Uncovering underlying physical principles and driving forces of cell differentiation and reprogramming from single-cell transcriptomics.

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.

The interferon γ pathway enhances pluripotency and X-chromosome reactivation in iPSC reprogramming.

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.

Maternal Innate Immune Reprogramming After Complicated Pregnancy.

PROBLEM: Preeclampsia (PE) and fetal growth restriction (FGR) are often associated with maternal inflammation and an increased risk of cardiovascular and metabolic disease in the affected mothers. The mechanism responsible for this increased risk of subsequent disease may involve reprogramming of innate immune cells, characterized by epigenetic modifications. METHOD OF STUDY: Circulating monocytes from women with PE, FGR, or uncomplicated pregnancies (control) were isolated before labor. Cytokine release from monocytes following exposure to lipopolysaccharide (LPS) and the presence of lysine 4-trimethylated histone 3 (H3K4me3) within TNF promoter sequences were evaluated. Single-cell transcriptomic profiles of circulating monocytes from women with PE or uncomplicated pregnancies were assessed. RESULTS: Monocytes from women with PE or FGR exhibited increased IL-10 secretion and decreased IL-1ß and GM-CSF secretion in response to LPS. While TNFα secretion was not significantly different in cultures of control monocytes versus those from complicated pregnancies with or without LPS exposure, monocytes from complicated pregnancies had significantly decreased levels of H3K4me3 associated with TNF promoter sequences. Cluster quantification and pathway analysis of differentially expressed genes revealed an increased proportion of anti-inflammatory myeloid cells and a lower proportion of inflammatory non-classical monocytes among the circulating monocyte population in women with PE. CONCLUSIONS: Monocytes from women with PE and FGR exhibit an immune tolerance phenotype before initiation of labor. Further investigation is required to determine whether this tolerogenic phenotype persists after the affected pregnancy and contributes to increased risk of subsequent disease.

Epigenetic reprogramming driving successful and failed repair in acute kidney injury.

Acute kidney injury (AKI) causes epithelial damage followed by subsequent repair. While successful repair restores kidney function, this process is often incomplete and can lead to chronic kidney disease (CKD) in a process called failed repair. To better understand the epigenetic reprogramming driving this AKI-to-CKD transition, we generated a single-nucleus multiomic atlas for the full mouse AKI time course, consisting of ~280,000 single-nucleus transcriptomes and epigenomes. We reveal cell-specific dynamic alterations in gene regulatory landscapes reflecting, especially, activation of proinflammatory pathways. We further generated single-nucleus multiomic data from four human AKI samples including validation by genome-wide identification of nuclear factor κB binding sites. A regularized regression analysis identifies key regulators involved in both successful and failed repair cell fate, identifying the transcription factor CREB5 as a regulator of both successful and failed tubular repair that also drives proximal tubular cell proliferation after injury. Our interspecies multiomic approach provides a foundation to comprehensively understand cell states in AKI.

Metabolic states influence chicken retinal pigment epithelium cell fate decisions.

During tissue regeneration, proliferation, dedifferentiation and reprogramming are necessary to restore lost structures. However, it is not fully understood how metabolism intersects with these processes. Chicken embryos can regenerate their retina through retinal pigment epithelium (RPE) reprogramming when treated with fibroblast factor 2 (FGF2). Using transcriptome profiling, we uncovered extensive regulation of gene sets pertaining to proliferation, neurogenesis and glycolysis throughout RPE-to-neural retina reprogramming. By manipulating cell media composition, we determined that glucose, glutamine or pyruvate are individually sufficient to support RPE reprogramming, identifying glycolysis as a requisite. Conversely, the activation of pyruvate dehydrogenase by inhibition of pyruvate dehydrogenase kinases, induces epithelial-to-mesenchymal transition, while simultaneously blocking the activation of neural retina fate. We also identified that epithelial-to-mesenchymal transition fate is partially driven by an oxidative environment. Our findings provide evidence that metabolism controls RPE cell fate decisions and provide insights into the metabolic state of RPE cells, which are prone to fate changes in regeneration and pathologies, such as proliferative vitreoretinopathy.

Generation of Human-Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells of C9ORF72-Associated Amyotrophic Lateral Sclerosis Patients.

Disease modeling of neuromuscular disorders, such as amyotrophic lateral sclerosis (ALS), is hindered by limited accessibility of affected cells. This problem can be overcome by generation of human induced pluripotent stem cells (hiPSC), which can be then differentiated into required cells. Here, we describe the detailed protocol of hiPSC establishment from peripheral blood mononuclear cells (PBMC) of two ALS patients with detected expansion of G4C2 (GGGGCC) repeats in the first intron of C9ORF72 gene, known to be linked with the most common form of familial ALS.Successful PBMC reprogramming with non-integrating Sendai vectors was confirmed by expression of pluripotency markers: OCT4, NANOG, SSEA4, and TRA-1-60 in obtained hiPSC and their ability to differentiate into cells of three germ layers.The generated ALS-patient-specific hiPSC create a possibility for deciphering molecular basis of this devastating neuromuscular disease.

A Bibliometric Analysis of Metabolic Reprogramming in the Tumor Microenvironment From 2003 to 2022.

BACKGROUND: Despite considerable progress in cancer immunotherapy, it is not available for many patients. Resistance to immune checkpoint blockers arises from the intricate interactions between cancer and its microenvironment. Metabolic reprogramming in tumor and immune cells in the tumor microenvironment (TME) influences anti-tumor immune responses by remodeling the immune microenvironment. Metabolic reprogramming has emerged as an important hallmark of tumorigenesis. However, few studies have focused on the TME and metabolic reprogramming. Therefore, we aimed to explore the current research status and popular topics in TME-related metabolic reprogramming over a 20 years using a bibliometric approach. METHODS: Studies focusing on metabolic reprogramming and TME were searched using the Web of Science Core Collection database. Bibliometric and visual analyses of the articles and reviews were performed using Bibliometrix, VOSviewer, and CiteSpace. RESULTS: In total, 4726 articles published between 2003 and 2022 were selected. The number of publications and citations has increased annually. Cooperation network analysis indicated that the United States holds the foremost position in metabolic reprogramming and TME research with the highest volume of publications and citations, thus exerting the greatest influence. Among these institutions, Fudan University displayed the highest level of productivity. Frontiers in Immunology showed the highest degree of productivity in this field. Ho Ping-Chih made the most article contributions, and Pearce Edward J. was the most co-cited author. Four clusters were obtained after a cluster analysis of the authors' keywords: TME, metabolic reprogramming, immunometabolism, and immunity. Immunometabolism, glycolysis, immune cells, and tumor-associated macrophages are relatively recent keywords that have attracted increasing attention. CONCLUSIONS: A comprehensive landscape of advancements in metabolic reprogramming and the TME was evaluated, which provided crucial information for scholars to further advance this promising field. Further research should explore new topics related to immunometabolism in the TME using a transdisciplinary approach.

Generation of Induced Pluripotent Stem Cells from Erythroid Progenitor Cells.

Induced pluripotent stem cells (iPSCs) are generated through the reprogramming of somatic cells to an embryonic-like state by activating specific genes. They closely resemble embryonic stem cells (ESCs), in various aspects, including the expression of key stem cell genes, potency, and differentiation capabilities. iPSCs can be derived from various cell types such as fibroblasts, keratinocytes, and peripheral blood mononuclear cells (PBMCs). The ease of obtaining origin cells through non-invasive methods simplifies the generation of human iPSCs. Therefore, PBMCs are commonly preferred, with erythroid progenitor cells (EPCs) obtained through EPC enrichment being used as origin cells in this protocol. The EPC enrichment performed in this protocol not only reduces costs but also increases efficiency by enhancing the percentage of reprogrammable cells with progenitor characteristics. Human iPSCs are incredibly valuable for in vitro research, cell therapy, drug discovery, and tissue engineering. The outlined procedures below provide a general framework for inducing iPSCs from erythroid progenitor cells, pluripotency confirmation experiments, and cultivating them for downstream experiments.

Generation of Directly Reprogrammed Human Endothelial Cells.

Direct reprogramming provides a novel breakthrough for generating functional endothelial cells (ECs) without the need for intermediate stem or progenitor states, offering a promising resource for cardiovascular research and treatment. ETV2 is a key transcription factor that has been identified as a pioneering factor for specifying endothelial lineage. Achieving precise ETV2 induction is essential for effective endothelial reprogramming, and maintaining the reprogrammed cellular phenotype relies on a specific combination of growth factors and small molecules. Thus, we hereby provide a straightforward and comprehensive protocol for generating two distinct types of reprogrammed ECs (rECs) from human dermal fibroblasts (HDFs). Early rECs demonstrate a robust neovascularization property but lack the mature EC phenotype, while late rECs exhibit phenotypical similarity to human postnatal ECs and have a neovascularization capacity similar to early rECs. Both cell types can be derived from human somatic source cells, making them suitable for personalized disease investigations, drug discovery, and disease therapy.

Metabolic reprogramming of poly(morpho)nuclear giant cells determines glioblastoma recovery from doxorubicin-induced stress.

BACKGROUND: Multi-drug resistance of poly(morpho)nuclear giant cells (PGCs) determines their cytoprotective and generative potential in cancer ecosystems. However, mechanisms underlying the involvement of PGCs in glioblastoma multiforme (GBM) adaptation to chemotherapeutic regimes remain largely obscure. In particular, metabolic reprogramming of PGCs has not yet been considered in terms of GBM recovery from doxorubicin (DOX)-induced stress. METHODS: Long-term proteomic and metabolic cell profiling was applied to trace the phenotypic dynamics of GBM populations subjected to pulse DOX treatment in vitro, with a particular focus on PGC formation and its metabolic background. The links between metabolic reprogramming, drug resistance and drug retention capacity of PGCs were assessed, along with their significance for GBM recovery from DOX-induced stress. RESULTS: Pulse DOX treatment triggered the transient formation of PGCs, followed by the appearance of small expanding cell (SEC) clusters. Development of PGCs was accompanied by the mobilization of their metabolic proteome, transient induction of oxidative phosphorylation (OXPHOS), and differential intracellular accumulation of NADH, NADPH, and ATP. The metabolic background of PGC formation was confirmed by the attenuation of GBM recovery from DOX-induced stress following the chemical inhibition of GSK-3ß, OXPHOS, and the pentose phosphate pathway. Concurrently, the mobilization of reactive oxygen species (ROS) scavenging systems and fine-tuning of NADPH-dependent ROS production systems in PGCs was observed. These processes were accompanied by perinuclear mobilization of ABCB1 and ABCG2 transporters and DOX retention in the perinuclear PGC compartments. CONCLUSIONS: These data demonstrate the cooperative pattern of GBM recovery from DOX-induced stress and the crucial role of metabolic reprogramming of PGCs in this process. Metabolic reprogramming enhances the efficiency of self-defense systems and increases the DOX retention capacity of PGCs, potentially reducing DOX bioavailability in the proximity of SECs. Consequently, the modulation of PGC metabolism is highlighted as a potential target for intervention in glioblastoma treatment.

Modeling late-onset Alzheimer's disease neuropathology via direct neuronal reprogramming.

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-ß (Aß) 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 Aß-dependent neurodegeneration, and treatment with ß- or γ-secretase inhibitors before (but not subsequent to) Aß deposit formation mitigated neuronal death. Moreover inhibiting age-associated retrotransposable elements in LOAD neurons reduced both Aß deposition and neurodegeneration. Our study underscores the efficacy of modeling late-onset neuropathology of LOAD through high-efficiency miRNA-based neuronal reprogramming.

The significance of lipid metabolism reprogramming of tumor-associated macrophages in hepatocellular carcinoma.

In the intricate landscape of the tumor microenvironment, tumor-associated macrophages (TAMs) emerge as a ubiquitous cellular component that profoundly affects the oncogenic process. The microenvironment of hepatocellular carcinoma (HCC) is characterized by a pronounced infiltration of TAMs, underscoring their pivotal role in modulating the trajectory of the disease. Amidst the evolving therapeutic paradigms for HCC, the strategic reprogramming of metabolic pathways presents a promising avenue for intervention, garnering escalating interest within the scientific community. Previous investigations have predominantly focused on elucidating the mechanisms of metabolic reprogramming in cancer cells without paying sufficient attention to understanding how TAM metabolic reprogramming, particularly lipid metabolism, affects the progression of HCC. In this review article, we intend to elucidate how TAMs exert their regulatory effects via diverse pathways such as E2F1-E2F2-CPT2, LKB1-AMPK, and mTORC1-SREBP, and discuss correlations of TAMs with these processes and the characteristics of relevant pathways in HCC progression by consolidating various studies on TAM lipid uptake, storage, synthesis, and catabolism. It is our hope that our summary could delineate the impact of specific mechanisms underlying TAM lipid metabolic reprogramming on HCC progression and provide useful information for future research on HCC and the development of new treatment strategies.

Global changes of miRNA expression indicates an increased reprogramming efficiency of induced mammary epithelial cells by repression of miR-222-3p in fibroblasts.

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-ß 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.

Robust reprogramming of glia into neurons by inhibition of Notch signaling and nuclear factor I (NFI) factors in adult mammalian retina.

Generation of neurons through direct reprogramming has emerged as a promising therapeutic approach for treating neurodegenerative diseases. In this study, we present an efficient method for reprogramming retinal glial cells into neurons. By suppressing Notch signaling by disrupting either Rbpj or Notch1/2, we induced mature Müller glial cells to reprogram into bipolar- and amacrine-like neurons. We demonstrate that Rbpj directly activates both Notch effector genes and genes specific to mature Müller glia while indirectly repressing expression of neurogenic basic helix-loop-helix (bHLH) factors. Combined loss of function of Rbpj and Nfia/b/x resulted in conversion of nearly all Müller glia to neurons. Last, inducing Müller glial proliferation by overexpression of dominant-active Yap promotes neurogenesis in both Rbpj- and Nfia/b/x/Rbpj-deficient Müller glia. These findings demonstrate that Notch signaling and NFI factors act in parallel to inhibit neurogenic competence in mammalian Müller glia and help clarify potential strategies for regenerative therapies aimed at treating retinal dystrophies.

Reprogramming Tumor-Associated Macrophages with a Se-Based Core-Satellite Nanoassembly to Enhance Cancer Immunotherapy.

Tumor-associated macrophages (TAMs), as the most prevalent immune cells in the tumor microenvironment, play a pivotal role in promoting tumor development through various signaling pathways. Herein, we have engineered a Se@ZIF-8 core-satellite nanoassembly to reprogram TAMs, thereby enhancing immunotherapy outcomes. When the nanoassembly reaches the tumor tissue, selenium nanoparticles and Zn2+ are released in response to the acidic tumor microenvironment, resulting in a collaborative effort to promote the production of reactive oxygen species (ROS). The generated ROS, in turn, activate the nuclear factor κB (NF-κB) signaling pathway, driving the repolarization of TAMs from M2-type to M1-type, effectively eliminating cancer cells. Moreover, the nanoassembly can induce the immunogenic death of cancer cells through excess ROS to expose calreticulin and boost macrophage phagocytosis. The Se@ZIF-8 core-satellite nanoassembly provides a potential paradigm for cancer immunotherapy by reversing the immunosuppressive microenvironment.

Decoding single-cell molecular mechanisms in astrocyte-to-iN reprogramming via Ngn2- and Pax6-mediated direct lineage switching.

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.

The significant role of amino acid metabolic reprogramming in cancer.

Amino acid metabolism plays a pivotal role in tumor microenvironment, influencing various aspects of cancer progression. The metabolic reprogramming of amino acids in tumor cells is intricately linked to protein synthesis, nucleotide synthesis, modulation of signaling pathways, regulation of tumor cell metabolism, maintenance of oxidative stress homeostasis, and epigenetic modifications. Furthermore, the dysregulation of amino acid metabolism also impacts tumor microenvironment and tumor immunity. Amino acids can act as signaling molecules that modulate immune cell function and immune tolerance within the tumor microenvironment, reshaping the anti-tumor immune response and promoting immune evasion by cancer cells. Moreover, amino acid metabolism can influence the behavior of stromal cells, such as cancer-associated fibroblasts, regulate ECM remodeling and promote angiogenesis, thereby facilitating tumor growth and metastasis. Understanding the intricate interplay between amino acid metabolism and the tumor microenvironment is of crucial significance. Expanding our knowledge of the multifaceted roles of amino acid metabolism in tumor microenvironment holds significant promise for the development of more effective cancer therapies aimed at disrupting the metabolic dependencies of cancer cells and modulating the tumor microenvironment to enhance anti-tumor immune responses and inhibit tumor progression.

Mycb and Mych stimulate Müller glial cell reprogramming and proliferation in the uninjured and injured zebrafish retina.

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.

Atf3-mediated metabolic reprogramming in hepatic macrophage orchestrates metabolic dysfunction-associated steatohepatitis.

Metabolic dysfunction-associated steatohepatitis (MASH) is regulated by complex interplay between the macrophages and surrounding cells in the liver. Here, we show that Atf3 regulates glucose-fatty acid cycle in macrophages attenuates hepatocyte steatosis, and fibrogenesis in hepatic stellate cells (HSCs). Overexpression of Atf3 in macrophages protects against the development of MASH in Western diet-fed mice, whereas Atf3 ablation has the opposite effect. Mechanistically, Atf3 improves the reduction of fatty acid oxidation induced by glucose via forkhead box O1 (FoxO1) and Cd36. Atf3 inhibits FoxO1 activity via blocking Hdac1-mediated FoxO1 deacetylation at K242, K245, and K262 and increases Zdhhc4/5-mediated CD36 palmitoylation at C3, C7, C464, and C466; furthermore, macrophage Atf3 decreases hepatocytes lipogenesis and HSCs activation via retinol binding protein 4 (Rbp4). Anti-Rbp4 can prevent MASH progression that is induced by Atf3 deficiency in macrophages. This study identifies Atf3 as a regulator of glucose-fatty acid cycle. Targeting macrophage Atf3 or Rbp4 may be a plausible therapeutic strategy for MASH.