Allison Mayle

Less Is More: Unveiling the Functional Core of Hematopoietic Stem Cells through Knockout Mice

Hematopoietic stem cells (HSCs) represent one of the first recognized somatic stem cell types. As such, nearly 200 genes have been examined for roles in HSC function in knockout mice. In this review, we compile the majority of these reports to provide a broad overview of the functional modules revealed by these genetic analyses and highlight some key regulatory pathways involved, including cell cycle control, Tgf-β signaling, Pten/Akt signaling, Wnt signaling, and cytokine signaling. Finally, we propose recommendations for characterization of HSC function in knockout mice to facilitate cross-study comparisons that would generate a more cohesive picture of HSC biology.

Flow cytometry analysis of murine hematopoietic stem cells

Hematopoietic stem cells (HSCs) remain the most well‐characterized adult stem cell population both in terms of markers for purification and assays to assess functional potential. However, despite over 40 years of research, working with HSCs in the mouse remains challenging because of the relative abundance (or lack thereof) of these cells in the bone marrow. The frequency of HSCs in murine bone marrow is about 0.01% of total nucleated cells and ∼5,000 can be isolated from an individual mouse depending on the age, sex, and strain of mice as well as purification scheme utilized. Adding to the challenge is the continual reporting of new markers for HSC purification, which makes it difficult for the uninitiated in the field to know which purification strategies yield the highest proportion of long‐term, multilineage HSCs. In this updated version of our review from 2009, we review different strategies for hematopoietic stem and progenitor cell identification and purification. We will also discuss methods for rapid flow cytometric analysis of peripheral blood cell types, and novel strategies for working with rare cell populations such as HSCs in the analysis of cell cycle status by BrdU, Ki‐67, and Pyronin Y staining. The purpose of updating this review is to provide insight into some of the recent experimental and technical advances in mouse hematopoietic stem cell biology.

Highly Efficient Genome Editing of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Our understanding of the mechanisms that regulate hematopoietic stem/progenitor cells (HSPCs) has been advanced by the ability to genetically manipulate mice; however, germline modification is time consuming and expensive. Here, we describe fast, efficient, and cost-effective methods to directly modify the genomes of mouse and human HSPCs using the CRISPR/Cas9 system. Using plasmid and virus-free delivery of guide RNAs alone into Cas9-expressing HSPCs or Cas9-guide RNA ribonucleoprotein (RNP) complexes into wild-type cells, we have achieved extremely efficient gene disruption in primary HSPCs from mouse (>60%) and human (∼75%). These techniques enabled rapid evaluation of the functional effects of gene loss of Eed, Suz12, and DNMT3A. We also achieved homology-directed repair in primary human HSPCs (>20%). These methods will significantly expand applications for CRISPR/Cas9 technologies for studying normal and malignant hematopoiesis.

DNMT3A Loss Drives Enhancer Hypomethylation in FLT3-ITD-Associated Leukemias

DNMT3A, the gene encoding the de novo DNA methyltransferase 3A, is among the most frequently mutated genes in hematologic malignancies. However, the mechanisms through which DNMT3A normally suppresses malignancy development are unknown. Here, we show that DNMT3A loss synergizes with the FLT3 internal tandem duplication in a dose-influenced fashion to generate rapid lethal lymphoid or myeloid leukemias similar to their human counterparts. Loss of DNMT3A leads to reduced DNA methylation, predominantly at hematopoietic enhancer regions in both mouse and human samples. Myeloid and lymphoid diseases arise from transformed murine hematopoietic stem cells. Broadly, our findings support a role for DNMT3A as a guardian of the epigenetic state at enhancer regions, critical for inhibition of leukemic transformation.

Hematopoietic stem cell development: an epigenetic journey.

Hematopoietic development and homeostasis are based on hematopoietic stem cells (HSCs), a pool of ancestor cells characterized by the unique combination of self-renewal and multilineage potential. These two opposing forces are finely orchestrated by several regulatory mechanisms, comprising both extrinsic and intrinsic factors. Over the past decades, several studies have contributed to dissect the key role of niche factors, signaling transduction pathways, and transcription factors in HSC development and maintenance. Accumulating evidence, however, suggests that a higher level of intrinsic regulation exists; epigenetic marks, by controlling chromatin accessibility, directly shape HSC developmental cascades, including their emergence during embryonic development, maintenance of self-renewal, lineage commitment, and aging. In addition, aberrant epigenetic marks have been found in several hematological malignancies, consistent with clinical findings that mutations targeting epigenetic regulators promote leukemogenesis. In this review, we will focus on both normal and malignant hematopoiesis, covering recent findings that illuminate the epigenetic life of HSCs.

Erratum: DNMT3A Loss Drives Enhancer Hypomethylation in FLT3-ITD-Associated Leukemias (Cancer Cell (2016) 29(6) (922–934) (S1535610816302082) (10.1016/j.ccell.2016.05.003))

1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA 2 Dan L. Duncan Cancer Center and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA 3 Department of Bioinformatics, School of Life sciences and Technology, Tongji University, Shanghai 20092, China. 4 Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, Texas 77030, USA 5 Department of Pathology and Immunology, Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas 77030, USA 6 Department of Systems Biology, Division of Cancer Medicine, University of Texas MD Anderson Cancer Center, Houston, Texas, USA. 7 Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030, USA 8 Division of Oncology, Washington University School of Medicine, St. Louis, Missouri 63110, USA 9 Wellcome Trust/MRC Stem Cell Institute, Cambridge, UK 10 Greehey Childrens Cancer Research Institute and Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA 11 Rice University, Houston, Texas 77030

Contribution of LPP copy number and sequence changes to esophageal atresia, tracheoesophageal fistula, and VACTERL association

Contribution of LPP Copy Number and Sequence Changes to Esophageal Atresia, Tracheoesophageal Fistula, and VACTERL Association Andr es Hern andez-Garc ıa, Erwin Brosens, Hitisha P. Zaveri, Elisabeth M. de Jong, Zhiyin Yu, Maria Namwanje, Allison Mayle, Caraciolo J. Fernandes, Brendan Lee, Maria Blazo, Seema R. Lalani, Dick Tibboel, Annelies de Klein, and Daryl A. Scott* Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas Universidad Aut onoma de Coahuila, Saltillo, Coahuila, M exico Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands Department of Pediatric Surgery, Erasmus Medical Center, Rotterdam, The Netherlands Department of Pediatrics, Baylor College of Medicine, Houston, Texas Howard Hughes Medical Institute, Houston, Texas Texas A&M Health Science Center College of Medicine, Temple, Texas Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas