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Cancer research remains clinically unmet in many areas due to limited access to patient samples and the lack of reliable model systems that truly reflect human cancer biology. The emergence of patient-derived induced pluripotent stem cells and engineered human pluripotent stem cells (hPSCs) has helped overcome these challenges, offering a versatile alternative platform for advancing cancer research. These hPSCs are already proving to be valuable models for studying specific cancer driver mutations, offering insights into cancer origins, pathogenesis, tumor heterogeneity, clonal evolution, and facilitating drug discovery and testing. This article reviews recent progress in utilizing hPSCs for clinically relevant cancer models and highlights efforts to deepen our understanding of fundamental cancer biology., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Antigen-presenting cells (APCs) are a heterogenous group of immune cells composed by dendritic cells (DCs) and macrophages (Mϕ), which are critical for orchestrating immunity against cancer or infections. Several strategies have been explored to generate APC subsets, including enrichment from peripheral blood and differentiation from pluripotent or multipotent cells. During development, the generation of APC subsets is instructed by transcription factors (TFs). Direct cell reprogramming, also known as transdifferentiation, offers an approach to harness combinations of TFs to generate APCs from unrelated somatic cells, including cancer cells. In this review, we summarize the transcriptional specification of DC subsets, highlight transcriptional networks for their generation, and discuss future applications of DC reprogramming in cancer immunotherapy., Competing Interests: Declaration of Competing Interest C.-F.P. has equity interest and serves in management positions at Asgard Therapeutics AB, which develops cancer immunotherapies based on in vivo DC reprogramming technologies. C.-F.P. is an inventor on granted patents U.S. 11345891, JP 7303743, CN ZL201880005047.3, patent application WO 2018/185709, and patent application WO 2022/243448 (together with E.A.) held by Asgard Therapeutics that covers the direct cell reprogramming approach described here., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Sidedness and staging are major sources of tumor microenvironment (TME) differences in colorectal cancer (CRC). Subpopulation compositions of stromal cells and immune cells, and interactions between cells collectively constitute the immunosuppressive microenvironment of CRC. In this study, we comprehensively collected single-cell RNA sequencing data from public databases. We filtered out 126,279 cells from 55 CRC samples to characterize the differences in cellular composition, and to elucidate the transcriptional features and potential functions of cell types, temporally and positionally. We observed an increased degree of hypoxia in right side-specific cancer cells compared to left-sided cancer. Cancer-associated fibroblasts (CAFs) illustrated molecular signatures tremendously tended to be associated with functions that orchestrate extracellular matrix remodeling and angiogenesis, and right-sided CAFs characterized the stronger cancer invasion signals. Crosstalk between side-specific cancer cells and stromal together with immune cells characterized CRC via different sample groups, and was pertinent to worse prognosis. Our study captured immunosuppressive pattern exhibiting more intricate intercellular interactions in right-sided CRC. Additionally, during malignant progression of CRC, the transformation of CD8+ T cell cytotoxic and exhausted properties and macrophage pro-inflammatory and anti-inflammatory properties epitomized the cellular reprogramming phenomenon that the function of TME shifted from promoting immunity to suppressive immunity. Our study shed lights on refining personalized therapeutic regimens during malignant progression in left- and right-sided CRCs., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intercellular transmission of messenger RNA (mRNA) is being explored in mammalian species using immortal cell lines. Here, we uncover an intercellular mRNA transfer phenomenon that allows for the adaptation and reprogramming of human primed pluripotent stem cells (hPSCs). This process is induced by the direct cell contact-mediated coculture with mouse embryonic stem cells under the condition impermissible for primed hPSC culture. Mouse-derived mRNA contents are transmitted into adapted hPSCs only in the coculture. Transfer-specific mRNA analysis shows the enrichment for divergent biological pathways involving transcription/translational machinery and stress-coping mechanisms, wherein such transfer is diminished when direct cell contacts are lost. After 5 d of coculture with mouse embryonic stem cells, surface marker analysis and global gene profiling confirmed that mRNA transfer-prone hPSC efficiently gains a naïve-like state. Furthermore, transfer-specific knockdown experiments targeting mouse-specific transcription factor-coding mRNAs in hPSC show that mouse-derived Tfcp2l1 , Tfap2c, and Klf4 are indispensable for human naïve-like conversion. Thus, interspecies mRNA transfer triggers cellular reprogramming in mammalian cells. Our results support that episodic mRNA transfer can occur in cell cooperative and competitive processes, which provides a fresh perspective on understanding the roles of mRNA mobility for intra- and interspecies cellular communications., Competing Interests: Competing interests statement:The authors declare no competing interest.

Chronic liver diseases are highly linked with mitochondrial dysfunction and macrophage infiltration. Mallory-Denk bodies (MDBs) are protein aggregates associated with hepatic inflammation, and MDBs pathogenesis could be induced in mice by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Here, we investigate the macrophage heterogeneity and the role of macrophage during MDBs pathogenesis on DDC-induced MDBs mouse model by single-nucleus RNA sequencing (snRNA-seq). We defined liver macrophages into four distinct subsets including monocyte-derived macrophages (MDMs) subset and three Kupffer cells (KCs) subsets (Gpnmb high KCs, Peam1 high KCs, and Gpnmb low Pecam1 low KCs). Particularly, we identified a novel Gpnmb high KCs subset as lipid-associated macrophage (LAM) with high expression of Trem2, CD63, and CD9. Interestingly, LAM showed a potential immunosuppressive characteristic by expressing anti-inflammatory genes IL-7R during the MDBs formation. Using contact and transwell co-culture systems, the released mtDNA from hepatocytes was found to induce the activation of inflammasome in macrophages. Furthermore, we revealed the damaged DNA could activate the NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasome and subsequently form apoptosis-associated speck-like protein containing a caspase recruit domain (ASC) specks of liver macrophages. Collectively, our results firstly revealed macrophage heterogeneity and inflammasome activation by mtDNA from injured liver during MDBs pathogenesis, providing crucial understanding of pathogenesis of chronic liver disease., Competing Interests: Declarations. Conflict of interest: The authors of this manuscript have no conflict of interest to disclose., (© 2024. The Author(s).)

Early events in the reprogramming of fibroblasts to cardiac muscle cells are unclear. While various histone undergo modification and re-positioning, and these correlate with the activity of certain genes, it is unknown if these events are causal or happen in response to reprogramming. Histone modification and re-positioning would be expected to open up chromatin on lineage-specific genes and this can be ascertained by studying nucleosome architecture. We have recently developed a set of tools to identify significant changes in nucleosome architecture which we used to study skeletal muscle differentiation. In this report, we have applied these tools to understand nucleosome architectural changes during fibroblast to cardiac muscle reprogramming. We found that nucleosomes surrounding the transcription start sites of cardiac muscle genes induced during reprogramming were insensitive to reprogramming factors as well as to agents which enhance reprogramming efficacy. In contrast, significant changes in nucleosome architecture were observed distal to the transcription start site. These regions were associated with nucleosome build-up. In summary, investigations into nucleosome structure do not support the notion that fibroblasts to cardiac muscle cell reprogramming involves chromatin opening and suggests instead long-range effects such as breaking closed-loop inhibition., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Harris et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Tumor initiation represents the first step in tumorigenesis during which normal progenitor cells undergo cell fate transition to cancer. Capturing this process as it occurs in vivo, however, remains elusive. Here we employ spatiotemporally controlled oncogene activation and tumor suppressor inhibition together with multiomics to unveil the processes underlying oral epithelial progenitor cell reprogramming into tumor initiating cells at single cell resolution. Tumor initiating cells displayed a distinct stem-like state, defined by aberrant proliferative, hypoxic, squamous differentiation, and partial epithelial to mesenchymal invasive gene programs. YAP-mediated tumor initiating cell programs included activation of oncogenic transcriptional networks and mTOR signaling, and recruitment of myeloid cells to the invasive front contributing to tumor infiltration. Tumor initiating cell transcriptional programs are conserved in human head and neck cancer and associated with poor patient survival. These findings illuminate processes underlying cancer initiation at single cell resolution, and identify candidate targets for early cancer detection and prevention., Competing Interests: Competing interests: J.S.G. has received other commercial research support from Kura Oncology, Mavupharma, Dracen, Verastem, and SpringWorks Therapeutics, and is a consultant/advisory board member for Domain Therapeutics, Pangea Therapeutics, and io9, and founder of Kadima Pharmaceuticals. The remaining authors declare no competing interests., (© 2025. The Author(s).)

Background: Anti-citrullinated peptide antibodies (ACPA)-negative (ACPA-) rheumatoid arthritis (RA) presents significant diagnostic and therapeutic challenges due to the absence of specific biomarkers, underscoring the need to elucidate its distinctive cellular and metabolic profiles for more targeted interventions., Methods: Single-cell RNA sequencing data from peripheral blood mononuclear cells (PBMCs) and synovial tissues of patients with ACPA- and ACPA+ RA, as well as healthy controls, were analyzed. Immune cell populations were classified based on clustering and marker gene expression, with pseudotime trajectory analysis, weighted gene co-expression network analysis (WGCNA), and transcription factor network inference providing further insights. Cell-cell communication was explored using CellChat and MEBOCOST, while scFEA enabled metabolic flux estimation. A neural network model incorporating key genes was constructed to differentiate patients with ACPA- RA from healthy controls., Results: Patients with ACPA- RA demonstrated a pronounced increase in classical monocytes in PBMCs and C1QChigh macrophages (p < 0.001 and p < 0.05). Synovial macrophages exhibited increased heterogeneity and were enriched in distinct metabolic pathways, including complement cascades and glutathione metabolism. The neural network model achieved reliable differentiation between patients with ACPA- RA and healthy controls (AUC = 0.81). CellChat analysis identified CD45 and CCL5 as key pathways facilitating macrophage-monocyte interactions in ACPA- RA, prominently involving iron-mediated metabolite communication. Metabolic flux analysis indicated elevated beta-alanine and glutathione metabolism in ACPA- RA macrophages., Conclusion: These findings underscore that ACPA-negative rheumatoid arthritis is marked by elevated classical monocytes in circulation and metabolic reprogramming of synovial macrophages, particularly in complement cascade and glutathione metabolism pathways. By integrating single-cell RNA sequencing with machine learning, this study established a neural network model that robustly differentiates patients with ACPA- RA from healthy controls, highlighting promising diagnostic biomarkers and therapeutic targets centered on immune cell metabolism., Competing Interests: The authors declare that this research was conducted without any commercial or financial relationships that could present a potential conflict of interest., (Copyright © 2025 Jiang, Hu, Huang, Ho, Wang and Kang.)

Basal cells (BCs) are the progenitor cells responsible for tracheal epithelium integrity. Here, we demonstrate that mitochondrial pyruvate carriers (MPCs) act as metabolic checkpoints that are essential for BC fate decision. Inhibition of MPCs enables long-term expansion of BCs from both mice and humans. Genetic inactivation of Mpc2 in mice leads to BC hyperplasia and reduced ciliated cells during homeostasis, as well as delayed epithelial regeneration and accumulation of intermediate cells following injury. Mechanistically, MPC2 links glycolysis to ATP citrate lyase (ACLY)-dependent cytosolic acetyl-coenzyme A (CoA) generation, which is required for the epigenetic control of differentiation-related gene transcription. Modulating this metabolic-epigenetic axis partially rescues Yes-associated protein (YAP)-dysfunction-induced changes in BCs. Importantly, exogenous citrate promotes the differentiation of BCs from chronic obstructive lung disease (COPD) patients. Thus, beyond demonstrating the role of pyruvate metabolism in BC fate decision, our study suggests that targeting pyruvate-citrate metabolism may serve as a potential strategy to rectify abnormal BC behavior in lung diseases., Competing Interests: Declaration of interests T.R., P.S., Y.L., and Q.Z. have filed a patent application regarding targeting MPCs for stem cell expansion., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Somatic cells can be transformed into induced pluripotent stem cells (iPSCs) using a technique called reprogramming. This process involves introducing Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc) to the cells through retroviral supernatants. This chapter outlines a protocol for reprogramming mouse embryonic fibroblasts (MEFs) using the hormone triiodo-L-thyronine (T3) to enhance the generation of iPSCs. It also describes how to analyze these iPSCs by colony staining for alkaline phosphatase activity, a standard marker for identifying pluripotent embryonic stem cells. To further study iPSCs, individual colonies must be selected and expanded, and pluripotency is examined by analyzing gene expression profiles using quantitative real-time PCR to measure the endogenous expression of pluripotency genes. Integrating T3 into reprogramming methods may significantly improve the production of functional iPSCs. This advancement could open new avenues for research in cell plasticity, disease modeling, and regenerative therapies., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

DNA methylation is a widely studied epigenetic mark which in mammals involves the incorporation of a methyl group to the fifth carbon of cytosines, mainly those belonging to CpG dinucleotides. It has been linked to context-dependent regulatory functions ranging from gene and repetitive DNA silencing to gene body transcriptional activity. Because of its important roles during embryonic development and cell differentiation, DNA methylation can be used to track cell reprogramming by measuring the methylation levels of pluripotency-associated factors. In this scenario, bisulfite pyrosequencing is a simple, robust, and widely used technique which allows for the quantification of DNA methylation levels at small, specific regions of the genome. It involves the amplification and biotin tagging of bisulfite-converted DNA. Single amplified strands are then purified using streptavidin and finally pyrosequenced using a sequencing primer. Thus, it is an ideal method for the quantitative profiling of specific genomic regions, with applications ranging from biomarker discovery and epigenetic clock tracking to omic validation studies., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

As cancers progress, they become increasingly aggressive-metastatic tumours are less responsive to first-line therapies than primary tumours, they acquire resistance to successive therapies and eventually cause death 1,2 . Mutations are largely conserved between primary and metastatic tumours from the same patients, suggesting that non-genetic phenotypic plasticity has a major role in cancer progression and therapy resistance 3-5 . However, we lack an understanding of metastatic cell states and the mechanisms by which they transition. Here, in a cohort of biospecimen trios from same-patient normal colon, primary and metastatic colorectal cancer, we show that, although primary tumours largely adopt LGR5 + intestinal stem-like states, metastases display progressive plasticity. Cancer cells lose intestinal cell identities and reprogram into a highly conserved fetal progenitor state before undergoing non-canonical differentiation into divergent squamous and neuroendocrine-like states, a process that is exacerbated in metastasis and by chemotherapy and is associated with poor patient survival. Using matched patient-derived organoids, we demonstrate that metastatic cells exhibit greater cell-autonomous multilineage differentiation potential in response to microenvironment cues compared with their intestinal lineage-restricted primary tumour counterparts. We identify PROX1 as a repressor of non-intestinal lineage in the fetal progenitor state, and show that downregulation of PROX1 licenses non-canonical reprogramming., Competing Interests: Competing interests: K.G. is listed as an inventor on US patent 11,464,874, and US provisional patent applications 63/478,809 and 63/478,829 on targeting L1CAM to treat cancer, submitted by MSKCC. D.P. is on the scientific advisory board of Insitro. J.S. is a consultant for Paige AI. R.Y. has served on the advisory board for Pfizer, Mirati Therapeutics, Revolution Medicine, Loxo@Lilly and Amgen, received a speaker’s honorarium from Zai Lab, and has received research support from Pfizer, Boehringer Ingelheim, Mirati Therapeutics, Daiichi Sankyo, FogPharma and Boundless Bio. J.G.-A. owns stock in Intuitive Surgical. R.C. is a consultant for Sanavia Oncology, S2 Genomics and LevitasBio. The other authors declare no competing interests., (© 2024. The Author(s).)

Somatic cells can be reprogrammed into pluripotent stem cells (iPSCs) by overexpressing defined transcription factors. Specifically, overexpression of OCT4 alone has been demonstrated to reprogram mouse fibroblasts into iPSCs. However, it remains unclear whether any other single factor can induce iPSCs formation. Here, we report that SALL4 alone, under an optimized reprogramming medium iCD4, is capable of reprogramming mouse fibroblasts into iPSCs. Mechanistically, SALL4 facilitates reprogramming by inhibiting somatic genes and activating pluripotent genes, such as Esrrb and Tfap2c. Furthermore, we demonstrate that co-overexpressing SALL4 and OCT4 synergistically enhances reprogramming efficiency. Specifically, the activation of Rsk1/Esrrb/Tfap2c by SALL4, alongside OCT4's activation of Sox2 and the suppression of Mndal by SALL4 and Sbsn by OCT4, cooperate to facilitate SALL4+OCT4-mediated reprogramming. Overall, our study not only establishes an efficient method for iPSCs induction using the SALL4 single factor but also provides insights into the synergistic effects of SALL4 and OCT4 in reprogramming., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Plants possess a high potential for somatic cell reprogramming, enabling the transition from differentiated tissue to pluripotent callus, followed by the formation of de novo shoots during plant regeneration. Despite extensive studies on the molecular network and key genetic factors involved in this process, the underlying epigenetic landscape remains incompletely understood., Results: Here, we explored the dynamics of the methylome and transcriptome during the two-step plant regeneration process. During the leaf-to-callus transition in Arabidopsis Ler, CG methylation shifted across genic regions, exhibiting a similar number of differentially methylated regions (DMRs) for both hypo- and hypermethylation. Pericentromeric regions underwent substantial CG and extensive CHH hypomethylation, alongside some CHG hypermethylation. Upon shoot regeneration from callus, genic regions displayed extensive reconfiguration of CG methylation, while pericentromeric methylation levels highly increased across all cytosine contexts, coinciding with the activation of the RNA-directed DNA methylation (RdDM) pathway. However, mutation in DEMETER (DME) DNA demethylase gene resulted in significant genic CG redistribution and global non-CG hypomethylation in pericentromeric regions, particularly during shoot regeneration. This non-CG hypomethylation observed in dme-2 mutants was, at least partly, due to RdDM downregulation. The dme-2 mutants affected gene expression involved in pluripotency and shoot meristem development, resulting in enhanced shoot regeneration through a reprogrammed state established by pericentromeric hypomethylation compared to wild type., Conclusion: Our study demonstrates epigenetic changes, accompanied by transcriptome alterations, during pluripotency acquisition (leaf-to-callus) and regeneration (callus-to-de novo shoot). Additionally, it highlights the functions of the DME demethylase, particularly its close association with the RdDM pathway, which underlies pericentromeric non-CG methylation maintenance. These results provide important insights into the epigenetic reconfiguration associated with cell identity transition during somatic cell reprogramming., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

N6-methyladenosine is one of the most common and reversible post-transcriptional modifications in eukaryotes, and it is involved in alternative splicing and RNA transcription, degradation, and translation. It is well known that cancer cells acquire energy through metabolic reprogramming to exhibit various biological behaviors. Moreover, numerous studies have demonstrated that m6A induces cancer metabolic reprogramming by regulating the expression of core metabolic genes or by activating metabolic signaling pathways. Meanwhile, m6A modifications and related regulators are key targets in the regulation of immune effects. We further summarize how m6A modifications contribute to tumor metabolism, and how these events affect the tumor immune microenvironment, with a specific focus on different cell types. Finally, we focus on the specific applications of this field to tumor immunotherapy. We review the potential role of m6A in metabolic reprogramming of tumor immune microenvironment and its regulatory mechanism, with the aim of providing new targets for tumor metabolic regulation and immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2024 Li, Peng, Yang, Luo, Wang, Mou, Zhou, Luo and Xiang.)

Cutaneous wound healing is a slow process that often terminates with permanent scarring while oral wounds, in contrast, regenerate after damage faster. Unique molecular networks in epidermal and oral epithelial keratinocytes contribute to the tissue-specific response to wounding, but key factors that establish those networks and how the keratinocytes interact with their cellular environment remain to be elucidated. The transcription factor PITX1 is highly expressed in the oral epithelium but is undetectable in cutaneous keratinocytes. To delineate if PITX1 contributes to oral keratinocyte identity, cell-cell interactions, and the improved wound healing capabilities, we ectopically expressed PITX1 in the epidermis of murine skin. Using comparative analysis of murine skin and oral (buccal) mucosa with single-cell RNA-Seq and spatial transcriptomics, we found that PITX1 expression enhances epidermal keratinocyte migration and proliferation and alters differentiation to a quasi-oral keratinocyte state. PITX1+ keratinocytes reprogrammed intercellular communication between skin-resident cells to mirror buccal tissue while stimulating the influx of neutrophils that establish a pro-inflammatory environment. Furthermore, PITX1+ skin healed significantly faster than control skin via increased keratinocyte activation and migration and a tunable inflammatory environment. These results illustrate that PITX1 programs oral keratinocyte identity and cellular interactions while revealing critical downstream networks that promote wound closure.

Regulative development, demonstrated by many animal embryos, is the ability to replace missing cells or parts. The underlying molecular mechanism(s) of that ability is not well understood. If sea urchin micromeres (skeletogenic cell progenitors) are removed at the 16-cell stage, early endoderm initiates a sequential switch in cell fates, called transfating. Without micromeres, other mesoderm cells are absent as well, because their specification depends on signaling from micromeres. Most mesoderm cells later return by transfating, but pigment cells do not. Single-cell RNA sequencing, tracked over time, reveals the reprogramming sequence of those replacements. Beginning with an early endoderm specification state, cells progress through endomesoderm, then mesoderm, and finally distinct skeletogenic and blastocoelar cell specification states emerge, but pigment cells do not. Rescue of pigment cells was found to be a consequence of signal timing: if Delta is expressed prior to Nodal, pigment cells return. Thus, transfating operates through a series of gene regulatory state transitions, and reprogramming fails if endogenous negative signals occur prior to positive signals in the reprogramming sequence., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The proneural transcription factor ASCL1 regulates neurogenesis and drives somatic cell reprogramming into neurons. However, not all cell types can be reprogrammed by ASCL1, raising the questions of what provides competence and how we can overcome barriers to enable directed differentiation. Here, we investigate how levels of ASCL1 and its phosphorylation modulate its activity over progressive lineage restriction of mouse embryonic stem cells. We find that inhibition of ASCL1 phosphorylation enhances reprogramming of both mesodermal and neuroectodermal cells, while pluripotent cells remain refractory to ASCL1-directed neuronal differentiation. By performing RNA-seq and ATAC-seq in neuroectoderm, we find that un(der)phosphorylated ASCL1 causes increased chromatin accessibility at sites proximal to neuronal genes, accompanied by their increased expression. Combined analysis of protein stability and proneural function of phosphomutant and phosphomimetic ASCL1 reveals that protein stability plays only a marginal role in regulating activity, while changes in amino acid charge cannot fully explain enhanced activity of the serine-proline mutant variants of ASCL1. Our work provides new insights into proneural factor activity and regulation, and suggests ways to optimize reprogramming protocols in cancer and regenerative medicine., Competing Interests: Competing interests S.G. is now employed at bit.bio. The other authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The MCF-7R breast cancer cell line, developed by treating the parental MCF-7 cells with increasing doses of doxorubicin, serves as a model for studying acquired multidrug resistance (MDR). MDR is a major challenge in cancer therapy, often driven by overexpression of the efflux pump P-glycoprotein (P-gp) and epigenetic modifications. While many P-gp inhibitors show promise in vitro, their nonspecific effects on the efflux pump limit in vivo application. Curcumin, a natural compound with pleiotropic action, is a nontoxic P-gp inhibitor capable of modulating multiple pathways. To explore curcumin's molecular effects on MCF-7R cells, we analyzed the expression of genes involved in DNA methylation and transcription regulation, including ABCB1/MDR1 . Reduced representation bisulfite sequencing further unveiled key epigenetic changes induced by curcumin. Our findings indicate that curcumin treatment not only modulates critical cellular processes, such as ribosome biogenesis and cytoskeletal dynamics, but also reverses the resistant phenotype, toward that of sensitive cells. This study highlights curcumin's potential as an adjuvant therapy to overcome chemoresistance, offering new avenues for pharmacological strategies targeting epigenetic regulation to re-sensitize resistant cancer cells.

Retinal degeneration in mammals causes permanent loss of vision, due to an inability to regenerate naturally. Some non-mammalian vertebrates show robust regeneration, via Muller glia (MG). We have recently made significant progress in stimulating adult mouse MG to regenerate functional neurons by transgenic expression of the proneural transcription factor Ascl1. While these results showed that MG can serve as an endogenous source of neuronal replacement, the efficacy of this process is limited. With the goal of improving this in mammals, we designed a small molecule screen using sci-Plex, a method to multiplex up to thousands of single-nucleus RNA-seq conditions into a single experiment. We used this technology to screen a library of 92 compounds, identified, and validated two that promote neurogenesis in vivo. Our results demonstrate that high-throughput single-cell molecular profiling can substantially improve the discovery process for molecules and pathways that can stimulate neural regeneration and further demonstrate the potential for this approach to restore vision in patients with retinal disease., Competing Interests: AT, MH, LT, FK, NB No competing interests declared, CT SAB member, consultant and/or co-founder of Algen Biotechnologies, Altius Therapeutics, and Scale Biosciences, TR SAB member, consultant and/or co-founder of Tenpoint Therapeutics, (© 2023, Tresenrider et al.)

BRG1 has been found to promote the generation of induced pluripotent stem cells (iPSCs) by regulating epigenetic modifications or binding to transcription factors, however, the role of BRG1 on the cellular metabolism during reprogramming has not been reported. In this study, we found that BRG1 improved the efficiency of porcine iPSC generation, and upregulated the expression of pluripotency-related factors. Further analysis revealed that BRG1 promoted cellular glycolysis, and increased levels of glycolysis-related metabolites. It enhanced the transcriptional activity of glycolysis-related gene HK2, PKM2, and PFK-1 promoters, and decreased the enrichment of H3K9me3 in glycolysis- and pluripotency-related gene promoters. BRG1 also increased the phosphorylation level at the Ser473 site of AKT protein. The specific PI3K/AKT signaling pathway inhibitor, LY294002, impaired the generation of porcine iPSCs, downregulated the expression of pluripotency-related factors, and inhibited cellular glycolysis, overexpressing BRG1 rescued those changes caused by LY294002 treatment. In addition, the glycolysis inhibitor 2-DG and BRG1 inhibitor PFI-3 had similar effects to LY294002. The above results suggest that overexpression of BRG1 promotes the generation of porcine iPSCs by facilitating glycolytic reprogramming through the PI3K/AKT signaling pathway., Competing Interests: Declaration. Conflict of interest: The authors declare that they have no competing interests. Ethical approval and consent to participate: Not applicable. Consent for publication: Not applicable., (© 2024. The Author(s).)

Cellular reprogramming of somatic cells towards induced pluripotency is a multistep stochastic process mediated by the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM), which orchestrate global epigenetic and transcriptional changes. We performed a large-scale analysis of integrated ChIP-seq, ATAC-seq and RNA-seq data and revealed the spatiotemporal highly dynamic pattern of OSKM DNA binding during reprogramming. We found that OSKM show distinct temporal patterns of binding to different classes of pluripotency-related enhancers. Genes involved in reprogramming are regulated by the coordinated activity of multiple enhancers, which are sequentially bound by OSKM for strict transcriptional control. Based on these findings, we developed an unbiased approach to identify Reprogramming-Inducible Enhancers (RIEs), constructed enhancer-traps and isolated cells undergoing reprogramming in real time. We used a representative RIE taken from the Upp1 gene fused to Gfp and isolated cells at different time-points during reprogramming and found that they have unique developmental capacities as they are reprogrammed with high efficiency due to their distinct molecular signatures. In conclusion, our experiments have led to the development of an unbiased method to identify and isolate reprogrammable cells in real time by exploiting the functional dynamics of OSKM, which can be used as efficient reprogramming biomarkers.

Gene dysregulations are associated with many human diseases, such as cancers and hereditary diseases. Artificial transcription factors (ATFs) are synthetic molecular tools to regulate the expression of disease-associated genes, which is of great significance in basic biological research and biomedical applications. Recent advances in the engineering of ATFs for regulating endogenous gene expression provide an expanded set of tools for understanding and treating diseases. However, the potential immunogenicity, large size, inefficient delivery, and off-target effects persist as obstacles for ATFs to be developed into therapeutics. Moreover, the activation of an endogenous gene following ATF activity lacks durability. In this review, we first describe the functional components of ATFs, including DNA-binding domains, transcriptional effector domains, and control switches. We then highlight examples of applications of ATFs, including cell reprogramming and differentiation, pathogenic gene screening, and disease treatment. Finally, we analyze and summarize major challenges for the clinical translation of ATFs and propose potential strategies to improve these useful molecular tools., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

To increase antibody affinity against pathogens, positively selected GC-B cells initiate cell division in the light zone (LZ) of germinal centers (GCs). Among these, higher-affinity clones migrate to the dark zone (DZ) and vigorously proliferate by utilizing energy provided by oxidative phosphorylation (OXPHOS). However, it remains unknown how positively selected GC-B cells adapt their metabolism for cell division in the glycolysis-dominant, cell cycle arrest-inducing, hypoxic LZ microenvironment. Here, we show that microRNA (miR)-155 mediates metabolic reprogramming during positive selection to protect high-affinity clones. Mechanistically, miR-155 regulates H3K36me2 levels in hypoxic conditions by directly repressing the histone lysine demethylase, Kdm2a, whose expression increases in response to hypoxia. The miR-155-Kdm2a interaction is crucial for enhancing OXPHOS through optimizing the expression of vital nuclear mitochondrial genes under hypoxia, thereby preventing excessive production of reactive oxygen species and subsequent apoptosis. Thus, miR-155-mediated epigenetic regulation promotes mitochondrial fitness in high-affinity GC-B cells, ensuring their expansion and consequently affinity maturation., Competing Interests: Competing interests: The authors declare no direct competing financial or nonfinancial interests., (© 2024. The Author(s).)

Primordial germ cells (PGCs) are the precursors of sperm and eggs. They undergo genome-wide epigenetic reprogramming to erase epigenetic memory and reset the genomic potential for totipotency. Global DNA methylation erasure is a crucial part of epigenetic resetting when DNA methylation levels decrease across the genome to <5%. However, certain localized regions exhibit slower demethylation or resistance to reprogramming. Since DNA methylation plays a crucial role in transcriptional regulation, this depletion in PGCs requires mechanisms independent of DNA methylation to regulate transcriptional control during PGC reprogramming. Histone modifications are predicted to compensate for the loss of DNA methylation in gene regulation. Different histone modifications exhibit distinct patterns in PGCs undergoing epigenetic programming at the genomic level during PGC development in conjunction with changes in DNA methylation. Together, they contribute to PGC-specific genomic regulation. Recent findings related to these processes provide a comprehensive overview of germline epigenetic reprogramming and its importance in mouse and human PGC development. Additionally, we evaluated the extent to which in vitro culture techniques have replicated the development processes of human PGCs., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Induced pluripotent stem cells (iPSCs) are a new type of pluripotent cells reprogrammed from somatic cells back into an embryonic-like pluripotent state of stem cells to study development, disease and potential gene therapies. The induction and regulation mechanisms of iPSCs in fish are still unclear. By using the transfection technique, we investigated the crucial function of the OSKMNL factor co-expression for somatic reprogramming in the muscle cell line of large yellow croaker (Larimichthys crocea) (LYCMs) and successfully established a stable iPSCs line (Lc-OSNL-iPSCs). Stable culturing of iPSCs with high alkaline phosphatase activity and a stable karyotype was achieved. The qRT-PCR and immunofluorescence labeling results revealed that Lc-OSNL-iPSCs displayed a high expression level of pluripotent marker genes such as Nanog, Oct4, and Sox2. There were significant differences between Lc-OSNL-iPSCs, Lc-OSKMNL-iPSCs, and LYCMs, and the expression of several genes in maintaining cell pluripotency was up-regulated when the pluripotency signal pathway of stem cells was activated. The technical system for inducing iPSCs of Larimichthys crocea was constructed in this study. This system can serve as a basic model to understand germ cell differentiation mechanism, gender control, genetics, and breeding of large yellow croaker and a platform for studying iPSCs in fish. Interestingly, the acquired iPSCs serves as a useful material for the directional induction of muscle stem cells, thereby establishing the groundwork for obtaining "artificial fish" in the future., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

When PIWIL2 expression is restored via heterogeneous integration of human papillomavirus, cellular reprogramming is initiated to form tumor-initiating cells (TICs), which triggers cervical squamous intraepithelial lesions (SIL). TIC stemness is critical for the prognosis of SIL. However, the mechanisms underlying TIC stemness maintenance and tumorigenicity remain unclear. Here, it is revealed that aberrant pyruvate dehydrogenase kinase 1 (PDK1) expression is closely related to aerobic glycolysis in SIL and poor survival in patients with cervical cancer. Mechanistically, that PIWIL2, which induced by stable transfection of either PIWIL2 or HPV16 oncogene E6 in human primary cervical basal epithelial cells and keratinocyte cell line HaCaT, upregulates PDK1 expression via the LIN28/let-7 axis, hence reprogramming metabolism to activate glycolysis and synchronize with TIC formation. It is further demonstrate that PDK1 is critical for TIC stemness maintenance and tumorigenicity via the PI3K/AKT/mTOR pathway both in vitro and in vivo, revealing a previously unclear mechanism for SIL progression, regression or relapse. Therefore, this findings suggest a potential rationale for prognostic predictions and selecting targeted therapy for cervical lesions., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

The memory of cellular identity is crucial for the correct development of an individual and is maintained throughout life by the epigenome. Chromatin marks, such as DNA methylation and histone modifications, ensure the stability of gene expression programmes over time and through cell division. Loss of these marks can lead to severe pathologies, including cancer and developmental syndromes. However, reprogramming of cellular identity is also a natural phenomenon that occurs early in mammalian development, particularly in the germ line, which enables the production of mature and functional gametes. The germ line transmits genetic and epigenetic information to the next generation, contributing to the survival of the species. Primordial germ cells (PGCs) undergo extensive chromatin remodelling, including global DNA demethylation and erasure of the parental imprints. This review introduces the concept of epigenetic reprogramming, its discovery and key steps, as well as the transcriptional and chromatin changes that accompany germ cell formation in mice. Finally, we discuss the epigenetic mechanisms of genomic imprinting, its discovery, regulation and relevance to human disease., (© 2024 médecine/sciences – Inserm.)

Adult mammals are susceptible to substantial cardiomyocyte (CM) loss following various cardiac diseases due to the limited capacity of CM proliferation and regeneration. Recently, direct cardiac reprogramming, converting fibroblasts into induced CMs, has been achieved both in vitro and in vivo through forced expression of transcription factors (TFs). This review encapsulates the advancements made in enhancing reprogramming efficiency and underlying molecular mechanisms. It covers the optimization of TF-based reprogramming cocktails and in vivo delivery platform and recently identified regulators in enhancing reprogramming efficiency. In addition, we discuss recent insights into the molecular mechanisms of direct cardiac reprogramming from single-cell omics analyses. Finally, we briefly touch on remaining challenges and prospective direction of this field., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Organoids have a wide range of potential applications in areas such as organ development, precision medicine, regenerative medicine, drug screening, disease modeling, and gene editing. Currently, most organoids are generated through three-dimensional (3D) in vitro culture of adult stem cells or pluripotent stem cells. However, this method of generating organoids still has several limitations and challenges, including complex manipulations, costly culturing materials, extended time requirements, and certain heterogeneity. Recently, we have found that fibroblasts, when overexpressing several key regulatory transcription factors, are able to directly and rapidly generate two types of ganglion organoids: sensory ganglion (SG) and autonomic ganglion (AG) organoids. They have structures and electrophysiological properties similar to those of endogenous organs in the body. Here, we provide a brief overview of organoid development, focusing on direct reprogramming of SG and AG organoids and their transplantation and regeneration. Finally, the advantages and prospects of direct reprogramming of organoids are discussed., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Tissue nanotransfection (TNT) topically delivers Etv2, Foxc2, and Fli1 (EFF) plasmids increasing vasculogenic fibroblasts (VF) and promoting vascularization in ischemic murine skin. Human dermal fibroblasts respond to EFF nanoelectroporation with elevated expression of endothelial genes in vitro, which is linked to increased ten-eleven translocase 1/2/3 (TET) expression. Single cell RNA sequencing dependent validation of VF induction reveals a TET-dependent transcript signature. TNT EFF also induces TET expression in vivo, and fibroblast-specific EFF overexpression leads to VF-transition, with TET-activation correlating with higher 5-hydroxymethylcytosine (5-hmC) levels in VF. VF emergence requires TET-dependent demethylation of endothelial genes in vivo, enhancing VF abundance and restoring perfusion in diabetic ischemic limbs. TNT EFF improves perfusion and wound closure in diabetic mice, while increasing VF in cultured human skin explants. Suppressed in diabetes, TET1/2/3 play a critical role in TNT-mediated VF formation which supports de novo blood vessel development to rescue diabetic ischemic tissue., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Cardiac differentiation of human pluripotent stem cells is not only a new strategy of regenerative therapy for cardiovascular disease treatment but also provides unique opportunities for the study of in vitro disease models and human heart development. To elucidate the dynamic gene regulatory networks and pivotal regulators involved in the cardiomyocyte differentiation process, we conducted an analysis of single-cell RNA sequencing data obtained from the reprogramming of two human induced pluripotent stem cell (iPSC) lines into cardiomyocytes. The data were collected from 32,365 cells at 4 stages of this process. We successfully identified cardiomyocyte clusters and several other cell clusters with different molecular characteristics derived from iPSC and described the differentiation trajectory of cardiomyocytes during differentiation in vitro. Through differential gene analysis and SCENIC analysis, we identified several candidate genes including CREG and NR2F2 that play an important regulatory role in cardiomyocyte lineage commitment. This study provides the key differentiation trajectory of heart differentiation in vitro at single-cell resolution and reveals the molecular basis of heart development and differentiation of iPSC-derived cardiomyocytes., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Talpid3 is a basal body protein required for the formation of primary cilia, an organelle involved in signal transduction. Here, we asked if Talpid3 has a role in the regulation of differentiation and/or self-renewal of ES cells and whether cells lacking cilia due to a deletion in Talpid3 can be reprogrammed to induced pluripotent stem (iPS) cells. We show that mouse embryonic limb fibroblasts which lack primary cilia with a targeted deletion in the Talpid3 ( Ta3 ) gene can be efficiently reprogrammed to iPS cells. Furthermore, vector-free Ta3 -/- iPS cells retain ES cell features and are able to self-renew. However, both Ta3 -/- iPS and ES cells are unable to form visceral endoderm and differentiate poorly into neurons. The observed defects are not a consequence of reprogramming since Ta3 -/- ES cells also exhibit this phenotype. Thus, Talpid3 and primary cilia are required for some differentiation events but appear to be dispensable for stem cell self-renewal and reprogramming.

Mesenchymal stem/stromal cells (MSCs) are becoming increasingly important for biomedical applications, such as cell therapy, disease modeling, and drug screening. At the same time, long-term cultivation, which is necessary to prepare a sufficient amount of cellular material for therapeutic and research purposes, is accompanied by the development of replicative senescence. Partial reprogramming emerged as a novel method that shows promising results in the rejuvenation of cells in vitro and in vivo; however, it has not yet been applied for human MSCs that have undergone replicative senescence in culture. In the present study, we subjected senescent human endometrial MSCs to partial reprogramming using Sendai virus vectors containing genes encoding Yamanaka transcription factors Oct4, Sox2, Klf4, and c-Myc. Characterization of the MSCs 5 days after transduction showed the loss of key markers of senescence: the youthful morphology was restored, the expression of senescent-associated β-galactosidase and the number of double-strand DNA breaks decreased, proliferation was activated, and the DNA damage response was enhanced. Further, using an in vitro wound-healing assay, we demonstrated that conditioned medium from partially reprogrammed MSCs showed higher therapeutic activity than that from senescent cells. However, a biosafety test revealed the presence of viral components in conditioned medium, which caused the agglutination of erythrocytes. Collectively, our data suggest that partial reprogramming is a potentially effective strategy for the rejuvenation of cultured MSCs in late passages but requires the use of virus-free protocols, such as chemical reprogramming.

ETV2 is an essential transcription factor as Etv2 null murine embryos lack all vasculature, blood and are lethal early during embryogenesis. Previous studies have established that ETV2 functions as a pioneer factor and directly reprograms fibroblasts to endothelial cells. However, the underlying molecular mechanisms regulating this reprogramming process remain incompletely defined. In the present study, we examined the ETV2-RIG1 cascade as regulators that govern ETV2-mediated reprogramming. Mouse embryonic fibroblasts (MEFs) harboring an inducible ETV2 expression system were used to overexpress ETV2 and reprogram these somatic cells to the endothelial lineage. Single-cell RNA-seq from reprogrammed fibroblasts defined the induction of the transcriptional network involved in Rig1-like receptor signaling pathways. Studies using ChIP-seq, electrophoretic mobility shift assays, and transcriptional assays demonstrated that ETV2 was a direct upstream activator of Rig1 gene expression. We further demonstrated that the knockdown of Rig1 and separately, Nfκb1 using shRNA significantly reduced the efficiency of endothelial cell reprogramming. These results highlight that ETV2 reprograms fibroblasts to endothelial cells by directly activating RIG1. These findings extend our current understanding of the molecular mechanisms underlying ETV2-mediated reprogramming and will be important in the design of revascularization strategies for the treatment of ischemic tissues such as ischemic heart disease., Competing Interests: Declarations Competing interests Drs. Daniel J. Garry and Mary G. Garry are co-founders of NorthStar Genomics, LLC., (© 2024. The Author(s).)

Direct pro-neural reprogramming is a conversion of differentiated somatic cells to neural cells without an intermediate pluripotency stage. It is usually achieved via ectopic expression (EE) of certain transcription factors (TFs) or other reprogramming factors (RFs). Determining the transcriptional changes (TCs) caused by particular RFs in a given cell line enables an informed approach to reprogramming initiation. Here, we characterized TCs in the human fibroblast cell line LF1 on the 5th day after EE of the single well-known pro-neural RFs NGN2, ASCL1, SOX2, and MSI1. As assessed by expression analysis of the bona fide neuronal markers nestin and beta-III tubulin, all four RFs initiated pro-neuronal phenotype conversion; analysis by RNA-seq revealed striking differences in the resulting TCs, although some pathways were overlapping. ASCL1 and SOX2 were not sufficient to induce significant pro-neural phenotype switches using our EE system. NGN2 induced TCs indicative of cell phenotype changes towards neural crest cells, neural stem cells, mature neurons, as well as radial glia, astrocytes, and oligodendrocyte precursors and their mature forms. MSI1 mainly induced a switch towards early stem-like cells, such as radial glia., Competing Interests: The authors declare no conflicts of interest.

Stem cells are a hallmark of animal multicellularity. Sox and POU transcription factors are associated with stemness and were believed to be animal innovations, reported absent in their unicellular relatives. Here we describe unicellular Sox and POU factors. Choanoflagellate and filasterean Sox proteins have DNA-binding specificity similar to mammalian Sox2. Choanoflagellate-but not filasterean-Sox can replace Sox2 to reprogram mouse somatic cells into induced pluripotent stem cells (iPSCs) through interacting with the mouse POU member Oct4. In contrast, choanoflagellate POU has a distinct DNA-binding profile and cannot generate iPSCs. Ancestrally reconstructed Sox proteins indicate that iPSC formation capacity is pervasive among resurrected sequences, thus loss of Sox2-like properties fostered Sox family subfunctionalization. Our findings imply that the evolution of animal stem cells might have involved the exaptation of a pre-existing set of transcription factors, where pre-animal Sox was biochemically similar to extant Sox, whilst POU factors required evolutionary innovations., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

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.)

Induced Pluripotent Stem Cells (iPSCs) are nowadays a common starting point for wide-ranging applications including 3D disease modeling (i.e. organoids) and in future regenerative medicine. Physiological processes like homeostasis, cell differentiation, development and reproduction are tightly regulated by hormones through binding to their transmembrane or nuclear receptors of target cells. Considering their pleiotropic effect, take into account also their expression in an iPSCs-based disease modeling would better recapitulate the molecular events leading to 3D organoid development and disease study. Here we reported the expression pattern of estrogen receptor (ERα) and progesterone receptor (PR) in four different iPSCs, obtained from CD34 + progenitor cells and skin fibroblasts with four different methods. Expression of ERα and PR mRNA were significantly downregulated in iPSCs as well as fibroblasts compared to MCF7 positive control. Immunofluorescence (IF) staining detected only the expression of PR protein in all the different iPSCs cell lines, while ERα was not detectable. By flow cytometry analysis we observed that the ~ 65% of the total population of iPSCs cells expressed only PR, with 100% fold increase compared to HSPCs and fibroblasts, while ERα was not expressed. Our results collectively demonstrated for the first time that the reprogramming of somatic cells into iPSCs leads to the expression of PR receptor., Competing Interests: Declarations Conflicts of Interest The authors declare no conflict of interest. Data Availability The data supporting the findings of this study are contained within the contents of this article. The datasets generated during this study will be freely provided by the corresponding author upon request. Informed Consent Informed consent was obtained from all subjects involved in the study. Institutional Review Board Statement The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committees of ASST Spedali Civili of Brescia (NP3426)., (© 2024. The Author(s).)

Objective: Mammalian somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) via the forced expression of Yamanaka reprogramming factors. However, only a limited population of the cells that pass through a particular pathway can metamorphose into iPSCs, while the others do not. This study aimed to clarify the pathways that chondrocytes follow during the reprogramming process., Design: The fate of human articular chondrocytes under reprogramming was investigated through a time-coursed single-cell transcriptomic analysis, which we termed an inverse genetic approach. The iPS interference technique was also employed to verify that chondrocytes inversely return to pluripotency following the proper differentiation pathway., Results: We confirmed that human chondrocytes could be converted into cells with an iPSC phenotype. Moreover, it was clarified that a limited population that underwent the silencing of SOX9, a master gene for chondrogenesis, at a specific point during the proper transcriptome transition pathway, could eventually become iPSCs. Interestingly, the other cells, which failed to be reprogrammed, followed a distinct pathway toward cells with a surface zone chondrocyte phenotype. The critical involvement of cellular communication network factors (CCNs) in this process was indicated. The idea that chondrocytes, when reprogrammed into iPSCs, follow the differentiation pathway backward was supported by the successful iPS interference using SOX9., Conclusions: This inverse genetic strategy may be useful for seeking candidates for the master genes for the differentiation of various somatic cells. The utility of CCNs in articular cartilage regeneration is also supported., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.)

Epigenetic aging clocks have been widely used to validate rejuvenation effects during cellular reprogramming. However, these predictions are unverifiable because the true biological age of reprogrammed cells remains unknown. We present an analytical framework to consider rejuvenation predictions from the uncertainty perspective. Our analysis reveals that the DNA methylation profiles across reprogramming are poorly represented in the aging data used to train clock models, thus introducing high epistemic uncertainty in age estimations. Moreover, predictions of different published clocks are inconsistent, with some even suggesting zero or negative rejuvenation. While not questioning the possibility of age reversal, we show that the high clock uncertainty challenges the reliability of rejuvenation effects observed during in vitro reprogramming before pluripotency and throughout embryogenesis. Conversely, our method reveals a significant age increase after in vivo reprogramming. We recommend including uncertainty estimation in future aging clock models to avoid the risk of misinterpreting the results of biological age prediction., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Human induced pluripotent stem cells (hiPSCs) are an invaluable tool to study molecular mechanisms on a human background. Culturing stem cells at an oxygen level different from their microenvironmental niche impacts their viability. To understand this mechanistically, dermal skin fibroblasts of 52 probands were reprogrammed into hiPSCs, followed by either hyperoxic (20 % O 2 ) or physioxic (5 % O 2 ) culture and proteomic profiling. Analysis of chromosomal stability by Giemsa-banding revealed that physioxic -cultured hiPSC clones exhibited less pathological karyotypes than hyperoxic (e.g. 6 % vs. 32 % mosaicism), higher pluripotency as evidenced by higher Stage-Specific Embryonic Antigen 3 positivity, higher glucose consumption and lactate production. Global proteomic analysis demonstrated lower abundance of several subunits of NADH:ubiquinone oxidoreductase (complex I) and an underrepresentation of pathways linked to oxidative phosphorylation and cellular senescence. Accordingly, release of the pro-senescent factor IGFBP3 and β-galactosidase staining were lower in physioxic hiPSCs. RNA- and ATAC-seq profiling revealed a distinct hypoxic transcription factor-binding footprint, amongst others higher expression of the HIF1α-regulated target NDUFA4L2 along with increased chromatin accessibility of the NDUFA4L2 gene locus. While mitochondrial DNA content did not differ between groups, physioxic hiPSCs revealed lower polarized mitochondrial membrane potential, altered mitochondrial network appearance and reduced basal respiration and electron transfer capacity. Blue-native polyacrylamide gel electrophoresis coupled to mass spectrometry of the mitochondrial complexes detected higher abundance of NDUFA4L2 and ATP5IF1 and loss of incorporation into complex IV or V, respectively. Taken together, physioxic culture of hiPSCs improved chromosomal stability, which was associated with downregulation of oxidative phosphorylation and senescence and extensive re-wiring of mitochondrial complex composition., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: TE is a shareholder and a member of the scientific advisory board of DiNAQOR, which is not relating to this manuscript. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. 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.)

The extracellular isoform of superoxide dismutase (SOD3) is decreased in patients and animals with pulmonary hypertension (PH). The human R213G single-nucleotide polymorphism (SNP) in SOD3 causes its release from tissue extracellular matrix (ECM) into extracellular fluids, without modulating enzyme activity, increasing cardiovascular disease risk in humans and exacerbating chronic hypoxic PH in mice. Given the importance of interstitial macrophages (IMs) to PH pathogenesis, this study aimed to determine whether R213G SOD3 increases IM accumulation and alters IM reprogramming in response to hypoxia. R213G mice and wild-type (WT) controls were exposed to hypobaric hypoxia for 4 or 14 days compared with normoxia. Flow cytometry demonstrated a transient increase in IMs at day 4 in both strains. Contrary to our hypothesis, the R213G SNP did not augment IM accumulation. To determine strain differences in the IM reprogramming response to hypoxia, we performed RNAsequencing on IMs isolated at each timepoint. We found that IMs from R213G mice exposed to hypoxia activated ECM-related pathways and a combination of alternative macrophage and proinflammatory signaling. Furthermore, when compared with WT responses, IMs from R213G mice lacked metabolic remodeling and demonstrated a blunted anti-inflammatory response between the early ( day 4 ) and later ( day 14 ) timepoints. We confirmed metabolic responses using Agilent Seahorse assays, whereby WT, but not R213G, IMs upregulated glycolysis at day 4 that returned to baseline at day 14 . Finally, we identify differential regulation of several redox-sensitive upstream regulators that could be investigated in future studies. NEW & NOTEWORTHY Redistributed expression of SOD3 out of tissue ECM due to the human R213G SNP exacerbates chronic hypoxic PH. Highlighting the importance of macrophage phenotype, our findings reveal that the R213G SNP does not exacerbate pulmonary macrophage accumulation in response to hypoxia but influences their metabolic and phenotypic reprogramming. We demonstrate a deficiency in the metabolic response to hypoxic stress in R213G macrophages, associated with weakened inflammatory resolution and activation of profibrotic pathways implicated in PH.

The tumor microenvironment (TME) is a complex and dynamic ecosystem composed of tumor cells, immune cells, supporting cells, and the extracellular matrix. Typically, the TME is characterized by an immunosuppressive state. To meet the demands of rapid proliferation, cancer cells undergo metabolic reprogramming, which enhances their biosynthesis and bioenergy supply. Immune cells require similar nutrients for activation and proliferation, leading to competition and immunosuppression within the TME. Additionally, tumor metabolites inhibit immune cell activation and function. Consequently, an immunosuppressed and immune-tolerant TME promotes cancer cell proliferation and metastasis. Long non-coding RNAs (lncRNAs), a category of non-coding RNA longer than 200 nucleotides, regulate tumor metabolic reprogramming by interacting with key enzymes, transporters, and related signaling pathways involved in tumor metabolism. Furthermore, lncRNAs can interact with both cellular and non-cellular components in the TME, thereby facilitating tumor growth, metastasis, drug resistance, and inducing immunosuppression. Recent studies have demonstrated that lncRNAs play a crucial role in reshaping the TME by regulating tumor metabolic reprogramming. In this discussion, we explore the potential mechanisms through which lncRNAs regulate tumor metabolic reprogramming to remodel the TME. Additionally, we examine the prospects of lncRNAs as targets for anti-tumor therapy and as biomarkers for tumor prognosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jiao, Zhao, Li, Jin and Liu.)

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).)

Peripheral Nerve Injuries (PNI) affect more than 20 million Americans and severely impact quality of life by causing long-term disability. PNI is characterized by nerve degeneration distal to the site of nerve injury resulting in long periods of skeletal muscle denervation. During this period, muscle fibers atrophy and frequently become incapable of "accepting" innervation because of the slow speed of axon regeneration post injury. We hypothesize that reprogramming the skeletal muscle to an embryonic-like state may preserve its reinnervation capability following PNI. To this end, we generate a mouse model in which NANOG, a pluripotency-associated transcription factor is expressed locally upon delivery of doxycycline (Dox) in a polymeric vehicle. NANOG expression in the muscle upregulates the percentage of Pax7+ nuclei and expression of eMYHC along with other genes that are involved in muscle development. In a sciatic nerve transection model, NANOG expression leads to upregulation of key genes associated with myogenesis, neurogenesis and neuromuscular junction (NMJ) formation. Further, NANOG mice demonstrate extensive overlap between synaptic vesicles and NMJ acetylcholine receptors (AChRs) indicating restored innervation. Indeed, NANOG mice show greater improvement in motor function as compared to wild-type (WT) animals, as evidenced by improved toe-spread reflex, EMG responses and isometric force production. In conclusion, we demonstrate that reprogramming muscle can be an effective strategy to improve reinnervation and functional outcomes after PNI., (© 2024. The Author(s).)

Plant protoplasts provide starting material for of inducing pluripotent cell masses that are competent for tissue regeneration in vitro, analogous to animal induced pluripotent stem cells (iPSCs). Dedifferentiation is associated with large-scale chromatin reorganisation and massive transcriptome reprogramming, characterised by stochastic gene expression. How this cellular variability reflects on chromatin organisation in individual cells and what factors influence chromatin transitions during culturing are largely unknown. Here, we used high-throughput imaging and a custom supervised image analysis protocol extracting over 100 chromatin features of cultured protoplasts. The analysis revealed rapid, multiscale dynamics of chromatin patterns with a trajectory that strongly depended on nutrient availability. Decreased abundance in H1 (linker histones) is hallmark of chromatin transitions. We measured a high heterogeneity of chromatin patterns indicating intrinsic entropy as a hallmark of the initial cultures. We further measured an entropy decline over time, and an antagonistic influence by external and intrinsic factors, such as phytohormones and epigenetic modifiers, respectively. Collectively, our study benchmarks an approach to understand the variability and evolution of chromatin patterns underlying plant cell reprogramming in vitro., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)