Publications by authors named "George Q Daley"

336 Publications

EZH1 repression generates mature iPSC-derived CAR T cells with enhanced antitumor activity.

Cell Stem Cell 2022 Aug;29(8):1181-1196.e6

Stem Cell Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Division of Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Boston, MA 02115, USA; Developmental and Regenerative Biology Program, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.stem.2022.06.014DOI Listing
August 2022

Lifelong multilineage contribution by embryonic-born blood progenitors.

Nature 2022 06 15;606(7915):747-753. Epub 2022 Jun 15.

Stem Cell Program, Boston Children's Hospital, Boston, MA, USA.

Haematopoietic stem cells (HSCs) arise in the embryo from the arterial endothelium through a process known as the endothelial-to-haematopoietic transition (EHT). 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.
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http://dx.doi.org/10.1038/s41586-022-04804-zDOI Listing
June 2022

Anniversary reflections: ISSCR presidents on 20 years of progress.

Cell Stem Cell 2022 Jun;29(6):876-878

This year, Cell Stem Cell and the International Society for Stem Cell Research (ISSCR) are celebrating their 15 and 20 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.
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http://dx.doi.org/10.1016/j.stem.2022.05.003DOI Listing
June 2022

Hypoxic, glycolytic metabolism is a vulnerability of B-acute lymphoblastic leukemia-initiating cells.

Cell Rep 2022 04;39(4):110752

Stem Cell Program, Boston Children's Hospital, Boston, MA 02115, USA; Stem Cell Transplantation Program, Department of Hematology, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115, USA; Cancer and Blood Disorders Center, Dana-Farber Cancer Institute and Boston Children's Hospital, Boston, MA 02115, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2022.110752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9099058PMC
April 2022

CellComm infers cellular crosstalk that drives haematopoietic stem and progenitor cell development.

Nat Cell Biol 2022 04 12;24(4):579-589. Epub 2022 Apr 12.

Stem Cell Program, Boston Children's Hospital, Boston, MA, USA.

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.
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http://dx.doi.org/10.1038/s41556-022-00884-1DOI Listing
April 2022

Developmental maturation of the hematopoietic system controlled by a Lin28b-let-7-Cbx2 axis.

Cell Rep 2022 04;39(1):110587

Department of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02115, USA; Stem Cell Program, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115, USA; Dana-Farber Boston Children's Cancer and Blood Disorders Center, Boston, MA 02115, USA; Stem Cell Transplantation Program, Boston Children's Hospital, Boston, MA 02115, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2022.110587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9029260PMC
April 2022

LIN28B alters ribosomal dynamics to promote metastasis in MYCN-driven malignancy.

J Clin Invest 2021 11;131(22)

Stem Cell Program, Boston Children's Hospital, Boston, Massachusetts, USA.

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.
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http://dx.doi.org/10.1172/JCI145142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8592552PMC
November 2021

rRNA biogenesis regulates mouse 2C-like state by 3D structure reorganization of peri-nucleolar heterochromatin.

Nat Commun 2021 11 9;12(1):6365. Epub 2021 Nov 9.

Center of Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, 310003, Hangzhou, China.

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.
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http://dx.doi.org/10.1038/s41467-021-26576-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8578659PMC
November 2021

Oncogenic switch and single-agent MET inhibitor sensitivity in a subset of -mutant lung cancer.

Sci Transl Med 2021 Sep 1;13(609):eabb3738. Epub 2021 Sep 1.

Bioscience, Oncology R&D, AstraZeneca, 25 Gatehouse Park, Waltham, MA 02451, USA.

The clinical efficacy of epidermal growth factor receptor (EGFR)–targeted therapy in -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 () proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent mutation and 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 -mutant, -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 mRNA expression stoichiometry as associated with oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitor–resistant -mutant, -amplified patients also exhibited a reduced mRNA ratio. Our findings reveal that a subset of -mutant, -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.
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http://dx.doi.org/10.1126/scitranslmed.abb3738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8627689PMC
September 2021

LIN28 coordinately promotes nucleolar/ribosomal functions and represses the 2C-like transcriptional program in pluripotent stem cells.

Protein Cell 2022 07 31;13(7):490-512. Epub 2021 Jul 31.

Center for Stem Cell and Regenerative Medicine, Department of Basic Medical Sciences and the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310058, China.

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.
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http://dx.doi.org/10.1007/s13238-021-00864-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9226220PMC
July 2022

Lin28 paralogs regulate lung branching morphogenesis.

Cell Rep 2021 07;36(3):109408

Division of Pediatric Hematology/Oncology, Boston Children's Hospital Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2021.109408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8371695PMC
July 2021

Sequential regulation of hemogenic fate and hematopoietic stem and progenitor cell formation from arterial endothelium by Ezh1/2.

Stem Cell Reports 2021 07 17;16(7):1718-1734. Epub 2021 Jun 17.

Stem Cell Program, Department of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02115, USA; Developmental and Regenerative Biology Program, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.stemcr.2021.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8282472PMC
July 2021

ISSCR Guidelines for Stem Cell Research and Clinical Translation: The 2021 update.

Stem Cell Reports 2021 06 27;16(6):1398-1408. Epub 2021 May 27.

Histogen, San Diego, CA, USA.

The International Society for Stem Cell Research has updated its Guidelines for Stem Cell Research and Clinical Translation in order to address advances in stem cell science and other relevant fields, together with the associated ethical, social, and policy issues that have arisen since the last update in 2016. While growing to encompass the evolving science, clinical applications of stem cells, and the increasingly complex implications of stem cell research for society, the basic principles underlying the Guidelines remain unchanged, and they will continue to serve as the standard for the field and as a resource for scientists, regulators, funders, physicians, and members of the public, including patients. A summary of the key updates and issues is presented here.
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http://dx.doi.org/10.1016/j.stemcr.2021.05.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8190668PMC
June 2021

Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia.

Sci Transl Med 2020 10;12(566)

Stem Cell Program, Boston Children's Hospital and Harvard Stem Cell Institute, Boston, MA 02115, USA.

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 () 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.
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http://dx.doi.org/10.1126/scitranslmed.abb5831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709865PMC
October 2020

Mitochondrial and Redox Modifications in Huntington Disease Induced Pluripotent Stem Cells Rescued by CRISPR/Cas9 CAGs Targeting.

Front Cell Dev Biol 2020 22;8:576592. Epub 2020 Sep 22.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.

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 gene. In this study, we thoroughly investigated mitochondrial-based mechanisms in HD patient-derived iPSC (HD-iPSC) and differentiated neural stem cells (NSC) 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.
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http://dx.doi.org/10.3389/fcell.2020.576592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536317PMC
September 2020

An induced pluripotent stem cell model of Fanconi anemia reveals mechanisms of p53-driven progenitor cell differentiation.

Blood Adv 2020 10;4(19):4679-4692

Stem Cell Program, Boston Children's Hospital, Boston, MA.

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.
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http://dx.doi.org/10.1182/bloodadvances.2020001593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556119PMC
October 2020

Diversification of reprogramming trajectories revealed by parallel single-cell transcriptome and chromatin accessibility sequencing.

Sci Adv 2020 09 11;6(37). Epub 2020 Sep 11.

Epigenetics and Cell Fates Laboratory, Programme in Stem Cell, Regenerative Medicine and Aging, Institute of Molecular and Cell Biology, A*STAR, Singapore 138673, Singapore.

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.
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http://dx.doi.org/10.1126/sciadv.aba1190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486102PMC
September 2020

COVID Highlights Another Crisis: Lack of Black Physicians and Scientists.

Med (N Y) 2021 01 24;2(1):2-3. Epub 2020 Jun 24.

Connecticut Convergence Institute for Translation in Regenerative Engineering, University of Connecticut Health Center, Farmington, CT 06030, USA.

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http://dx.doi.org/10.1016/j.medj.2020.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311898PMC
January 2021

Metabolic Regulation of Inflammasome Activity Controls Embryonic Hematopoietic Stem and Progenitor Cell Production.

Dev Cell 2020 10 17;55(2):133-149.e6. Epub 2020 Aug 17.

Stem Cell Program, Department of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02115, USA; Developmental and Regenerative Biology Program, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Cambridge, MA 02138, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.devcel.2020.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008739PMC
October 2020

Pancreatic circulating tumor cell profiling identifies LIN28B as a metastasis driver and drug target.

Nat Commun 2020 07 3;11(1):3303. Epub 2020 Jul 3.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, 02114, USA.

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.
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http://dx.doi.org/10.1038/s41467-020-17150-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335061PMC
July 2020

LIN28B regulates transcription and potentiates MYCN-induced neuroblastoma through binding to ZNF143 at target gene promotors.

Proc Natl Acad Sci U S A 2020 07 29;117(28):16516-16526. Epub 2020 Jun 29.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215;

LIN28B is highly expressed in neuroblastoma and promotes tumorigenesis, at least, in part, through inhibition of microRNA biogenesis. Here, we report that overexpression of either wild-type (WT) LIN28B or a LIN28B mutant that is unable to inhibit 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 and that are involved in neuronal cell adhesion and migration. These findings reveal an unexpected -independent function of LIN28B in transcriptional regulation during neuroblastoma pathogenesis.
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http://dx.doi.org/10.1073/pnas.1922692117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368283PMC
July 2020

Oral health care in the 21st century: It is time for the integration of dental and medical education.

J Dent Educ 2020 Sep 20;84(9):999-1002. Epub 2020 May 20.

Faculty of Medicine, Harvard Medical School, Boston, Massachusetts, USA.

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.
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http://dx.doi.org/10.1002/jdd.12191DOI Listing
September 2020

Transcriptome Dynamics of Hematopoietic Stem Cell Formation Revealed Using a Combinatorial Runx1 and Ly6a Reporter System.

Stem Cell Reports 2020 05 16;14(5):956-971. Epub 2020 Apr 16.

Division of Pediatric Hematology/Oncology, Boston Children's Hospital and Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Stem Cell Program, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Harvard University, Boston, MA, USA. Electronic address:

Studies of hematopoietic stem cell (HSC) development from pre-HSC-producing hemogenic endothelial cells (HECs) are hampered by the rarity of these cells and the presence of other cell types with overlapping marker expression profiles. We generated a Tg(Runx1-mKO2; Ly6a-GFP) dual reporter mouse to visualize hematopoietic commitment and study pre-HSC emergence and maturation. Runx1-mKO2 marked all intra-arterial HECs and hematopoietic cluster cells (HCCs), including pre-HSCs, myeloid- and lymphoid progenitors, and HSCs themselves. However, HSC and lymphoid potential were almost exclusively found in reporter double-positive (DP) cells. Robust HSC activity was first detected in DP cells of the placenta, reflecting the importance of this niche for (pre-)HSC maturation and expansion before the fetal liver stage. A time course analysis by single-cell RNA sequencing revealed that as pre-HSCs mature into fetal liver stage HSCs, they show signs of interferon exposure, exhibit signatures of multi-lineage differentiation gene expression, and develop a prolonged cell cycle reminiscent of quiescent adult HSCs.
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http://dx.doi.org/10.1016/j.stemcr.2020.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7220988PMC
May 2020

The Toughest Triage - Allocating Ventilators in a Pandemic.

N Engl J Med 2020 May 23;382(21):1973-1975. Epub 2020 Mar 23.

From the Center for Bioethics (R.D.T., C.M.), the Department of Global Health and Social Medicine (R.D.T., C.M.), the Office of the Dean of the Faculty of Medicine (G.Q.D.), Harvard Medical School, and the Department of Anesthesiology, Critical Care, and Pain Medicine, Boston Children's Hospital (R.D.T.) - both in Boston.

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http://dx.doi.org/10.1056/NEJMp2005689DOI Listing
May 2020

Author Correction: Lin28 and let-7 regulate the timing of cessation of murine nephrogenesis.

Nat Commun 2020 Mar 9;11(1):1327. Epub 2020 Mar 9.

Division of Pediatric Hematology/Oncology, Children's Hospital Boston, Boston, MA, 02115, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-14944-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062780PMC
March 2020

Introduction to the Special Issue on CRISPR.

Authors:
George Q Daley

Perspect Biol Med 2020 ;63(1):1-13

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http://dx.doi.org/10.1353/pbm.2020.0000DOI Listing
December 2020

A nanobody targeting the LIN28:let-7 interaction fragment of TUT4 blocks uridylation of let-7.

Proc Natl Acad Sci U S A 2020 03 14;117(9):4653-4663. Epub 2020 Feb 14.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115;

The LIN28:pre-let-7:TUTase ternary complex regulates pluripotency and oncogenesis by controlling processing of the let-7 family of microRNAs. The complex oligouridylates the 3' ends of pre-let-7 molecules, leading to their degradation via the DIS3L2 exonuclease. Previous studies suggest that components of this complex are potential therapeutic targets in malignancies that aberrantly express LIN28. In this study we developed a functional epitope selection approach to identify nanobody inhibitors of the LIN28:pre-let-7:TUT4 complex. We demonstrate that one of the identified nanobodies, Nb-S2A4, targets the 106-residue LIN28:let-7 interaction (LLI) fragment of TUT4. Nb-S2A4 can effectively inhibit oligouridylation and monouridylation of pre-let-7g in vitro. Expressing Nb-S2A4 allows maturation of the let-7 species in cells expressing LIN28, highlighting the therapeutic potential of targeting the LLI fragment.
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http://dx.doi.org/10.1073/pnas.1919409117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060709PMC
March 2020

YAP Regulates Hematopoietic Stem Cell Formation in Response to the Biomechanical Forces of Blood Flow.

Dev Cell 2020 02 6;52(4):446-460.e5. Epub 2020 Feb 6.

Stem Cell Program, Division of Pediatric Hematology and Oncology, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address:

Hematopoietic stem and progenitor cells (HSPCs), first specified from hemogenic endothelium (HE) in the ventral dorsal aorta (VDA), support lifelong hematopoiesis. Their de novo production promises significant therapeutic value; however, current in vitro approaches cannot efficiently generate multipotent long-lived HSPCs. Presuming this reflects a lack of extrinsic cues normally impacting the VDA, we devised a human dorsal aorta-on-a-chip platform that identified Yes-activated protein (YAP) as a cyclic stretch-induced regulator of HSPC formation. In the zebrafish VDA, inducible Yap overexpression significantly increased runx1 expression in vivo and the number of CD41 HSPCs downstream of HE specification. Endogenous Yap activation by lats1/2 knockdown or Rho-GTPase stimulation mimicked Yap overexpression and induced HSPCs in embryos lacking blood flow. Notably, in static human induced pluripotent stem cell (iPSC)-derived HE culture, compound-mediated YAP activation enhanced RUNX1 levels and hematopoietic colony-forming potential. Together, our findings reveal a potent impact of hemodynamic Rho-YAP mechanotransduction on HE fate, relevant to de novo human HSPC production.
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http://dx.doi.org/10.1016/j.devcel.2020.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398148PMC
February 2020

Author Correction: Epoxyeicosatrienoic acids enhance embryonic haematopoiesis and adult marrow engraftment.

Nature 2019 Sep;573(7772):E1

Stem Cell Program and Division of Haematology/Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, 02115, USA.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41586-019-1489-4DOI Listing
September 2019
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