Differentiation of stem and progenitor cells is a highly regulated process that involves the coordinated action of multiple layers of regulation. Here we show how the post-transcriptional regulatory layer instructs the level of chromatin regulation via miR-144 and its targets to orchestrate chromatin condensation during erythropoiesis. The loss of miR-144 leads to impaired chromatin condensation during erythrocyte maturation.
View Article and Find Full Text PDFHemogenic endothelial cells (HECs) are specialized cells that undergo endothelial-to-hematopoietic transition (EHT) to give rise to the earliest precursors of hematopoietic progenitors that will eventually sustain hematopoiesis throughout the lifetime of an organism. Although HECs are thought to be primarily limited to the aorta-gonad-mesonephros (AGM) during early development, EHT has been described in various other hematopoietic organs and embryonic vessels. Though not defined as a hematopoietic organ, the lung houses many resident hematopoietic cells, aids in platelet biogenesis, and is a reservoir for hematopoietic stem and progenitor cells (HSPCs).
View Article and Find Full Text PDFRed blood cells (RBCs) (erythrocytes) are the simplest primary human cells, lacking nuclei and major organelles and instead employing about a thousand proteins to dynamically control cellular function and morphology in response to physiological cues. In this study, we define a canonical RBC proteome and interactome using quantitative mass spectrometry and machine learning. Our data reveal an RBC interactome dominated by protein homeostasis, redox biology, cytoskeletal dynamics, and carbon metabolism.
View Article and Find Full Text PDFPolycomb Repressive Complex 2 (PRC2) is an epigenetic regulator required for gene silencing during development. Although PRC2 is a well-established RNA-binding complex, the biological function of PRC2-RNA interaction has been controversial. Here, we study the gene-regulatory role of the inhibitory PRC2-RNA interactions.
View Article and Find Full Text PDFHematopoietic stem cells (HSCs) reside at the top of the hematopoietic hierarchy and can give rise to all the mature blood cell types in our body, while at the same time maintaining a pool of HSCs through self-renewing divisions. This potential is reflected in their functional definition as cells that are capable of long-term multi-lineage engraftment upon transplantation. While all HSCs meet these criteria, subtle differences exist between developmentally different populations of these cells.
View Article and Find Full Text PDFThe human hematopoietic stem cell harbors remarkable regenerative potential that can be harnessed therapeutically. During early development, hematopoietic stem cells in the fetal liver undergo active expansion while simultaneously retaining robust engraftment capacity, yet the underlying molecular program responsible for their efficient engraftment remains unclear. Here, we profile 26,407 fetal liver cells at both the transcriptional and protein level including ~7,000 highly enriched and functional fetal liver hematopoietic stem cells to establish a detailed molecular signature of engraftment potential.
View Article and Find Full Text PDFA workshop entitled "Stem Cells, Cell Therapies and Bioengineering in Lung Biology and Diseases" was hosted by the University of Vermont Larner College of Medicine in collaboration with the National Heart, Lung and Blood Institute, the Alpha-1 Foundation, the Cystic Fibrosis Foundation, the International Society for Cell and Gene Therapy and the Pulmonary Fibrosis Foundation. The event was held from July 15 to 18, 2019 at the University of Vermont, Burlington, Vermont. The objectives of the conference were to review and discuss the current status of the following active areas of research: 1) technological advancements in the analysis and visualisation of lung stem and progenitor cells; 2) evaluation of lung stem and progenitor cells in the context of their interactions with the niche; 3) progress toward the application and delivery of stem and progenitor cells for the treatment of lung diseases such as cystic fibrosis; 4) progress in induced pluripotent stem cell models and application for disease modelling; and 5) the emerging roles of cell therapy and extracellular vesicles in immunomodulation of the lung.
View Article and Find Full Text PDFThe protocols herein outline the use of qRT-PCR to detect the presence of SARS-CoV-2 genomic RNA in patient samples. In order to cope with potential fluctuations in supply chain and testing demands and to enable expedient adaptation of reagents and assays on hand, we include details for three parallel methodologies (one- and two-step singleplex and one-step multiplex assays). The diagnostic platforms described can be easily adapted by basic science research laboratories for SARS-CoV-2 diagnostic testing with relatively short turnaround time.
View Article and Find Full Text PDFBackground: Significant delays in the rapid development and distribution of diagnostic testing for SARS-CoV-2 (COVID-19) infection have prevented adequate public health management of the disease, impacting the timely mapping of viral spread and the conservation of personal protective equipment. Furthermore, vulnerable populations, such as those served by the Boston Medical Center (BMC), the largest safety net hospital in New England, represent a high-risk group across multiple dimensions, including a higher prevalence of pre-existing conditions and substance use disorders, lower health maintenance, unstable housing, and a propensity for rapid community spread, highlighting the urgent need for expedient and reliable in-house testing.
Methods: We developed a SARS-CoV-2 diagnostic medium-throughput qRT-PCR assay with rapid turnaround time and utilized this Clinical Laboratory Improvement Amendments (CLIA)-certified assay for testing nasopharyngeal swab samples from BMC patients, with emergency authorization from the Food and Drug Administration (FDA) and the Massachusetts Department of Public Health.
Expert Rev Precis Med Drug Dev
November 2019
Introduction: Sickle cell anemia is a mendelian disease that is noted for the heterogeneity of its clinical expression. Because of this, providing an accurate prognosis has been a longtime quest.
Areas Covered: Reviewed are the benefits and shortcomings of testing for the major modulators of the severity of disease, like fetal hemoglobin and α thalassemia, along with studies that have attempted to link genetic variation with sub-phenotypes of disease in a predictive fashion.
Robust β-globin expression in erythroid cells derived from induced pluripotent stem cells (iPSCs) increases the resolution with which red blood cell disorders such as sickle cell disease and β thalassemia can be modeled in vitro. To better quantify efforts in augmenting β-globin expression, we report the creation of a β-globin reporter iPSC line that allows for the mapping of β-globin expression throughout human erythropoietic development in real time at single-cell resolution. Coupling this tool with single-cell RNA sequencing (scRNAseq) identified features that distinguish β-globin-expressing cells and allowed for the dissection of the developmental and maturational statuses of iPSC-derived erythroid lineage cells.
View Article and Find Full Text PDFInduced pluripotent stem cells (iPSCs) stand to revolutionize the way we study human development, model disease, and eventually, treat patients. However, these cell sources produce progeny that retain embryonic and/or fetal characteristics. The failure to mature to definitive, adult-type cells is a major barrier for iPSC-based disease modeling and drug discovery.
View Article and Find Full Text PDFSickle cell anemia affects millions of people worldwide and is an emerging global health burden. As part of a large NIH-funded NextGen Consortium, we generated a diverse, comprehensive, and fully characterized library of sickle-cell-disease-specific induced pluripotent stem cells (iPSCs) from patients of different ethnicities, β-globin gene (HBB) haplotypes, and fetal hemoglobin (HbF) levels. iPSCs stand to revolutionize the way we study human development, model disease, and perhaps eventually, treat patients.
View Article and Find Full Text PDFEven with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells (hPSCs) is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects.
View Article and Find Full Text PDFTools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage.
View Article and Find Full Text PDFTo understand how haploinsufficiency of progranulin (PGRN) causes frontotemporal dementia (FTD), we created induced pluripotent stem cells (iPSCs) from patients carrying the GRN(IVS1+5G > C) mutation (FTD-iPSCs). FTD-iPSCs were fated to cortical neurons, the cells most affected in FTD. Although generation of neuroprogenitors was unaffected, their further differentiation into CTIP2-, FOXP2-, or TBR1-TUJ1 double-positive cortical neurons, but not motorneurons, was significantly decreased in FTD-neural progeny.
View Article and Find Full Text PDFFanconi anemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure (BMF) during childhood, aside from numerous congenital abnormalities. FA mouse models have been generated; however, they do not fully mimic the hematopoietic phenotype. As there is mounting evidence that the hematopoietic impairment starts already in utero, a human pluripotent stem cell model would constitute a more appropriate system to investigate the mechanisms underlying BMF in FA and its developmental basis.
View Article and Find Full Text PDFPancreatic endocrine progenitors obtained from human embryonic stem cells (hESCs) represent a promising source to develop cell-based therapies for diabetes. Although endocrine pancreas progenitor cells have been isolated from mouse pancreata on the basis of Ngn3 expression, human endocrine progenitors have not been isolated yet. As substantial differences exist between human and murine pancreas biology, we investigated whether it is possible to isolate pancreatic endocrine progenitors from differentiating hESC cultures by lineage tracing of NGN3.
View Article and Find Full Text PDFAlthough Gurdon demonstrated already in 1958 that the nucleus of intestinal epithelial cells could be reprogrammed to give rise to adult frogs, the field of cellular reprogramming has only recently come of age with the description by Takahashi and Yamanaka in 2006, which defined transcription factors can reprogram fibroblasts to an embryonic stem cell-like fate. With the mounting interest in the use of human pluripotent stem cells and culture-expanded somatic stem/progenitor cells, such as mesenchymal stem cells, increasing attention has been given to the effect of changes in the in vitro microenvironment on the fate of stem cells. These studies have demonstrated that changes in culture conditions may change the potency of pluripotent stem cells or reprogram adult stem/progenitor cells to endow them with a broader differentiation potential.
View Article and Find Full Text PDFStem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes.
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