA expression in the early and middle-late stages, respectively. Additionally, we uncovered the mechanism of BRD4's regulation of rDNA transcription throughout reprogramming. Specifically, BRD4 interacts with UBF and co-localizes to both the rDNA promoter and enhancer regions. Ultimately, BRD4 facilitates rDNA transcription by promoting the enrichment of histone H3 lysine 27 acetylation in the surrounding chromatin. Moreover, we also discovered that early inhibition of BRD4 facilitates cells' transition out of the somatic cell state and activate pluripotent genes., Conclusions: In conclusion, our results demonstrate that early inhibition of BRD4 promotes sequential dynamic expression of rDNA, which improves iPSC reprogramming efficiency., (© 2024. The Author(s).)

Direct neuronal reprogramming is a promising approach to replace neurons lost due to disease via the conversion of endogenous glia reacting to brain injury into neurons. However, it is essential to demonstrate that the newly generated neurons originate from glial cells and/or show that they are not pre-existing endogenous neurons. Here, we use controls for both requirements while comparing two viral vector systems (Mo-MLVs and AAVs) for the expression of the same neurogenic factor, the phosphorylation-resistant form of Neurogenin2. Our results show that Mo-MLVs targeting proliferating glial cells after traumatic brain injury reliably convert astrocytes into neurons, as assessed by genetic fate mapping of astrocytes. Conversely, expressing the same neurogenic factor in a flexed AAV system results in artefactual labelling of endogenous neurons fatemapped by birthdating in development that are negative for the genetic fate mapping marker induced in astrocytes. These results are further corroborated by chronic live in vivo imaging. Taken together, the phosphorylation-resistant form of Neurogenin2 is more efficient in reprogramming reactive glia into neurons than its wildtype counterpart in vivo using retroviral vectors (Mo-MLVs) targeting proliferating glia. Conversely, AAV-mediated expression generates artefacts and is not sufficient to achieve fate conversion.

Recent advances in single-cell sequencing technology have revolutionized our ability to acquire whole transcriptome data. However, uncovering the underlying transcriptional drivers and nonequilibrium driving forces of cell function directly from these data remains challenging. We address this by learning cell state vector fields from discrete single-cell RNA velocity to quantify the single-cell global nonequilibrium driving forces as landscape and flux. From single-cell data, we quantified the Waddington landscape, showing that optimal paths for differentiation and reprogramming deviate from the naively expected landscape gradient paths and may not pass through landscape saddles at finite fluctuations, challenging conventional transition state estimation of kinetic rate for cell fate decisions due to the presence of the flux. A key insight from our study is that stem/progenitor cells necessitate greater energy dissipation for rapid cell cycles and self-renewal, maintaining pluripotency. We predict optimal developmental pathways and elucidate the nucleation mechanism of cell fate decisions, with transition states as nucleation sites and pioneer genes as nucleation seeds. The concept of loop flux quantifies the contributions of each cycle flux to cell state transitions, facilitating the understanding of cell dynamics and thermodynamic cost, and providing insights into optimizing biological functions. We also infer cell-cell interactions and cell-type-specific gene regulatory networks, encompassing feedback mechanisms and interaction intensities, predicting genetic perturbation effects on cell fate decisions from single-cell omics data. Essentially, our methodology validates the landscape and flux theory, along with its associated quantifications, offering a framework for exploring the physical principles underlying cellular differentiation and reprogramming and broader biological processes through high-throughput single-cell sequencing experiments., Competing Interests: Competing interests statement:The authors declare no competing interest.

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) requires activation of the pluripotency network and resetting of the epigenome by erasing the epigenetic memory of the somatic state. In female mouse cells, a critical epigenetic reprogramming step is the reactivation of the inactive X chromosome. Despite its importance, a systematic understanding of the regulatory networks linking pluripotency and X-reactivation is missing. Here, we reveal important pathways for pluripotency acquisition and X-reactivation using a genome-wide CRISPR screen during neural precursor to iPSC reprogramming. In particular, we discover that activation of the interferon γ (IFNγ) pathway early during reprogramming accelerates pluripotency acquisition and X-reactivation. IFNγ stimulates STAT3 signaling and the pluripotency network and leads to enhanced TET-mediated DNA demethylation, which consequently boosts X-reactivation. We therefore gain a mechanistic understanding of the role of IFNγ in reprogramming and X-reactivation and provide a comprehensive resource of the molecular networks involved in these processes.

The extensive degeneration of functional somatic cells and the depletion of endogenous stem/progenitor populations present significant challenges to tissue regeneration in degenerative diseases. Currently, a cellular reprogramming approach enabling directly generating corresponding progenitor populations from degenerative somatic cells remains elusive. The present study focused on intervertebral disc degeneration (IVDD) and identified a three-factor combination (OCT4, FOXA2, TBXT [OFT]) that could induce the dedifferentiation-like reprogramming of degenerative nucleus pulposus cells (dNPCs) toward induced notochordal-like cells (iNCs). Single-cell transcriptomics dissected the transitions of cell identity during reprogramming. Further, OCT4 was found to directly interact with bromodomain PHD-finger transcription factor to remodel the chromatin during the early phases, which was crucial for initiating this dedifferentiation-like reprogramming. In rat models, intradiscal injection of adeno-associated virus carrying OFT generated iNCs from in situ dNPCs and reversed IVDD. These results collectively present a proof-of-concept for dedifferentiation-like reprogramming of degenerated somatic cells into corresponding progenitors through the development of a factor-based strategy, providing a promising approach for regeneration in degenerative disc diseases., Competing Interests: Declaration of interests All authors in this work declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Late-onset Alzheimer's disease (LOAD) is the most common form of Alzheimer's disease (AD). However, modeling sporadic LOAD that endogenously captures hallmark neuronal pathologies such as amyloid-β (Aβ) deposition, tau tangles, and neuronal loss remains an unmet need. We demonstrate that neurons generated by microRNA (miRNA)-based direct reprogramming of fibroblasts from individuals affected by autosomal dominant AD (ADAD) and LOAD in a three-dimensional environment effectively recapitulate key neuropathological features of AD. Reprogrammed LOAD neurons exhibit Aβ-dependent neurodegeneration, and treatment with β- or γ-secretase inhibitors before (but not subsequent to) Aβ deposit formation mitigated neuronal death. Moreover inhibiting age-associated retrotransposable elements in LOAD neurons reduced both Aβ deposition and neurodegeneration. Our study underscores the efficacy of modeling late-onset neuropathology of LOAD through high-efficiency miRNA-based neuronal reprogramming.

Mammalian cochlear hair cells (HCs) are essential for hearing, and damage to HCs results in severe hearing impairment. Damaged HCs can be regenerated by neighboring supporting cells (SCs), thus the functional regeneration of HCs is the main goal for the restoration of auditory function in vivo. Here, cochlear SC trans-differentiation into outer and inner HC by the induced expression of the key transcription factors Atoh1 and its co-regulators Gfi1, Pou4f3, and Six1 (GPAS), which are necessary for SCs that are destined for HC development and maturation via the AAV-ie targeting the inner ear stem cells are successfully achieved. Single-cell nuclear sequencing and lineaging tracing results showed that the majority of new Atoh1-derived HCs are in a state of initiating differentiation, while GP (Gfi1, Pou4f3) and GPS (Gfi1, Pou4f3, and Six1) enhanced the Atoh1-induced new HCs into inner and outer HCs. Moreover, the patch-clamp analysis indicated that newborn inner HCs induced by GPAS forced expression have similar electrophysiological characteristics to those of native inner HCs. Also, GPAS can induce HC regeneration in the HC-damaged mice model. In summary, the study demonstrates that AAV-mediated co-regulation of multiple genes, such as GPAS, is an effective means to achieve functional HC regeneration in the mouse cochlea., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

The transformation from a fibroblast mesenchymal cell state to an epithelial-like state is critical for induced pluripotent stem cell (iPSC) reprogramming. In this report, we describe studies with PFI-3, a small-molecule inhibitor that specifically targets the bromodomains of SMARCA2/4 and PBRM1 subunits of SWI/SNF complex, as an enhancer of iPSC reprogramming efficiency. Our findings reveal that PFI-3 induces cellular plasticity in multiple human dermal fibroblasts, leading to a mesenchymal-epithelial transition during iPSC formation. This transition is characterized by the upregulation of E-cadherin expression, a key protein involved in epithelial cell adhesion. Additionally, we identified COL11A1 as a reprogramming barrier and demonstrated COL11A1 knockdown increased reprogramming efficiency. Notably, we found that PFI-3 significantly reduced the expression of numerous extracellular matrix (ECM) genes, particularly those involved in collagen assembly. Our research provides key insights into the early stages of iPSC reprogramming, highlighting the crucial role of ECM changes and cellular plasticity in this process., (Published by Oxford University Press 2024.)

Critical reprogramming factors resided predominantly in the oocyte or male pronucleus can enhance the efficiency or the quality of induced pluripotent stem cells (iPSCs) induction. However, few reprogramming factors exist in the male pronucleus had been verified. Here, we demonstrated that granulin (Grn), a factor enriched specifically in male pronucleus, can significantly improve the generation of iPSCs from mouse fibroblasts. Grn is highly expressed on Day 1, Day 3, Day 14 of reprogramming induced by four Yamanaka factors and functions at the initial stage of reprogramming. Transcriptome analysis indicates that Grn can promote the expression of lysosome-related genes, while inhibit the expression of genes involved in DNA replication and cell cycle at the early reprogramming stage. Further verification determined that Grn suppressed cell proliferation due to the arrest of cell cycle at G2/M phase. Moreover, ectopic Grn can enhance the lysosomes abundance and rescue the efficiency reduction of reprogramming resulted from lysosomal protease inhibition. Taken together, we conclude that Grn serves as an activator for somatic cell reprogramming through mitigating cell hyperproliferation and promoting the function of lysosomes., (© 2024 Wiley Periodicals LLC.)

Background: The limited regenerative capacity of damaged neurons in adult mammals severely restricts neural repair. Although stem cell transplantation is promising, its clinical application remains challenging. Direct reprogramming, which utilizes cell plasticity to regenerate neurons, is an emerging alternative approach., Methods: We utilized primary postnatal cortical astrocytes for reprogramming induced neurons (iNs) through the viral-mediated overexpression of the transcription factors Ngn2 and Pax6 (NP). Fluorescence-activated cell sorting (FACS) was used to enrich successfully transfected cells, followed by single-cell RNA sequencing (scRNA-seq) using the 10 × Genomics platform for comprehensive transcriptomic analysis., Results: The scRNA-seq revealed that NP overexpression led to the differentiation of astrocytes into iNs, with percentages of 36% and 39.3% on days 4 and 7 posttransduction, respectively. CytoTRACE predicted the developmental sequence, identifying astrocytes as the reprogramming starting point. Trajectory analysis depicted the dynamic changes in gene expression during the astrocyte-to-iN transition., Conclusions: This study elucidates the molecular dynamics underlying astrocyte reprogramming into iNs, revealing key genes and pathways involved in this process. Our research contributes novel insights into the molecular mechanisms of NP-mediated reprogramming, suggesting avenues for optimizing the efficiency of the reprogramming process., (© 2024. The Author(s).)

Neuronal reprogramming is a promising approach for making major advancement in regenerative medicine. Distinct from the approach of induced pluripotent stem cells, neuronal reprogramming converts non-neuronal cells to neurons without going through a primitive stem cell stage. In vivo neuronal reprogramming brings this approach to a higher level by changing the cell fate of glial cells to neurons in neural tissue through overexpressing reprogramming factors. Despite the ongoing debate over the validation and interpretation of newly generated neurons, in vivo neuronal reprogramming is still a feasible approach and has the potential to become clinical treatment with further optimization and refinement. Here, we discuss the major neuronal reprogramming factors (mostly pro-neurogenic transcription factors during development), especially the significance of their expression levels during neurogenesis and the reprogramming process focusing on NeuroD1. In the developing central nervous system, these pro-neurogenic transcription factors usually elicit distinct spatiotemporal expression patterns that are critical to their function in generating mature neurons. We argue that these dynamic expression patterns may be similarly needed in the process of reprogramming adult cells into neurons and further into mature neurons with subtype identities. We also summarize the existing approaches and propose new ones that control gene expression levels for a successful reprogramming outcome.

In the injured zebrafish retina, Müller glial cells (MG) reprogram to adopt retinal stem cell properties and regenerate damaged neurons. The strongest zebrafish reprogramming factors might be good candidates for stimulating a similar regenerative response by mammalian MG. Myc proteins are potent reprogramming factors that can stimulate cellular plasticity in differentiated cells; however, their role in MG reprogramming and retina regeneration remains poorly explored. Here, we report that retinal injury stimulates mycb and mych expression and that, although both Mycb and Mych stimulate MG reprogramming and proliferation, only Mych enhances retinal neuron apoptosis. RNA-sequencing analysis of wild-type, mychmut and mycbmut fish revealed that Mycb and Mych regulate ∼40% and ∼16%, respectively, of the genes contributing to the regeneration-associated transcriptome of MG. Of these genes, those that are induced are biased towards regulation of ribosome biogenesis, protein synthesis, DNA synthesis, and cell division, which are the top cellular processes affected by retinal injury, suggesting that Mycb and Mych are potent MG reprogramming factors. Consistent with this, forced expression of either of these proteins is sufficient to stimulate MG proliferation in the uninjured retina., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: Our previous studies have successfully reported the reprogramming of fibroblasts into induced mammary epithelial cells (iMECs). However, the regulatory relationships and functional roles of MicroRNAs (miRNAs) in the progression of fibroblasts achieving the cell fate of iMECs are insufficiently understood., Methods: First, we performed pre-and post-induction miRNAs sequencing analysis by using high-throughput sequencing. Following that, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment studies were used to determine the primary roles of the significantly distinct miRNAs and targeted genes. Finally, the effect of miR-222-3p on iMECs fate reprogramming in vitro by transfecting., Results: As a result goat ear fibroblasts (GEFs) reprogramming into iMECs activates a regulatory program, involving 79 differentially expressed miRNAs. Besides, the programming process involved changes in multiple signaling pathways such as adherens junction, TGF-β signaling pathway, GnRH secretion and the prolactin signaling pathway, etc . Furthermore, it was discovered that the expression of miR-222-3p downregulation by miR-222-3p inhibitor significantly increase the reprogramming efficiency and promoted lipid accumulation of iMECs., Competing Interests: The authors declare that they have no competing interests., (© 2024 Liu et al.)

Epigenetic reprogramming resets parental epigenetic memories and differentiates primordial germ cells (PGCs) into mitotic pro-spermatogonia or oogonia. This process ensures sexually dimorphic germ cell development for totipotency 1 . In vitro reconstitution of epigenetic reprogramming in humans remains a fundamental challenge. Here we establish a strategy for inducing epigenetic reprogramming and differentiation of pluripotent stem-cell-derived human PGC-like cells (hPGCLCs) into mitotic pro-spermatogonia or oogonia, coupled with their extensive amplification (about >10 10 -fold). Bone morphogenetic protein (BMP) signalling is a key driver of these processes. BMP-driven hPGCLC differentiation involves attenuation of the MAPK (ERK) pathway and both de novo and maintenance DNA methyltransferase activities, which probably promote replication-coupled, passive DNA demethylation. hPGCLCs deficient in TET1, an active DNA demethylase abundant in human germ cells 2,3 , differentiate into extraembryonic cells, including amnion, with de-repression of key genes that bear bivalent promoters. These cells fail to fully activate genes vital for spermatogenesis and oogenesis, and their promoters remain methylated. Our study provides a framework for epigenetic reprogramming in humans and an important advance in human biology. Through the generation of abundant mitotic pro-spermatogonia and oogonia-like cells, our results also represent a milestone for human in vitro gametogenesis research and its potential translation into reproductive medicine., (© 2024. The Author(s).)

The process of induced pluripotent stem cells (iPSCs) reprogramming involves several crucial events, including the mesenchymal-epithelial transition (MET), activation of pluripotent genes, metabolic reprogramming, and epigenetic rewiring. Although these events intricately interact and influence each other, the specific element that regulates the reprogramming network remains unclear. Dux, a factor known to promote totipotency during the transition from embryonic stem cells (ESC) to 2C-like ESC (2CLC), has not been extensively studied in the context of iPSC reprogramming. In this study, we demonstrate that the modification of H3K18la induced by Dux overexpression controls the metabolism-H3K18la-MET network, enhancing the efficiency of iPSC reprogramming through a metabolic switch and the recruitment of p300 via its C-terminal domain. Furthermore, our proteomic analysis of H3K18la immunoprecipitation experiment uncovers the specific recruitment of Brg1 during reprogramming, with both H3K18la and Brg1 being enriched on the promoters of genes associated with pluripotency and epithelial junction. In summary, our study has demonstrated the significant role of Dux-induced H3K18la in the early reprogramming process, highlighting its function as a potent trigger. Additionally, our research has revealed, for the first time, the binding of Brg1 to H3K18la, indicating its role as a reader of histone lactylation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Prostate cancer (PCa) remains a leading cause of mortality among American men, with metastatic and recurrent disease posing significant therapeutic challenges due to a limited comprehension of the underlying biological processes governing disease initiation, dormancy, and progression. The conventional use of PCa cell lines has proven inadequate in elucidating the intricate molecular mechanisms driving PCa carcinogenesis, hindering the development of effective treatments. To address this gap, patient-derived primary cell cultures have been developed and play a pivotal role in unraveling the pathophysiological intricacies unique to PCa in each individual, offering valuable insights for translational research. This review explores the applications of the conditional reprogramming (CR) cell culture approach, showcasing its capability to rapidly and effectively cultivate patient-derived normal and tumor cells. The CR strategy facilitates the acquisition of stem cell properties by primary cells, precisely recapitulating the human pathophysiology of PCa. This nuanced understanding enables the identification of novel therapeutics. Specifically, our discussion encompasses the utility of CR cells in elucidating PCa initiation and progression, unraveling the molecular pathogenesis of metastatic PCa, addressing health disparities, and advancing personalized medicine. Coupled with the tumor organoid approach and patient-derived xenografts (PDXs), CR cells present a promising avenue for comprehending cancer biology, exploring new treatment modalities, and advancing precision medicine in the context of PCa. These approaches have been used for two NCI initiatives (PDMR: patient-derived model repositories; HCMI: human cancer models initiatives).

Patients with chronic hypoxia show a higher tumor incidence; however, no primary common cause has been recognized. Given the similarities between cellular reprogramming and oncogenic transformation, we directly compared these processes in human cells subjected to hypoxia. Mouse embryonic fibroblasts were employed as controls to compare transfection and reprogramming efficiency; human adipose-derived mesenchymal stem cells were employed as controls in human cells. Easily obtainable human peripheral blood mononuclear cells (PBMCs) were chosen to establish a standard protocol to compare cell reprogramming (into induced pluripotent stem cells (iPSCs)) and oncogenic focus formation efficiency. Cell reprogramming was achieved for all three cell types, generating actual pluripotent cells capable for differentiating into the three germ layers. The efficiencies of the cell reprogramming and oncogenic transformation were similar. Hypoxia slightly increased the reprogramming efficiency in all the cell types but with no statistical significance for PBMCs. Various PBMC types can respond to hypoxia differently; lymphocytes and monocytes were, therefore, reprogrammed separately, finding a significant difference between normoxia and hypoxia in monocytes in vitro. These differences were then searched for in vivo. The iPSCs and oncogenic foci were generated from healthy volunteers and patients with chronic obstructive pulmonary disease (COPD). Although higher iPSC generation efficiency in the patients with COPD was found for lymphocytes, this increase was not statistically significant for oncogenic foci. Remarkably, a higher statistically significant efficiency in COPD monocytes was demonstrated for both processes, suggesting that physiological hypoxia exerts an effect on cell reprogramming and oncogenic transformation in vivo in at least some cell types.

Myocardial infarction results in extensive cardiomyocyte apoptosis, leading to the formation of noncontractile scar tissue. Given the limited regenerative capacity of adult mammalian cardiomyocytes, direct reprogramming of cardiac fibroblasts (CFs) into cardiomyocytes represents a promising therapeutic strategy for myocardial repair, and small molecule drugs might offer a more attractive alternative to gene editing approaches in terms of safety and clinical feasibility. This study aimed to reprogram rat CFs into cardiomyocytes using a small molecular chemical mixture comprising CHIR99021, Valproic acid, Dorsomorphin, SB431542, and Forskolin. Immunofluorescence analysis revealed a significant increase in the expression of cardiomyocyte-specific markers, including cardiac troponin T (cTnT), Connexin 43 (Cx43), α-actinin, and Tbx5. Changes in intracellular calcium ion levels and Ca 2+ signal transfer between adjacent cells were monitored using a calcium ion fluorescence probe. mRNA sequencing analysis demonstrated the upregulation of genes associated with cardiac morphogenesis, myocardial differentiation, and muscle fiber contraction during CF differentiation induced by the small-molecule compounds. Conversely, the expression of fibroblast-related genes was downregulated. These findings suggest that chemical-induced cell fate conversion of rat CFs into cardiomyocyte-like cells is feasible, offering a potential therapeutic solution for myocardial injury., (© 2024 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Glioblastoma (GBM) is the most common yet uniformly fatal adult brain cancer. Intra-tumoral molecular and cellular heterogeneities are major contributory factors to therapeutic refractoriness and futility in GBM. Molecular heterogeneity is represented through molecular subtype clusters whereby the proneural (PN) subtype is associated with significantly increased long-term survival compared to the highly resistant mesenchymal (MES) subtype. Furthermore, it is universally recognized that a small subset of GBM cells known as GBM stem cells (GSCs) serve as reservoirs for tumor recurrence and progression. The clonal evolution of GSC molecular subtypes in response to therapy drives intra-tumoral heterogeneity and remains a critical determinant of GBM outcomes. In particular, the intra-tumoral MES reprogramming of GSCs using current GBM therapies has emerged as a leading hypothesis for therapeutic refractoriness. Preventing the intra-tumoral divergent evolution of GBM toward the MES subtype via new treatments would dramatically improve long-term survival for GBM patients and have a significant impact on GBM outcomes. In this review, we examine the challenges of the role of MES reprogramming in the malignant clonal evolution of glioblastoma and provide future perspectives for addressing the unmet therapeutic need to overcome resistance in GBM.

During cell fate transitions, cells remodel their transcriptome, chromatin, and epigenome; however, it has been difficult to determine the temporal dynamics and cause-effect relationship between these changes at the single-cell level. Here, we employ the heterokaryon-mediated reprogramming system as a single-cell model to dissect key temporal events during early stages of pluripotency conversion using super-resolution imaging. We reveal that, following heterokaryon formation, the somatic nucleus undergoes global chromatin decompaction and removal of repressive histone modifications H3K9me3 and H3K27me3 without acquisition of active modifications H3K4me3 and H3K9ac. The pluripotency gene OCT4 (POU5F1) shows nascent and mature RNA transcription within the first 24 h after cell fusion without requiring an initial open chromatin configuration at its locus. NANOG, conversely, has significant nascent RNA transcription only at 48 h after cell fusion but, strikingly, exhibits genomic reopening early on. These findings suggest that the temporal relationship between chromatin compaction and gene activation during cellular reprogramming is gene context dependent., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we used domain swapping and mutagenesis to study Oct4's reprogramming ability, identifying a redox-sensitive DNA binding domain, cysteine residue (Cys48), as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1 C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs) but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression, and aberrant differentiation. Pou5f1 C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1 C48S ( Janky ) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency., (© 2024 Shen et al.; Published by Cold Spring Harbor Laboratory Press.)

Endothelial cells are a heterogeneous population with various organ-specific and conserved functions that are critical to organ development, function, and regeneration. Here we report a Sox17-Erg direct reprogramming approach that uses cardiac fibroblasts to create differentiated endothelial cells that demonstrate endothelial-like molecular and physiological functions in vitro and in vivo. Injection of these induced endothelial cells into myocardial infarct sites after injury results in improved vascular perfusion of the scar region. Furthermore, we use genomic analyses to illustrate that Sox17-Erg reprogramming instructs cardiac fibroblasts toward an arterial-like identity. This results in a more efficient direct conversion of fibroblasts into endothelial-like cells when compared to traditional Etv2-based reprogramming. Overall, this Sox17-Erg direct reprogramming strategy offers a robust tool to generate endothelial cells both in vitro and in vivo, and has the potential to be used in repairing injured tissue., (© 2024. The Author(s).)

Heterogeneity among both primed and naive pluripotent stem cell lines remains a major unresolved problem. Here we show that expressing the maternal-specific linker histone H1FOO fused to a destabilizing domain (H1FOO-DD), together with OCT4, SOX2, KLF4, and LMYC, in human somatic cells improves the quality of reprogramming to both primed and naive pluripotency. H1FOO-DD expression was associated with altered chromatin accessibility around pluripotency genes and with suppression of the innate immune response. Notably, H1FOO-DD generates naive induced pluripotent stem cells with lower variation in transcriptome and methylome among clones and a more uniform and superior differentiation potency. Furthermore, we elucidated that upregulation of FKBP1A, driven by these five factors, plays a key role in H1FOO-DD-mediated reprogramming., Competing Interests: Declaration of interests A.K. and K.F. are co-inventors on a patent describing the method for producing human iPSCs from somatic cells using H1FOO-DD. K.F. is a co-founder and CEO of Heartseed Inc., and S.T., S. Yuasa, and K.F. own equity in Heartseed Inc. S.T. is an advisor of Heartseed Inc. J.K. and H.H. are employees and K.K. is a board member of ID Pharma Co., Ltd., without compensation relating to this study. S. Yamanaka is a scientific advisor to iPS Academia Japan without salary., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mammalian development commences with the zygote, which can differentiate into both embryonic and extraembryonic tissues, a capability known as totipotency. Only the zygote and embryos around zygotic genome activation (ZGA) (two-cell embryo stage in mice and eight-cell embryo in humans) are totipotent cells. Epigenetic modifications undergo extremely extensive changes during the acquisition of totipotency and subsequent development of differentiation. However, the underlying molecular mechanisms remain elusive. Recently, the discovery of mouse two-cell embryo-like cells, human eight-cell embryo-like cells, extended pluripotent stem cells and totipotent-like stem cells with extra-embryonic developmental potential has greatly expanded our understanding of totipotency. Experiments with these in vitro models have led to insights into epigenetic changes in the reprogramming of pluri-to-totipotency, which have informed the exploration of preimplantation development. In this review, we highlight the recent findings in understanding the mechanisms of epigenetic remodeling during totipotency capture, including RNA splicing, DNA methylation, chromatin configuration, histone modifications, and nuclear organization., (© 2024 Wiley Periodicals LLC.)

We have demonstrated that directly reprogramming cardiac fibroblasts into new cardiomyocytes via miR combo improves cardiac function in the infarcted heart. However, major challenges exist with delivery and efficacy. During a screening based approach to improve delivery, we discovered that C166-derived EVs were effective delivery agents for miR combo both in vitro and in vivo. In the latter, EV mediated delivery of miR combo induced significant conversion of cardiac fibroblasts into cardiomyocytes (∼20%), reduced fibrosis and improved cardiac function in a myocardial infarction injury model. When compared to lipid-based transfection, C166 EV mediated delivery of miR combo enhanced reprogramming efficacy. Improved reprogramming efficacy was found to result from a miRNA within the exosome: miR-148a-3p. The target of miR-148a-3p was identified as Mdfic. Over-expression and targeted knockdown studies demonstrated that Mdfic was a repressor of cardiomyocyte specific gene expression. In conclusion, we have demonstrated that C166-derived EVs are an effective method for delivering reprogramming factors to cardiac fibroblasts and we have identified a novel miRNA contained within C166-derived EVs which enhances reprogramming efficacy., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Ltd.)

Induced pluripotent stem cells (iPSCs) are a powerful tool for biomedical research, but their production presents challenges and safety concerns. Yamanaka and Takahashi revolutionised the field by demonstrating that somatic cells could be reprogrammed into pluripotent cells by overexpressing four key factors for a sufficient time. iPSCs are typically generated using viruses or virus-based methods, which have drawbacks such as vector persistence, risk of insertional mutagenesis, and oncogenesis. The application of less harmful nonviral vectors is limited as conventional plasmids cannot deliver the levels or duration of the factors necessary from a single transfection. Hence, plasmids that are most often used for reprogramming employ the potentially oncogenic Epstein-Barr nuclear antigen 1 (EBNA-1) system to ensure adequate levels and persistence of expression. In this study, we explored the use of nonviral SMAR DNA vectors to reprogram human fibroblasts into iPSCs. We show for the first time that iPSCs can be generated using nonviral plasmids without the use of EBNA-1 and that these DNA vectors can provide sufficient expression to induce pluripotency. We describe an optimised reprogramming protocol using these vectors that can produce high-quality iPSCs with comparable pluripotency and cellular function to those generated with viruses or EBNA-1 vectors.

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., (© 2024. The Author(s).)

H19 is an essential imprinted gene that is expressed to govern normal embryonic development. During reprogramming, the parental pronuclei have asymmetric reprogramming capacities and the critical reprogramming factors predominantly reside in the male pronucleus. After inhibiting the expression of H19 and Gtl2, androgenetic haploid ESCs (AG-haESCs) can efficiently and stably support the generation of healthy SC pups at a rate of ~20%, and double-knockout parthenogenetic haESCs can also produce efficiently. Induced pluripotent stem (iPS) cell reprogramming is thought to have a characteristic epigenetic pattern that is the reverse of its developmental potential; however, it is unclear how H19 participates in iPS cell reprogramming. Here, we showed that the expression of H19 was transiently increased during iPSC reprogramming. H19 knockdown resulted in greater reprogramming efficiency. The genes associated with pluripotency showed enhanced expression during the early reprogramming process, and the Oct4 promoter was demethylated by bisulfite genomic sequencing analysis. Moreover, expression analysis revealed that the mesenchymal master regulators associated with epithelial-to-mesenchymal transition (EMT) were downregulated during reprogramming in H19 knockdown. These findings provide functional insight into the role of H19 as a barrier to the early reprogramming process.

Although lineage reprogramming from one cell type to another is becoming a breakthrough technology for cell-based therapy, several limitations remain to be overcome, including the low conversion efficiency and subtype specificity. To address these, many studies have been conducted using genetics, chemistry, physics, and cell biology to control transcriptional networks, signaling cascades, and epigenetic modifications during reprogramming. Here, we summarize recent advances in cellular reprogramming and discuss future directions.

Previous studies have shown that hepatocyte-like cells can be generated from fibroblasts using either lineage-specific transcription factors or chemical induction methods. However, these methods have their own deficiencies that restrict the therapeutic applications of such induced hepatocytes. In this study, we present a transgene-free, highly efficient chemical-induced direct reprogramming approach to generate hepatocyte-like cells from mouse embryonic fibroblasts (MEFs). Using a small molecule cocktail (SMC) as an inducer, MEFs can be directly reprogrammed into hepatocyte-like cells, bypassing the intermediate stages of pluripotent and immature hepatoblasts. These chemical-induced hepatocyte-like cells (ciHeps) closely resemble mature primary hepatocytes in terms of morphology, biological behavior, gene expression patterns, marker expression levels, and hepatic functions. Furthermore, transplanted ciHeps can integrate into the liver, promote liver regeneration, and improve survival rates in mice with acute liver damage. ciHeps can also ameliorate liver fibrosis caused by chronic injuries and enhance liver function. Notably, ciHeps exhibit no tumorigenic potential either in vitro or in vivo. Mechanistically, SMC-induced mesenchymal-to-epithelial transition and suppression of SNAI1 contribute to the fate conversion of fibroblasts into ciHeps. These results indicate that this transgene-free, chemical-induced direct reprogramming technique has the potential to serve as a valuable means of producing alternative hepatocytes for both research and therapeutic purposes. Additionally, this method also sheds light on the direct reprogramming of other cell types under chemical induction., (© The Author(s) (2023). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

Recent developments in synthetic biology, next-generation sequencing, and machine learning provide an unprecedented opportunity to rationally design new disease treatments based on measured responses to gene perturbations and drugs to reprogram cells. The main challenges to seizing this opportunity are the incomplete knowledge of the cellular network and the combinatorial explosion of possible interventions, both of which are insurmountable by experiments. To address these challenges, we develop a transfer learning approach to control cell behavior that is pre-trained on transcriptomic data associated with human cell fates, thereby generating a model of the network dynamics that can be transferred to specific reprogramming goals. The approach combines transcriptional responses to gene perturbations to minimize the difference between a given pair of initial and target transcriptional states. We demonstrate our approach's versatility by applying it to a microarray dataset comprising >9,000 microarrays across 54 cell types and 227 unique perturbations, and an RNASeq dataset consisting of >10,000 sequencing runs across 36 cell types and 138 perturbations. Our approach reproduces known reprogramming protocols with an AUROC of 0.91 while innovating over existing methods by pre-training an adaptable model that can be tailored to specific reprogramming transitions. We show that the number of gene perturbations required to steer from one fate to another increases with decreasing developmental relatedness and that fewer genes are needed to progress along developmental paths than to regress. These findings establish a proof-of-concept for our approach to computationally design control strategies and provide insights into how gene regulatory networks govern phenotype., Competing Interests: Competing interests statement:The authors declare no competing interest.

Organismal aging is inherently connected to the aging of its constituent cells and systems. Reducing the biological age of the organism may be assisted by reducing the age of its cells - an approach exemplified by partial cell reprogramming through the expression of Yamanaka factors or exposure to chemical cocktails. It is crucial to protect cell type identity during partial reprogramming, as cells need to retain or rapidly regain their functions following the treatment. Another critical issue is the ability to quantify biological age as reprogrammed older cells acquire younger states. We discuss recent advances in reprogramming-induced rejuvenation and offer a critical review of this procedure and its relationship to the fundamental nature of aging. We further comparatively analyze partial reprogramming, full reprogramming and transdifferentiation approaches, assess safety concerns and emphasize the importance of distinguishing rejuvenation from dedifferentiation. Finally, we highlight translational opportunities that the reprogramming-induced rejuvenation approach offers., (© 2024. The Author(s).)

The multistep process of in vivo reprogramming, mediated by the transcription factors (TFs) Oct4, Sox2, Klf4, and c-Myc (OSKM), holds great promise for the development of rejuvenating and regenerative strategies. However, most of the approaches developed so far are accompanied by a persistent risk of tumorigenicity. Here, we review the groundbreaking effects of in vivo reprogramming with a particular focus on rejuvenation and regeneration. We discuss how the activity of pioneer TFs generates cellular plasticity that may be critical for inducing not only reprogramming and regeneration, but also cancer initiation. Finally, we highlight how a better understanding of the uncoupled control of cellular identity, plasticity, and aging during reprogramming might pave the way to the development of rejuvenating/regenerating strategies in a nontumorigenic manner., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate "reprogramming" by opening new chromatin sites for expression that can attract transcription factors from the starting cell's enhancers. Here we report that SOX4 is sufficient to initiate hepatobiliary metaplasia in the adult mouse liver, closely mimicking metaplasia initiated by toxic damage to the liver. In lineage-traced cells, we assessed the timing of SOX4-mediated opening of enhancer chromatin versus enhancer decommissioning. Initially, SOX4 directly binds to and closes hepatocyte regulatory sequences via an overlapping motif with HNF4A, a hepatocyte master regulatory transcription factor. Subsequently, SOX4 exerts pioneer factor activity to open biliary regulatory sequences. The results delineate a hierarchy by which gene networks become reprogrammed under physiological conditions, providing deeper insight into the basis for cell fate transitions in animals., (© 2024. The Author(s).)

We investigate how matrix stiffness regulates chromatin reorganization and cell reprogramming and find that matrix stiffness acts as a biphasic regulator of epigenetic state and fibroblast-to-neuron conversion efficiency, maximized at an intermediate stiffness of 20 kPa. ATAC sequencing analysis shows the same trend of chromatin accessibility to neuronal genes at these stiffness levels. Concurrently, we observe peak levels of histone acetylation and histone acetyltransferase (HAT) activity in the nucleus on 20 kPa matrices, and inhibiting HAT activity abolishes matrix stiffness effects. G-actin and cofilin, the cotransporters shuttling HAT into the nucleus, rises with decreasing matrix stiffness; however, reduced importin-9 on soft matrices limits nuclear transport. These two factors result in a biphasic regulation of HAT transport into nucleus, which is directly demonstrated on matrices with dynamically tunable stiffness. Our findings unravel a mechanism of the mechano-epigenetic regulation that is valuable for cell engineering in disease modeling and regenerative medicine applications.

Somatic cell reprogramming is a complex feature that allows differentiated cells to undergo fate changes into different cell types. This process, which is conserved between plants and animals, is often achieved via dedifferentiation into pluripotent stem cells, which have the ability to generate all other types of cells and tissues of a given organism. Cellular reprogramming is thus a complex process that requires extensive modification at the epigenetic and transcriptional level, unlocking cellular programs that allow cells to acquire pluripotency. In addition to alterations in the gene expression profile, cellular reprogramming requires rearrangement of the proteome, organelles and metabolism, but these changes are comparatively less studied. In this context, autophagy, a cellular catabolic process that participates in the recycling of intracellular constituents, has the capacity to affect different aspects of cellular reprogramming, including the removal of protein signatures that might hamper reprogramming, mitophagy associated with metabolic reprogramming, and the supply of energy and metabolic building blocks to cells that undergo fate changes. In this Review, we discuss advances in our understanding of the role of autophagy during cellular reprogramming by drawing comparisons between plant and animal studies, as well as highlighting aspects of the topic that warrant further research., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Aging and age-associated disease are a major medical and societal burden in need of effective treatments. Cellular reprogramming is a biological process capable of modulating cell fate and cellular age. Harnessing the rejuvenating benefits without altering cell identity via partial cellular reprogramming has emerged as a novel translational strategy with therapeutic potential and strong commercial interests. Here, we explore the aging-related benefits of partial cellular reprogramming while examining limitations and future directions for the field., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Massive loss of neurons following brain injury or disease is the primary cause of central nervous system dysfunction. Recently, much research has been conducted on how to compensate for neuronal loss in damaged parts of the nervous system and thus restore functional connectivity among neurons. Direct somatic cell differentiation into neurons using pro-neural transcription factors, small molecules, or microRNAs, individually or in association, is the most promising form of neural cell replacement therapy available. This method provides a potential remedy for cell loss in a variety of neurodegenerative illnesses, and the development of reprogramming technology has made this method feasible. This article provides a comprehensive review of reprogramming, including the selection and methods of reprogramming starting cell populations as well as the signaling methods involved in this process. Additionally, we thoroughly examine how reprogramming astrocytes into neurons can be applied to treat stroke and other neurodegenerative diseases. Finally, we discuss the challenges of neuronal reprogramming and offer insights about the field., (© 2024 The Author(s).)

Neutrophils are increasingly recognized as key players in the tumor immune response and are associated with poor clinical outcomes. Despite recent advances characterizing the diversity of neutrophil states in cancer, common trajectories and mechanisms governing the ontogeny and relationship between these neutrophil states remain undefined. Here, we demonstrate that immature and mature neutrophils that enter tumors undergo irreversible epigenetic, transcriptional, and proteomic modifications to converge into a distinct, terminally differentiated dcTRAIL-R1 + state. Reprogrammed dcTRAIL-R1 + neutrophils predominantly localize to a glycolytic and hypoxic niche at the tumor core and exert pro-angiogenic function that favors tumor growth. We found similar trajectories in neutrophils across multiple tumor types and in humans, suggesting that targeting this program may provide a means of enhancing certain cancer immunotherapies.

Experimental studies have shown that chromatin modifiers have a critical effect on cellular reprogramming, i.e., the conversion of differentiated cells to pluripotent stem cells. Here, we develop a model of the OCT4 gene regulatory network that includes genes expressing chromatin modifiers TET1 and JMJD2, and the chromatin modification circuit on which these modifiers act. We employ this model to compare three reprogramming approaches that have been considered in the literature with respect to reprogramming efficiency and latency variability. These approaches are overexpression of OCT4 alone, overexpression of OCT4 with TET1, and overexpression of OCT4 with JMJD2. Our results show more efficient and less variable reprogramming when also JMJD2 and TET1 are overexpressed, consistent with previous experimental data. Nevertheless, TET1 overexpression can lead to more efficient reprogramming compared to JMJD2 overexpression. This is the case when the recruitment of DNA methylation by H3K9me3 is weak and the methyl-CpG-binding domain (MBD) proteins are sufficiently scarce such that they do not hamper TET1 binding to methylated DNA. The model that we developed provides a mechanistic understanding of existing experimental results and is also a tool for designing optimized reprogramming approaches that combine overexpression of cell-fate specific transcription factors (TFs) with targeted recruitment of epigenetic modifiers., (© 2024. The Author(s).)