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Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.

In tumor cells from virtually all patients with chronic myelogenous leukemia, the Philadelphia chromosome, a fusion of chromosomes 9 and 22, directs the synthesis of the P210bcr/abl protein. The protein-tyrosine kinase activity and hybrid structure of P210bcr/abl are similar to the oncogene product of the Abelson murine leukemia virus, P160gag/v-abl, which induces acute lymphomas. To determine whether P210bcr/abl can induce chronic myelogenous leukemia, murine bone marrow was infected with a retrovirus encoding P210bcr/abl and transplanted into irradiated syngeneic recipients. Transplant recipients developed several hematologic malignancies; prominent among them was a myeloproliferative syndrome closely resembling the chronic phase of human chronic myelogenous leukemia. Tumor tissue from diseased mice harbored the provirus encoding P210bcr/abl. These results demonstrate that P210bcr/abl expression can induce chronic myelogenous leukemia. Retrovirus-mediated expression of the protein provides a murine model system for further analysis of the disease.

Elucidating mechanisms of T cell development can guide in vitro T cell differentiation from induced pluripotent stem cells (iPSCs) and facilitate off-the-shelf T cell-based immunotherapies. Using a stroma-free human iPSC-T cell differentiation platform, we screened for epigenetic modulators that influence T cell specification and identified the H3K9-directed histone methyltransferases G9a/GLP as repressors of T cell fate. We show that G9a/GLP inhibition during specific time windows of differentiation of hematopoietic stem and progenitor cells (HSPCs) skews cell fates toward lymphoid lineages. Inhibition of G9a/GLP promotes the production of lymphoid cells during zebrafish embryonic hematopoiesis, demonstrating the evolutionary conservation of G9a/GLP function. Importantly, chemical inhibition of G9a/GLP facilitates the generation of mature iPSC-T cells that bear transcriptional similarity to peripheral blood αβ T cells. When engineered to express chimeric antigen receptors, the epigenetically engineered iPSC-T cells exhibit enhanced effector functions in vitro and durable, persistent antitumor activity in a xenograft tumor-rechallenge model., Competing Interests: Declaration of interests R.J., G.Q.D., and Boston Children’s Hospital hold intellectual property relevant to the generation of iPSC-derived T cells. T.M.S. has received sponsored research support from Elevate Bio. G.Q.D. is a member of Cell Stem Cell’s advisory board., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Embryonic hematopoietic stem and progenitor cells (HSPCs) have the unique ability to undergo rapid proliferation while maintaining multipotency, a clinically-valuable quality which currently cannot be replicated in vitro. Here, we show that embryonic HSPCs achieve this state by precise spatio-temporal regulation of reactive oxygen species (ROS) via Bnip3lb-associated developmentally-programmed mitophagy, a distinct autophagic regulatory mechanism from that of adult HSPCs. While ROS drives HSPC specification in the dorsal aorta, scRNAseq and live-imaging of Tg(ubi:mitoQC) zebrafish indicate that mitophagy initiates as HSPCs undergo endothelial-to-hematopoietic transition and colonize the caudal hematopoietic tissue (CHT). Knockdown of bnip3lb reduced mitophagy and HSPC numbers in the CHT by promoting myeloid-biased differentiation and apoptosis, which was rescued by anti-oxidant exposure. Conversely, induction of mitophagy enhanced both embryonic HSPC and lymphoid progenitor numbers. Significantly, mitophagy activation improved ex vivo functional capacity of hematopoietic progenitors derived from human-induced pluripotent stem cells (hiPSCs), enhancing serial-replating hematopoietic colony forming potential., Highlights: ROS promotes HSPC formation in the dorsal aorta but negatively affects maintenance thereafter.HSPCs colonizing secondary niches control ROS levels via Bnip3lb-directed mitophagy.Mitophagy protects nascent HSPCs from ROS-associated apoptosis and maintains multipotency.Induction of mitophagy enhances long-term hematopoietic potential of iPSC-derived HSPCs.

It is unknown which posttranscriptional regulatory mechanisms are required for oncogenic competence. Here, we show that the LIN28 family of RNA-binding proteins (RBPs), which facilitate posttranscriptional RNA metabolism within ribonucleoprotein networks, is essential for the initiation of diverse oncotypes of hepatocellular carcinoma (HCC). In HCC models driven by NRASG12V/Tp53, CTNNB1/YAP/Tp53, or AKT/Tp53, mice without Lin28a and Lin28b were markedly impaired in cancer initiation. We biochemically defined an oncofetal regulon of 15 factors connected to LIN28 through direct mRNA and protein interactions. Interestingly, all were RBPs and only 1 of 15 was a Let-7 target. Polysome profiling and reporter assays showed that LIN28B directly increased the translation of 8 of these 15 RBPs. As expected, overexpression of LIN28B and IGFBP1-3 was able to genetically rescue cancer initiation. Using this platform to probe components downstream of LIN28, we found that 8 target RBPs were able to restore NRASG12V/Tp53 cancer formation in Lin28a/Lin28b-deficient mice. Furthermore, these LIN28B targets promote cancer initiation through an increase in protein synthesis. LIN28B, central to an RNP regulon that increases translation of RBPs, is important for tumor initiation in the liver.

A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFý, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFý. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo , revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.

LIN28A is important in somatic reprogramming and pluripotency regulation. Although previous studies addressed that LIN28A can repress let-7 microRNA maturation in the cytoplasm, few focused on its role within the nucleus. Here, we show that the nucleolus-localized LIN28A protein undergoes liquid-liquid phase separation (LLPS) in mouse embryonic stem cells (mESCs) and in vitro. The RNA binding domains (RBD) and intrinsically disordered regions (IDR) of LIN28A contribute to LIN28A and the other nucleolar proteins' phase-separated condensate establishment. S120A, S200A and R192G mutations in the IDR result in subcellular mislocalization of LIN28A and abnormal nucleolar phase separation. Moreover, we find that the naive-to-primed pluripotency state conversion and the reprogramming are associated with dynamic nucleolar remodeling, which depends on LIN28A's phase separation capacity, because the LIN28A IDR point mutations abolish its role in regulating nucleolus and in these cell fate decision processes, and an exogenous IDR rescues it. These findings shed light on the nucleolar function in pluripotent stem cell states and on a non-canonical RNA-independent role of LIN28A in phase separation and cell fate decisions., (© 2024. The Author(s).)

Competing Interests: Conflict-of-Interest Disclosures: Amy Abernethy reports personal fees from Verily/Alphabet, relationships with Georgiamune and EQRx, and personal investments in Iterative Health and One Health, outside the submitted work. Elliot Chaikof reports grants from the National Institutes of Health, outside the submitted work. George Q. Daley reports holding equity from Redona Therapeutics and from iTCells, outside the submitted work. Juan Enriquez reports investments with Excel Venture Management, outside the submitted work; investments in various life science technologies, including leading-edge brain technologies, and co-authoring a book on the impact of emerging brain technologies. Scott Gottlieb reports personal fees from Pfi zer, Inc, Illumina, Inc, Aetion, Tempus Labs, National Resilience, Inc, Cell-Carta, Parker Institute for Cancer Immunotherapy, Mount Sinai Health System, New Enterprise Associates, and American Enterprise Institute outside the submitted work. Sohail Tavazoie reports personal fees from Inspirna, outside the submitted work. Jacob S. Sherkow reports employment with the University of Illinois, grants from National Institutes of Health, and personal fees from Expert Consulting services, outside the submitted work.

Most organs have tissue-resident immune cells. Human organoids lack these immune cells, which limits their utility in modeling many normal and disease processes. Here, we describe that pluripotent stem cell-derived human colonic organoids (HCOs) co-develop a diverse population of immune cells, including hemogenic endothelium (HE)-like cells and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of functional macrophages. HCO macrophages acquired a transcriptional signature resembling human fetal small and large intestine tissue-resident macrophages. HCO macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals and were able to phagocytose and mount a robust response to pathogenic bacteria. When transplanted into mice, HCO macrophages were maintained within the colonic organoid tissue, established a close association with the colonic epithelium, and were not displaced by the host bone-marrow-derived macrophages. These studies suggest that HE in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Extracellular vesicles (EVs) carry bioactive molecules associated with various biological processes, including miRNAs. In both Huntington's disease (HD) models and human samples, altered expression of miRNAs involved in synapse regulation was reported. Recently, the use of EV cargo to reverse phenotypic alterations in disease models with synaptopathy as the end result of the pathophysiological cascade has become an interesting possibility. Methods: Here, we assessed the contribution of EVs to GABAergic synaptic alterations using a human HD model and studied the miRNA content of isolated EVs. Results: After differentiating human induced pluripotent stem cells into electrophysiologically active striatal-like GABAergic neurons, we found that HD-derived neurons displayed reduced density of inhibitory synapse markers and GABA receptor-mediated ionotropic signaling. Treatment with EVs secreted by control (CTR) fibroblasts reversed the deficits in GABAergic synaptic transmission and increased the density of inhibitory synapses in HD-derived neuron cultures, while EVs from HD-derived fibroblasts had the opposite effects on CTR-derived neurons. Moreover, analysis of miRNAs from purified EVs identified a set of differentially expressed miRNAs between manifest HD, premanifest, and CTR lines with predicted synaptic targets. Conclusion: The EV-mediated reversal of the abnormal GABAergic phenotype in HD-derived neurons reinforces the potential role of EV-miRNAs on synapse regulation., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Bone marrow adipocytes (BMAds) constitute the most abundant stromal component of adult human bone marrow. Two subtypes of BMAds have been described, the more labile regulated adipocytes (rBMAds) and the more stable constitutive adipocytes (cBMAds), which develop earlier in life and are more resilient to environmental and metabolic disruptions. In vivo , rBMAds are enriched in saturated fatty acids, contain smaller lipid droplets (LDs) and more readily provide hematopoietic support than their cBMAd counterparts. Mouse models have been used for BMAds research, but isolation of primary BMAds presents many challenges, and thus in vitro models remain the current standard to study nuances of adipocyte differentiation. No in vitro model has yet been described for the study of rBMAds/cBMAds. Here, we present an in vitro model of BM adipogenesis with differential rBMAd and cBMAd-like characteristics. We used OP9 BM stromal cells derived from a (C57BL/6xC3H)F2-op/op mouse, which have been extensively characterized as feeder layer for hematopoiesis research. We observed similar canonical adipogenesis transcriptional signatures for spontaneously-differentiated (sOP9) and induced (iOP9) cultures, while fatty acid composition and desaturase expression of Scd1 and Fads2 differed at the population level. To resolve differences at the single adipocyte level we tested Raman microspectroscopy and show it constitutes a high-resolution method for studying adipogenesis in vitro in a label-free manner, with resolution to individual LDs. We found sOP9 adipocytes have lower unsaturation ratios, smaller LDs and higher hematopoietic support than iOP9 adipocytes, thus functionally resembling rBMAds, while iOP9 more closely resembled cBMAds. Validation in human primary samples confirmed a higher unsaturation ratio for lipids extracted from stable cBMAd-rich sites (femoral head upon hip-replacement surgery) versus labile rBMAds (iliac crest after chemotherapy). As a result, the 16:1/16:0 fatty acid unsaturation ratio, which was already shown to discriminate BMAd subtypes in rabbit and rat marrow, was validated to discriminate cBMAds from rBMAd in both the OP9 model in vitro system and in human samples. We expect our model will be useful for cBMAd and rBMAd studies, particularly where isolation of primary BMAds is a limiting step., 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 © 2022 Tratwal, Falgayrac, During, Bertheaume, Bataclan, Tavakol, Campos, Duponchel, Daley, Penel, Chauveau and Naveiras.)

Human induced pluripotent stem cells (iPSCs) provide a potentially unlimited resource for cell therapies, but the derivation of mature cell types remains challenging. The histone methyltransferase EZH1 is a negative regulator of lymphoid potential during embryonic hematopoiesis. Here, we demonstrate that EZH1 repression facilitates in vitro differentiation and maturation of T cells from iPSCs. Coupling a stroma-free T cell differentiation system with EZH1-knockdown-mediated epigenetic reprogramming, we generated iPSC-derived T cells, termed EZ-T cells, which display a highly diverse T cell receptor (TCR) repertoire and mature molecular signatures similar to those of TCRαβ T cells from peripheral blood. Upon activation, EZ-T cells give rise to effector and memory T cell subsets. When transduced with chimeric antigen receptors (CARs), EZ-T cells exhibit potent antitumor activities in vitro and in xenograft models. Epigenetic remodeling via EZH1 repression allows efficient production of developmentally mature T cells from iPSCs for applications in adoptive cell therapy., Competing Interests: Declaration of interests R.J., G.Q.D., and Boston Children’s Hospital hold intellectual property and receive consulting fees and/or hold equity interest relevant to the generation of iPSC-derived T cells. T.M.S. receives sponsored research support from Elevate Bio. G.Q.D. is a member of Cell Stem Cell’s advisory board., (Copyright © 2022 Elsevier Inc. All rights reserved.)

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28's role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development., (© 2021. The Author(s).)

This year, Cell Stem Cell and the International Society for Stem Cell Research (ISSCR) are celebrating their 15 th and 20 th anniversaries, respectively. We took the opportunity to ask the current and four former ISSCR presidents to reflect on major stem cell advances during this time, the evolution of policy, clinical translation and ethical aspects, and future challenges for the field., Competing Interests: Declaration of interests S.J.M. is a founder and scientific advisory board member of Garuda Therapeutics and Kojin Therapeutics and a shareholder and former scientific advisory board member of G1 Therapeutics; serves on the scientific advisory boards of Frequency Therapeutics and ONA Therapeutics; and chairs the Public Policy Committee for the International Society for Stem Cell Research. M.L., S.J.M., J.R., D.S., and G.Q.D. are members of the Cell Stem Cell Advisory board. D.S. is a co-founder and member of the board of directors of Tenaya Therapeutics and has equity in Tenaya Therapeutics. G.Q.D. has filed invention disclosures and patents related to pluripotent stem cells and production of hematopoietic lineages, and he holds equity in and/or receives consulting fees from 28/7 Therapeutics and iTCells., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Haematopoietic stem cells (HSCs) arise in the embryo from the arterial endothelium through a process known as the endothelial-to-haematopoietic transition (EHT) 1-4 . This process generates hundreds of blood progenitors, of which a fraction go on to become definitive HSCs. It is generally thought that most adult blood is derived from those HSCs, but to what extent other progenitors contribute to adult haematopoiesis is not known. Here we use in situ barcoding and classical fate mapping to assess the developmental and clonal origins of adult blood in mice. Our analysis uncovers an early wave of progenitor specification-independent of traditional HSCs-that begins soon after EHT. These embryonic multipotent progenitors (eMPPs) predominantly drive haematopoiesis in the young adult, have a decreasing yet lifelong contribution over time and are the predominant source of lymphoid output. Putative eMPPs are specified within intra-arterial haematopoietic clusters and represent one fate of the earliest haematopoietic progenitors. Altogether, our results reveal functional heterogeneity during the definitive wave that leads to distinct sources of adult blood., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

High-risk forms of B-acute lymphoblastic leukemia (B-ALL) remain a therapeutic challenge. Leukemia-initiating cells (LICs) self-renew and spark relapse and therefore have been the subject of intensive investigation; however, the properties of LICs in high-risk B-ALL are not well understood. Here, we use single-cell transcriptomics and quantitative xenotransplantation to understand LICs in MLL-rearranged (MLL-r) B-ALL. Compared with reported LIC frequencies in acute myeloid leukemia (AML), engraftable LICs in MLL-r B-ALL are abundant. Although we find that multipotent, self-renewing LICs are enriched among phenotypically undifferentiated B-ALL cells, LICs with the capacity to replenish the leukemic cellular diversity can emerge from more mature fractions. While inhibiting oxidative phosphorylation blunts blast proliferation, this intervention promotes LIC emergence. Conversely, inhibiting hypoxia and glycolysis impairs MLL-r B-ALL LICs, providing a therapeutic benefit in xenotransplantation systems. These findings provide insight into the aggressive nature of MLL-r B-ALL and provide a rationale for therapeutic targeting of hypoxia and glycolysis., Competing Interests: Declaration of interests G.Q.D. holds equity in companies pursuing anti-cancer treatments (including Epizyme and 28-7 Therapeutics) and patents related to cancer therapeutics. A.K.S. reports compensation for consulting and/or science advisory board (SAB) membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Ochre Bio, Third Rock Ventures, Hovione, Relation Therapeutics Limited, Empress Therapeutics, FL82, and Dahlia Biosciences., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hematopoiesis changes over life to meet the demands of maturation and aging. Here, we find that the definitive hematopoietic stem and progenitor cell (HSPC) compartment is remodeled from gestation into adulthood, a process regulated by the heterochronic Lin28b/let-7 axis. Native fetal and neonatal HSPCs distribute with a pro-lymphoid/erythroid bias with a shift toward myeloid output in adulthood. By mining transcriptomic data comparing juvenile and adult HSPCs and reconstructing coordinately activated gene regulatory networks, we uncover the Polycomb repressor complex 1 (PRC1) component Cbx2 as an effector of Lin28b/let-7's control of hematopoietic maturation. We find that juvenile Cbx2 -/- hematopoietic tissues show impairment of B-lymphopoiesis, a precocious adult-like myeloid bias, and that Cbx2/PRC1 regulates developmental timing of expression of key hematopoietic transcription factors. These findings define a mechanism of regulation of HSPC output via chromatin modification as a function of age with potential impact on age-biased pediatric and adult blood disorders., Competing Interests: Declaration of interests G.Q.D. is a founder and shareholder of 28/7 Therapeutics., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intercellular communication orchestrates a multitude of physiologic and pathologic conditions. Algorithms to infer cell-cell communication and predict downstream signalling and regulatory networks are needed to illuminate mechanisms of stem cell differentiation and tissue development. Here, to fill this gap, we developed and applied CellComm to investigate how the aorta-gonad-mesonephros microenvironment dictates haematopoietic stem and progenitor cell emergence. We identified key microenvironmental signals and transcriptional networks that regulate haematopoietic development, including Stat3, Nr0b2, Ybx1 and App, and confirmed their roles using zebrafish, mouse and human models. Notably, CellComm revealed extensive crosstalk among signalling pathways and convergence on common transcriptional regulators, indicating a resilient developmental programme that ensures dynamic adaptation to changes in the embryonic environment. Our work provides an algorithm and data resource for the scientific community., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

High expression of LIN28B is associated with aggressive malignancy and poor survival. Here, probing MYCN-amplified neuroblastoma as a model system, we showed that LIN28B expression was associated with enhanced cell migration in vitro and invasive and metastatic behavior in murine xenografts. Sequence analysis of the polyribosome fraction of LIN28B-expressing neuroblastoma cells revealed let-7-independent enrichment of transcripts encoding components of the translational and ribosomal apparatus and depletion of transcripts of neuronal developmental programs. We further observed that LIN28B utilizes both its cold shock and zinc finger RNA binding domains to preferentially interact with MYCN-induced transcripts of the ribosomal complex, enhancing their translation. These data demonstrated that LIN28B couples the MYCN-driven transcriptional program to enhanced ribosomal translation, thereby implicating LIN28B as a posttranscriptional driver of the metastatic phenotype.

The nucleolus is the organelle for ribosome biogenesis and sensing various types of stress. However, its role in regulating stem cell fate remains unclear. Here, we present evidence that nucleolar stress induced by interfering rRNA biogenesis can drive the 2-cell stage embryo-like (2C-like) program and induce an expanded 2C-like cell population in mouse embryonic stem (mES) cells. Mechanistically, nucleolar integrity maintains normal liquid-liquid phase separation (LLPS) of the nucleolus and the formation of peri-nucleolar heterochromatin (PNH). Upon defects in rRNA biogenesis, the natural state of nucleolus LLPS is disrupted, causing dissociation of the NCL/TRIM28 complex from PNH and changes in epigenetic state and reorganization of the 3D structure of PNH, which leads to release of Dux, a 2C program transcription factor, from PNH to activate a 2C-like program. Correspondingly, embryos with rRNA biogenesis defect are unable to develop from 2-cell (2C) to 4-cell embryos, with delayed repression of 2C/ERV genes and a transcriptome skewed toward earlier cleavage embryo signatures. Our results highlight that rRNA-mediated nucleolar integrity and 3D structure reshaping of the PNH compartment regulates the fate transition of mES cells to 2C-like cells, and that rRNA biogenesis is a critical regulator during the 2-cell to 4-cell transition of murine pre-implantation embryo development., (© 2021. The Author(s).)

The clinical efficacy of epidermal growth factor receptor (EGFR)–targeted therapy in EGFR -mutant non–small cell lung cancer is limited by the development of drug resistance. One mechanism of EGFR inhibitor resistance occurs through amplification of the human growth factor receptor ( MET ) proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent EGFR mutation and MET amplification are historically thought to be codependent on the activation of both oncogenes. Hence, patients whose tumors harbor both alterations are commonly treated with a combination of EGFR and MET tyrosine kinase inhibitors (TKIs). Here, we identify and characterize six patient-derived models of EGFR -mutant, MET -amplified lung cancer that have switched oncogene dependence to rely exclusively on MET activation for survival. We demonstrate in this MET-driven subset of EGFR TKI-refractory cancers that canonical EGFR downstream signaling was governed by MET, even in the presence of sustained mutant EGFR expression and activation. In these models, combined EGFR and MET inhibition did not result in greater efficacy in vitro or in vivo compared to single-agent MET inhibition. We further identified a reduced EGFR:MET mRNA expression stoichiometry as associated with MET oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitor–resistant EGFR -mutant, MET -amplified patients also exhibited a reduced EGFR:MET mRNA ratio. Our findings reveal that a subset of EGFR -mutant, MET -amplified lung cancers develop dependence on MET activation alone, suggesting that such patients could be treated with a single-agent MET TKI rather than the current standard-of-care EGFR and MET inhibitor combination regimens.

The molecular mechanisms that govern the choreographed timing of organ development remain poorly understood. Our investigation of the role of the Lin28a and Lin28b paralogs during the developmental process of branching morphogenesis establishes that dysregulation of Lin28a/b leads to abnormal branching morphogenesis in the lung and other tissues. Additionally, we find that the Lin28 paralogs, which regulate post-transcriptional processing of both mRNAs and microRNAs (miRNAs), predominantly control mRNAs during the initial phases of lung organogenesis. Target mRNAs include Sox2, Sox9, and Etv5, which coordinate lung development and differentiation. Moreover, we find that functional interactions between Lin28a and Sox9 are capable of bypassing branching defects in Lin28a/b mutant lungs. Here, we identify Lin28a and Lin28b as regulators of early embryonic lung development, highlighting the importance of the timing of post-transcriptional regulation of both miRNAs and mRNAs at distinct stages of organogenesis., Competing Interests: Declaration of interests G.Q.D. holds equity in and receives consulting fees for service on the scientific advisory board of 28/7 Therapeutics, Inc. He holds a patent on technology related to LIN28, and J.K.O., A.V.Y., and G.Q.D. have filed a provisional patent related in part to the current work., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Across species, hematopoietic stem and progenitor cells (HSPCs) arise during embryogenesis from a specialized arterial population, termed hemogenic endothelium. Here, we describe a mechanistic role for the epigenetic regulator, Enhancer of zeste homolog-1 (Ezh1), in vertebrate HSPC production via regulation of hemogenic commitment. Loss of ezh1 in zebrafish embryos favored acquisition of hemogenic (gata2b) and HSPC (runx1) fate at the expense of the arterial program (ephrinb2a, dll4). In contrast, ezh1 overexpression blocked hematopoietic progression via maintenance of arterial gene expression. The related Polycomb group subunit, Ezh2, functioned in a non-redundant, sequential manner, whereby inhibition had no impact on arterial identity, but was capable of blocking ezh1-knockdown-associated HSPC expansion. Single-cell RNA sequencing across ezh1 genotypes revealed a dropout of ezh1 +/- cells among arterial endothelium associated with positive regulation of gene transcription. Exploitation of Ezh1/2 modulation has potential functional relevance for improving in vitro HSPC differentiation from induced pluripotent stem cell sources., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Embryonic hematopoietic stem and progenitor cells (HSPCs) robustly proliferate while maintaining multilineage potential in vivo; however, an incomplete understanding of spatiotemporal cues governing their generation has impeded robust production from human induced pluripotent stem cells (iPSCs) in vitro. Using the zebrafish model, we demonstrate that NLRP3 inflammasome-mediated interleukin-1-beta (IL1β) signaling drives HSPC production in response to metabolic activity. Genetic induction of active IL1β or pharmacologic inflammasome stimulation increased HSPC number as assessed by in situ hybridization for runx1/cmyb and flow cytometry. Loss of inflammasome components, including il1b, reduced CD41 + HSPCs and prevented their expansion in response to metabolic cues. Cell ablation studies indicated that macrophages were essential for initial inflammasome stimulation of Il1rl1 + HSPCs. Significantly, in human iPSC-derived hemogenic precursors, transient inflammasome stimulation increased multilineage hematopoietic colony-forming units and T cell progenitors. This work establishes the inflammasome as a conserved metabolic sensor that expands HSPC production in vivo and in vitro., Competing Interests: Declaration of Interests G.Q.D. holds equity in 28/7 Therapeutics and Epizyme, has received sponsored research support from Megakaryon (Japan), and holds patents pertaining to HSPC production from iPSCs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 ( rps29 ) mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34 + hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34 + cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Fanconi anemia (FA) is a disorder of DNA repair that manifests as bone marrow (BM) failure. The lack of accurate murine models of FA has refocused efforts toward differentiation of patient-derived induced pluripotent stem cells (IPSCs) to hematopoietic progenitor cells (HPCs). However, an intact FA DNA repair pathway is required for efficient IPSC derivation, hindering these efforts. To overcome this barrier, we used inducible complementation of FANCA-deficient IPSCs, which permitted robust maintenance of IPSCs. Modulation of FANCA during directed differentiation to HPCs enabled the production of FANCA-deficient human HPCs that recapitulated FA genotoxicity and hematopoietic phenotypes relative to isogenic FANCA-expressing HPCs. FANCA-deficient human HPCs underwent accelerated terminal differentiation driven by activation of p53/p21. We identified growth arrest specific 6 (GAS6) as a novel target of activated p53 in FANCA-deficient HPCs and modulate GAS6 signaling to rescue hematopoiesis in FANCA-deficient cells. This study validates our strategy to derive a sustainable, highly faithful human model of FA, uncovers a mechanism of HPC exhaustion in FA, and advances toward future cell therapy in FA., (© 2020 by The American Society of Hematology.)

Mitochondrial deregulation has gained increasing support as a pathological mechanism in Huntington's disease (HD), a genetic-based neurodegenerative disorder caused by CAG expansion in the HTT gene. In this study, we thoroughly investigated mitochondrial-based mechanisms in HD patient-derived iPSC (HD-iPSC) and differentiated neural stem cells (NSC) versus control cells, as well as in cells subjected to CRISPR/Cas9-CAG repeat deletion. We analyzed mitochondrial morphology, function and biogenesis, linked to exosomal release of mitochondrial components, glycolytic flux, ATP generation and cellular redox status. Mitochondria in HD cells exhibited round shape and fragmented morphology. Functionally, HD-iPSC and HD-NSC displayed lower mitochondrial respiration, exosomal release of cytochrome c, decreased ATP/ADP, reduced PGC-1α and complex III subunit expression and activity, and were highly dependent on glycolysis, supported by pyruvate dehydrogenase (PDH) inactivation. HD-iPSC and HD-NSC mitochondria showed ATP synthase reversal and increased calcium retention. Enhanced mitochondrial reactive oxygen species (ROS) were also observed in HD-iPSC and HD-NSC, along with decreased UCP2 mRNA levels. CRISPR/Cas9-CAG repeat deletion in HD-iPSC and derived HD-NSC ameliorated mitochondrial phenotypes. Data attests for intricate metabolic and mitochondrial dysfunction linked to transcriptional deregulation as early events in HD pathogenesis, which are alleviated following CAG deletion., (Copyright © 2020 Lopes, Tang, Anjo, Manadas, Onofre, de Almeida, Daley, Schlaeger and Rego.)

Cellular reprogramming suffers from low efficiency especially for the human cells. To deconstruct the heterogeneity and unravel the mechanisms for successful reprogramming, we adopted single-cell RNA sequencing (scRNA-Seq) and single-cell assay for transposase-accessible chromatin (scATAC-Seq) to profile reprogramming cells across various time points. Our analysis revealed that reprogramming cells proceed in an asynchronous trajectory and diversify into heterogeneous subpopulations. We identified fluorescent probes and surface markers to enrich for the early reprogrammed human cells. Furthermore, combinatory usage of the surface markers enabled the fine segregation of the early-intermediate cells with diverse reprogramming propensities. scATAC-Seq analysis further uncovered the genomic partitions and transcription factors responsible for the regulatory phasing of reprogramming process. Binary choice between a FOSL1 and a TEAD4-centric regulatory network determines the outcome of a successful reprogramming. Together, our study illuminates the multitude of diverse routes transversed by individual reprogramming cells and presents an integrative roadmap for identifying the mechanistic part list of the reprogramming machinery., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Major issues exist in the provision of oral health care in America, especially to underserved populations. Access to care, health disparities, an aging population with higher chronic disease burden, and rising healthcare costs continue to impact health outcomes for millions. The marginalization of oral health care, like that of behavioral health care, is a contributor. This perspective presents an idea whose time has come: putting the mouth back in the body. Several national reports stress the imperative to better integrate the practice of medicine and dentistry, including the first-ever Surgeon General's Report on Oral Health in 2000. A plan to lead a multifaceted integration of oral health into overall health is proposed. Leaders will come from new educational and practice models stressing teamwork, interprofessional education, innovative residency training programs and even dual degree options., (© 2020 American Dental Education Association.)

LIN28B is highly expressed in neuroblastoma and promotes tumorigenesis, at least, in part, through inhibition of let-7 microRNA biogenesis. Here, we report that overexpression of either wild-type (WT) LIN28B or a LIN28B mutant that is unable to inhibit let-7 processing increases the penetrance of MYCN-induced neuroblastoma, potentiates the invasion and migration of transformed sympathetic neuroblasts, and drives distant metastases in vivo. Genome-wide chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-seq) and coimmunoprecipitation experiments show that LIN28B binds active gene promoters in neuroblastoma cells through protein-protein interaction with the sequence-specific zinc-finger transcription factor ZNF143 and activates the expression of downstream targets, including transcription factors forming the adrenergic core regulatory circuitry that controls the malignant cell state in neuroblastoma as well as GSK3B and L1CAM that are involved in neuronal cell adhesion and migration. These findings reveal an unexpected let-7 -independent function of LIN28B in transcriptional regulation during neuroblastoma pathogenesis., Competing Interests: Competing interest statement: B.J.A. is a shareholder in Syros Pharmaceuticals, Inc. The other authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Pancreatic ductal adenocarcinoma (PDAC) lethality is due to metastatic dissemination. Characterization of rare, heterogeneous circulating tumor cells (CTCs) can provide insight into metastasis and guide development of novel therapies. Using the CTC-iChip to purify CTCs from PDAC patients for RNA-seq characterization, we identify three major correlated gene sets, with stemness genes LIN28B/KLF4, WNT5A, and LGALS3 enriched in each correlated gene set; only LIN28B CTC expression was prognostic. CRISPR knockout of LIN28B-an oncofetal RNA-binding protein exerting diverse effects via negative regulation of let-7 miRNAs and other RNA targets-in cell and animal models confers a less aggressive/metastatic phenotype. This correlates with de-repression of let-7 miRNAs and is mimicked by silencing of downstream let-7 target HMGA2 or chemical inhibition of LIN28B/let-7 binding. Molecular characterization of CTCs provides a unique opportunity to correlated gene set metastatic profiles, identify drivers of dissemination, and develop therapies targeting the "seeds" of metastasis.