2021
Lin28 Paralogs Regulate Lung Branching Morphogenesis
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...
Constrained Chromatin Accessibility in PU.1-mutated Agammaglobulinemia Patients
The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1...
2020
Transcriptome Dynamics of Hematopoietic Stem Cell Formation Revealed Using a Combinatorial Runx1 and Ly6a Reporter System
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...
T-reg Cell-based Therapies: Challenges and Perspectives
Cellular therapies using regulatory T (T-reg) cells are currently undergoing clinical trials for the treatment of autoimmune diseases, transplant rejection and graft-versus-host disease. In this Review, we discuss the biology of T-reg cells and describe new efforts in T-reg cell engineering to enhance specificity, stability, functional activity and delivery. Finally, we envision that the success of T-reg cell therapy in autoimmunity and transplantation will encourage the clinical use of adoptive T-reg cell therapy for non-immune diseases, such as neurological disorders and tissue repair...
Polymer-stabilized Cas9 Nanoparticles and Modified Repair Templates Increase Genome Editing Efficiency
Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies1–5. Here we report two improvements that increase the efficiency of CRISPR–Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR effi- ciency approximately two- to fourfold...
Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia
Diamond-Blackfan anemia (DBA) is a congenital anemia that generally presents in young children (1). The primary symptom is anemia due to a block in erythroid differentiation. DBA is also asso- ciated with an elevated risk of craniofacial anomalies, short stature, thumb abnormalities, and an increased cancer predisposition (2). Ribosomal protein S19 (RPS19) was the first gene found mutated in patients with DBA (3)...
2019
A Systems Biology Pipeline Identifies Regulatory Networks for Stem Cell Engineering
A major challenge for stem cell engineering is achieving a holistic understanding of the molecular networks and biological processes governing cell differentiation. To address this challenge, we describe a computational approach that combines gene expression analysis, previous knowledge from proteomic pathway informatics and cell signaling models to delineate key tran- sitional states of differentiating cells at high resolution...
2018
A CLK3-HMGA2 Alternative Splicing Axis Impacts Human Hematopoietic Stem Cell Molecular Identity throughout Development
While gene expression dynamics have been exten- sively cataloged during hematopoietic differentia- tion in the adult, less is known about transcriptome diversity of human hematopoietic stem cells (HSCs) during development. To characterize transcrip- tional and post-transcriptional changes in HSCs during development, we leveraged high-throughput genomic approaches to profile miRNAs, lincRNAs, and mRNAs...
Regulation of Embryonic Haematopoietic Multipotency by EZH1
All haematopoietic cell lineages that circulate in the blood of adult mammals derive from multipotent haematopoietic stem cells (HSCs)1. By contrast, in the blood of mammalian embryos, lineage-restricted progenitors arise first, independently of HSCs, which only emerge later in gestation2,3. As best defined in the mouse, ‘primitive’ progenitors first appear in the yolk sac at 7.5 days post-coitum2,3. Subsequently, erythroid–myeloid progenitors that express fetal haemoglobin4, as well as fetal lymphoid progenitors5, develop in the yolk sac and the embryo proper, but these cells lack HSC potential...
2017
Drug Discovery for Diamond-Blackfan Anemia using Reprogrammed Hematopoietic Progenitors
The blood system is a classical developmental hierarchy in which hematopoietic stem and progenitor cells (HPCs) continuously replen- ish a pool of short-lived mature cells. The discovery of induced pluri- potency has opened new avenues to regenerative medicine, including disease modeling, to gain insights into pathophysiology and drug screening against disease-relevant human cells. A large number of in- duced pluripotent stem cell (iPSC) models have been established from patients with hematological diseases...
2016
Developmental Vitamin D Availability Impacts Hematopoietic Stem Cell Production
Vitamin D insufficiency is a worldwide epidemic affecting billions of individuals, including pregnant women and children. Despite its high incidence, the impact of active vitamin D3 (1,25(OH)D3) on embry- onic development beyond osteo-regulation remains largely undefined. Here, we demonstrate that 1,25(OH)D3 availability modulates zebrafish hemato- poietic stem and progenitor cell (HSPC) production. Loss of Cyp27b1-mediated biosynthesis or vitamin D receptor (VDR) function by gene knockdown re- sulted in significantly reduced runx1 expression and Flk1+cMyb+ HSPC numbers...
2015
De Novo Generation of HSCs from Somatic and Pluripotent Stem Cell Sources
Generating human hematopoietic stem cells (HSCs) from autologous tissues, when coupled with genome editing technologies, is a promising approach for cellular transplantation therapy and for in vitro disease modeling, drug discovery, and toxicology studies. Human pluripotent stem cells (hPSCs) represent a potentially inexhaustible supply of autologous tissue; however, to date, directed differentiation from hPSCs has yielded hematopoietic cells that lack robust and sustained multi-lineage potential...
2013
Induction of Multipotential Hematopoietic Progenitors from Human Pluripotent Stem Cells via Respecification of Lineage-Restricted Precursors
Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. How- ever, the inability to derive engraftable human he- matopoietic stem and progenitor cells (HSPCs) has limited their characterization to in vitro assays. We report a strategy to respecify lineage-restricted CD34+CD45+ myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engrafted in vivo...
Corepressor-dependent Silencing of Fetal Hemoglobin Expression by BCL11A
Reactivation of fetal hemoglobin (HbF) in adults ameliorates the severity of the common β-globin disorders. The transcription factor BCL11A is a critical modulator of hemoglobin switching and HbF silencing, yet the molecular mechanism through which BCL11A coordinates the developmental switch is incompletely understood. Particularly, the identities of BCL11A cooperating protein complexes and their roles in HbF expression and erythroid development re- main largely unknown. Here we determine the interacting partner proteins of BCL11A in erythroid cells by a proteomic screen...
2011
Rapid and Efficient Clathrin-mediated Endocytosis Revealed in Genome-edited Mammalian Cells
clathrin-mediated endocytosis (cMe) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian cMe is a highly dynamic but inefficient and heterogeneous process. in contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient...
Enhancing Zinc-Finger-Nuclease Activity with Improved Obligate Heterodimeric Architectures
Zinc-finger nucleases (ZFns) drive efficient genome editing by introducing a double-strand break into the targeted gene. cleavage is induced when two custom-designed ZFns heterodimerize upon binding dnA to form a catalytically active nuclease complex. the importance of this dimerization event for subsequent cleavage activity has stimulated efforts to engineer the nuclease interface to prevent undesired homodimerization...
2010
Transient Cold Shock Enhances Zinc-Finger Nuclease–Mediated Gene Disruption
Zinc-finger nucleases (Zfns) are powerful tools for editing the genomes of cell lines and model organisms. Given the breadth of their potential application, simple methods that increase Zfn activity, thus ensuring genome modification, are highly attractive. here we show that transient hypothermia generally and robustly increased the level of stable, Zfn-induced gene disruption, thereby providing a simple technique to enhance the experimental efficacy of Zfns...
