Isolation and characterization of neural precursor cells from the Sox1-GFP reporter mouse.

We have made use of a reporter mouse line in which enhanced green fluorescence protein (GFP) is inserted into the Sox1 locus. We show that the GFP reporter is coexpressed with the Sox1 protein as well as with other known markers for neural stem and progenitor cells, and can be used to identify and isolate these cells by fluorescence-activated cell sorting (FACS) from the developing or adult brain and from neurosphere cultures. All neurosphere-forming cells with the capacity for multipotency and self-renewal reside in the Sox1-GFP-expressing population.

TGF-beta type II receptor-deficient thymocytes develop normally but demonstrate increased CD8+ proliferation in vivo.

We have taken advantage of the Cre/lox system to generate a mouse model with inducible deficiency of transforming growth factor beta receptor II (TbetaRII). Using this approach, transforming growth factor beta (TGF-beta) signaling deficiency can be restricted to the hematopoietic system by bone marrow transplantation. Mice that received transplants with TbetaRII-/- bone marrow develop a lethal inflammatory disorder closely resembling that of TGF-beta1-null mice.

The role of Smad signaling in hematopoiesis.

The TGF-beta family of ligands, including TGF-beta, bone morphogenetic protein (BMP) and activin, signal through Smad pathways to regulate the fate of hematopoietic progenitor and stem cells during development and postnatally. BMP regulates hematopoietic stem cell (HSC) specification during development, while TGF-beta1, 2 and 3 are not essential for the generation of HSCs. BMP4 can increase proliferation of human hematopoietic progenitors, while TGF-beta acts as a negative regulator of hematopoietic progenitor and stem cells in vitro.

Stroke-induced neurogenesis in aged brain.

Background And Purpose: Stroke induced by middle cerebral artery occlusion (MCAO) triggers increased neurogenesis in the damaged striatum and nondamaged hippocampus of young adult rodents. We explored whether stroke influences neurogenesis similarly in the aged brain.

Methods: Young adult (3 months) and old (15 months) rats were subjected to 1 hour of MCAO, and new cells were labeled by intraperitoneal injection of 5-bromo-2'-deoxyuridine 5'-monophosphate (BrdU), a marker for dividing cells, for 2 weeks thereafter.

Distinct patterns of hematopoietic stem cell involvement in acute lymphoblastic leukemia.

The cellular targets of primary mutations and malignant transformation remain elusive in most cancers. Here, we show that clinically and genetically different subtypes of acute lymphoblastic leukemia (ALL) originate and transform at distinct stages of hematopoietic development. Primary ETV6-RUNX1 (also known as TEL-AML1) fusions and subsequent leukemic transformations were targeted to committed B-cell progenitors.

Microglia-derived tumor necrosis factor-alpha exaggerates death of newborn hippocampal progenitor cells in vitro.

Production of new hippocampal neurons continues in adult mammals and different brain insults can significantly increase this process. However, many hippocampal progenitor cells (HPC) die shortly after birth. Here we investigated the possibility that increased release of cytokines by activated microglia contributes to the death of HPC.

DRhoGEF2 regulates actin organization and contractility in the Drosophila blastoderm embryo.

Morphogenesis of the Drosophila melanogaster embryo is associated with a dynamic reorganization of the actin cytoskeleton that is mediated by small GTPases of the Rho family. Often, Rho1 controls different aspects of cytoskeletal function in parallel, requiring a complex level of regulation. We show that the guanine triphosphate (GTP) exchange factor DRhoGEF2 is apically localized in epithelial cells throughout embryogenesis.

IFN-gamma negatively modulates self-renewal of repopulating human hemopoietic stem cells.

Whereas multiple growth-promoting cytokines have been demonstrated to be involved in regulation of the hemopoietic stem cell (HSC) pool, the potential role of negative regulators is less clear. However, IFN-gamma, if overexpressed, can mediate bone marrow suppression and has been directly implicated in a number of bone marrow failure syndromes, including graft-vs-host disease. Whether IFN-gamma might directly affect the function of repopulating HSCs has, however, not been investigated.

Deficiency of ribosomal protein S19 in CD34+ cells generated by siRNA blocks erythroid development and mimics defects seen in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia in which 25% of the patients have a mutation in the ribosomal protein S19 (RPS19) gene. To study effects of RPS19 deficiency in hematopoiesis we transduced CD34(+) umbilical cord blood (CB) and bone marrow (BM) cells with 3 lentiviral vectors expressing small interfering RNA (siRNA) against RPS19 and 1 scrambled control vector. All vectors also express green fluorescent protein (GFP).

Identification of Lin(-)Sca1(+)kit(+)CD34(+)Flt3- short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients.

In clinical bone marrow transplantation, the severe cytopenias induced by bone marrow ablation translate into high risks of developing fatal infections and bleedings, until transplanted hematopoietic stem and progenitor cells have replaced sufficient myeloerythroid offspring. Although adult long-term hematopoietic stem cells (LT-HSCs) are absolutely required and at the single-cell level sufficient for sustained reconstitution of all blood cell lineages, they have been suggested to be less efficient at rapidly reconstituting the hematopoietic system and rescuing myeloablated recipients. Such a function has been proposed to rather be mediated by less well-defined short-term hematopoietic stem cells (ST-HSCs).

Deficiency of oncoretrovirally transduced hematopoietic stem cells and correction through ex vivo expansion.

Background: Extensive efforts to develop hematopoietic stem cell (HSC) based gene therapy have been hampered by low gene marking. Major emphasis has so far been directed at improving gene transfer efficiency, but low gene marking in transplanted recipients might equally well reflect compromised repopulating activity of transduced cells, competing for reconstitution with endogenous and unmanipulated stem cells.

Methods: The autologous settings of clinical gene therapy protocols preclude evaluation of changes in repopulating ability following transduction; however, using a congenic mouse model, allowing for direct evaluation of gene marking of lympho-myeloid progeny, we show here that these issues can be accurately addressed.

Striatal neuron differentiation from neurosphere-expanded progenitors depends on Gsh2 expression.

Neural stem and progenitor cells from the embryonic forebrain can be expanded under growth factor stimulation in vitro, either as free-floating aggregates called neurospheres or as attached monolayer cultures. We have previously shown that despite the maintenance of important regulatory genes such as Gsh2, in vitro expansion of cells from the lateral ganglion eminence (LGE) restricts their differentiation potential. Specifically, their ability to differentiate into striatal projection neurons is compromised.

Bone marrow-derived hematopoietic cells generate cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation.

Recent studies have suggested that bone marrow cells might possess a much broader differentiation potential than previously appreciated. In most cases, the reported efficiency of such plasticity has been rather low and, at least in some instances, is a consequence of cell fusion. After myocardial infarction, however, bone marrow cells have been suggested to extensively regenerate cardiomyocytes through transdifferentiation.

Enforced expression of cyclin D2 enhances the proliferative potential of myeloid progenitors, accelerates in vivo myeloid reconstitution, and promotes rescue of mice from lethal myeloablation.

Severe and prolonged cytopenias represent a considerable problem in clinical stem cell transplantations. Cytokine-induced ex vivo expansion of hematopoietic stem and progenitor cells has been intensively explored as a means of accelerating hematopoietic recovery following transplantation but have so far had limited success. Herein, overexpression of D-type cyclins, promoting G0/G1 to S transition, was investigated as an alternative approach to accelerate myeloid reconstitution following stem cell transplantation.

Deceptive multilineage reconstitution analysis of mice transplanted with hemopoietic stem cells, and implications for assessment of stem cell numbers and lineage potentials.

Hemopoietic stem cells (HSC) are identified through their unique ability, at the single cell level, to long-term reconstitute all blood cell lineages. Sustained myeloid reconstitution is considered the hallmark of HSC, because myeloid progenitors and their progeny have very short half-lives. Here we demonstrate that the established practice of relying on RB6-8C5 as a myeloid specific Ab can result in overestimation of HSC frequencies because the RB6-8C5 Ab also detects Ags expressed on a sizeable population of CD3(+)CD8(+) T cells, constitutively as well as following transplantation.

Complementary signaling through flt3 and interleukin-7 receptor alpha is indispensable for fetal and adult B cell genesis.

Extensive studies of mice deficient in one or several cytokine receptors have failed to support an indispensable role of cytokines in development of multiple blood cell lineages. Whereas B1 B cells and Igs are sustained at normal levels throughout life of mice deficient in IL-7, IL-7Ralpha, common cytokine receptor gamma chain, or flt3 ligand (FL), we report here that adult mice double deficient in IL-7Ralpha and FL completely lack visible LNs, conventional IgM+ B cells, IgA+ plasma cells, and B1 cells, and consequently produce no Igs. All stages of committed B cell progenitors are undetectable in FL-/- x IL-7Ralpha-/- BM that also lacks expression of the B cell commitment factor Pax5 and its direct target genes.

Human CD34+ hematopoietic stem cells capable of multilineage engrafting NOD/SCID mice express flt3: distinct flt3 and c-kit expression and response patterns on mouse and candidate human hematopoietic stem cells.

The cytokine tyrosine kinase receptors c-kit and flt3 are expressed and function in early mouse and human hematopoiesis. Through its ability to promote ex vivo expansion and oncoretroviral transduction of primitive human hematopoietic progenitors, the flt3 ligand (FL) has emerged as a key stimulator of candidate human hematopoietic stem cells (HSCs). However, recent studies in the mouse suggest that though it is present on short-term repopulating cells, flt3 is not expressed on bone marrow long-term reconstituting HSCs, the ultimate target for the development of cell replacement and gene therapy.

Human reconstituting hematopoietic stem cells up-regulate Fas expression upon active cell cycling but remain resistant to Fas-induced suppression.

The Fas receptor and its ligand have been implicated in mediating the bone marrow (BM) suppression observed in graft-versus-host disease and a number of other BM-failure syndromes. However, previous studies have suggested that Fas is probably not expressed on human hematopoietic stem cells (HSCs), but up-regulated as a consequence of their commitment and differentiation, suggesting that progenitors or differentiated blood cells, rather than HSCs, are the targets of Fas-mediated suppression. The present studies confirm that candidate HSCs in human cord blood and BM lack constitutive expression of Fas, but demonstrate that Fas expression on CD34+ progenitor and stem cells is correlated to their cell cycle and activation status.