David Flowers

Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling

Hematopoietic stem cells give rise to progeny that either self-renew in an undifferentiated state or lose self-renewal capabilities and commit to lymphoid or myeloid lineages. Here we evaluated whether hematopoietic stem cell self-renewal is affected by the Notch pathway. Notch signaling controls cell fate choices in both invertebrates and vertebrates by inhibiting certain differentiation pathways, thereby permitting cells to either differentiate along an alternative pathway or to self-renew. Notch receptors are present in hematopoietic precursors and Notch signaling enhances the in vitro generation of human and mouse hematopoietic precursors, determines T- or B-cell lineage specification from a common lymphoid precursor and promotes expansion of CD8+ cells. Here, we demonstrate that constitutive Notch1 signaling in hematopoietic cells established immortalized, cytokine-dependent cell lines that generated progeny with either lymphoid or myeloid characteristics both in vitro and in vivo. These data support a role for Notch signaling in regulating hematopoietic stem cell self-renewal. Furthermore, the establishment of clonal, pluripotent cell lines provides the opportunity to assess mechanisms regulating stem cell commitment and demonstrates a general method for immortalizing stem cell populations for further analysis.

Wild-type microglia do not reverse pathology in mouse models of Rett syndrome.

arising from N. C. Derecki et al. 484, 105–109 (2012); doi:10.1038/nature10907Rett syndrome is a severe neurodevelopmental disorder caused by mutations in the X chromosomal gene MECP2 (ref. 1), and its treatment so far is symptomatic. Mecp2 disruption in mice phenocopies major features of the syndrome that can be reversed after Mecp2 re-expression. Recently, Derecki et al. reported that transplantation of wild-type bone marrow into lethally irradiated Mecp2-null (Mecp2tm1.1Jae/y) mice prevented neurological decline and early death by restoring microglial phagocytic activity against apoptotic targets, and clinical trials of bone marrow transplantation (BMT) for patients with Rett syndrome have thus been initiated. We aimed to replicate and extend the BMT experiments in three different Rett syndrome mouse models, but found that despite robust microglial engraftment, BMT from wild-type donors did not prevent early death or ameliorate neurological deficits. Furthermore, early and specific Mecp2 genetic expression in microglia did not rescue Mecp2-deficient mice.

SIRT1 is dispensable for function of hematopoietic stem cells in adult mice

SIRT1 is an NAD(+)-dependent histone deacetylase implicated in the establishment of the primitive hematopoietic system during mouse embryonic development. However, investigation of the role of SIRT1 in adult hematopoiesis has been complicated by the high perinatal mortality of SIRT1-deficient mice (SIRT1(-/-)). We performed a comprehensive in vivo study of the hematopoietic stem cell (HSC) compartment in adult SIRT1(-/-) mice and show that, apart from anemia and leukocytosis in older mice, the production of mature blood cells, lineage distribution within hematopoietic organs, and frequencies of the most primitive HSC populations are comparable to those of wild-type littermate controls. Furthermore, we show that SIRT1-deficient BM cells confer stable long-term reconstitution in competitive repopulation and serial transplantation experiments. The results of the present study rule out an essential physiologic role for cell-autonomous SIRT1 signaling in the maintenance of the adult HSC compartment in mice.

Regulation of colony forming cell generation by flt-3 ligand

The recently cloned ligand for the flt‐3/flk‐2 receptor was examined for its effect on colony formation by subpopulations of CD34+ cells including the least mature CD34+lin−CD38− small‐medium lymphocyte‐sized cell population. Flt‐3 ligand (flt‐3l) had little or no effect when added alone to cells. Isolated CD34+lin+ cells formed increased numbers of colony‐forming cells (CFC) when flt‐3l was added together with IL‐3, IL‐6, G‐CSF, GM‐CSF or c‐kit ligand (KL), or with the combination of IL‐3 and KL. Significant increases in CFC formation from CD34+lin− cells were consistently seen when flt‐3l was added to the IL‐3 and KL combination, with variable effects observed when it was added to individual growth factors. Studies of the generation of CFC from CD34+lin− cells in liquid cultures showed that cultures containing IL‐3 and KL continued to produce CFC after 3 weeks of culture, whereas cultures with IL‐3, KL and flt‐3l produced few CFC past 2 weeks of culture. Flt‐3l alone or the combination of IL‐3 and KL did not stimulate significant growth of CD34+lin−CD38− small‐medium lymphocyte‐sized cells, although these cells reproducibly generated CFC when grown in the combination of IL‐1β, IL‐3, IL‐6, G‐CSF, GM‐CSF and KL. Addition of flt‐3l to either IL‐3 and KL or to a combination of growth factors induced increased CFC in three of four experiments. These data therefore demonstrate a role for flt‐3l in the induction of myelopoiesis by haemopoietic precursors, including the least mature subpopulation population of CD34+ cells.

Breast cancer resistance protein (BCRP/ABCG2) does not confer resistance to gemtuzumab ozogamicin and calicheamicin-γ1 in acute myeloid leukemia cells

Breast cancer resistance protein (BCRP/ABCG2) does not confer resistance to gemtuzumab ozogamicin and calicheamicin- γ 1 in acute myeloid leukemia cells

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Corrigendum: Wild-type microglia do not reverse pathology in mouse models of Rett syndrome

This corrects the article DOI: 10.1038/nature14444

Erratum: Wild-type microglia do not reverse pathology in mouse models of Rett syndrome (Nature (2015) 521 (E1-E4) DOI:10.1038/nature14444)

This corrects the article DOI: 10.1038/nature14444

Erratum: Corrigendum: Wild-type microglia do not reverse pathology in mouse models of Rett syndrome

This corrects the article DOI: 10.1038/nature14444