Atsushi Kunisato

Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis

Excessive accumulation of smooth-muscle cells (SMCs) has a key role in the pathogenesis of vascular diseases. It has been assumed that SMCs derived from the outer medial layer migrate, proliferate and synthesize extracellular matrix components on the luminal side of the vessel. Although much effort has been devoted to targeting migration and proliferation of medial SMCs, there is no effective therapy that prevents occlusive vascular remodeling. We show here that in models of post-angioplasty restenosis, graft vasculopathy and hyperlipidemia-induced atherosclerosis, bone-marrow cells give rise to most of the SMCs that contribute to arterial remodeling. Notably, purified hematopoietic stem cells differentiate into SMCs in vitro and in vivo. Our findings indicate that somatic stem cells contribute to pathological remodeling of remote organs, and may provide the basis for the development of new therapeutic strategies for vascular diseases through targeting mobilization, homing, differentiation and proliferation of bone marrow-derived vascular progenitor cells.

Notch2 Is Preferentially Expressed in Mature B Cells and Indispensable for Marginal Zone B Lineage Development

The Notch genes play a key role in cellular differentiation. The significance of Notch1 during thymocyte development is well characterized, but the function of Notch2 is poorly understood. Here we demonstrate that Notch2 but no other Notch family member is preferentially expressed in mature B cells and that conditionally targeted deletion of Notch2 results in the defect of marginal zone B (MZB) cells and their presumed precursors, CD1d(hi) fraction of type 2 transitional B cells. Among Notch target genes, the expression level of Deltex1 is prominent in MZB cells and strictly dependent on that of Notch2, suggesting that Deltex1 may play a role in MZB cell differentiation.

Notch1 but Not Notch2 Is Essential for Generating Hematopoietic Stem Cells from Endothelial Cells

Hematopoietic stem cells (HSCs) are thought to arise in the aorta-gonad-mesonephros (AGM) region of embryo proper, although HSC activity can be detected in yolk sac (YS) and paraaortic splanchnopleura (P-Sp) when transplanted in newborn mice. We examined the role of Notch signaling in embryonic hematopoiesis. The activity of colony-forming cells in the YS from Notch1(-/-) embryos was comparable to that of wild-type embryos. However, in vitro and in vivo definitive hematopoietic activities from YS and P-Sp were severely impaired in Notch1(-/-) embryos. The population representing hemogenic endothelial cells, however, did not decrease. In contrast, Notch2(-/-) embryos showed no hematopoietic deficiency. These data indicate that Notch1, but not Notch2, is essential for generating hematopoietic stem cells from endothelial cells.

Expression of Notch ligands, Jagged1, 2 and Delta1 in antigen presenting cells in mice.

Notch1 is indispensable for T cell development. It is anticipated that Notch1 and other Notch receptors expressed on the surface of thymic T cell precursors are activated by ligands present on environmental cells, including antigen presenting cells (APCs), and involved in positive and negative selections. Notch receptors on peripheral T cells may also be activated by ligands on APCs. Here, we examined the expression pattern of three Notch ligands, Jagged1, 2 and Delta1 in APCs by an immunofluorescence cell staining method and a reverse transcriptase-polymerase chain reaction (RT-PCR) method. Peritoneal macrophages were strongly positive for Jagged1 staining. In contrast, macrophages separated from spleen and dendritic cells (DCs) separated from spleen and thymus showed positive staining for all the three ligands at a similar intensity. An analysis by RT-PCR revealed that peritoneal and splenic macrophages and splenic and thymic DCs, show a distinct pattern in Notch ligand expression. These findings may represent that expression of various Notch ligands in APCs has a physiological relevance in each organ.

Exogenous gene expression and growth regulation of hematopoietic cells via a novel human artificial chromosome

AbstractA number of gene delivery systems are currently being developed for potential use in gene therapy. Here, we demonstrate the feasibility of 21ΔqHAC, a newly developed human artificial chromosome (HAC), as a gene delivery system. We first introduced a 21ΔqHAC carrying an EGFP reporter gene and a geneticin-resistant gene (EGFP-21ΔqHAC) into hematopoietic cells by microcell-mediated chromosome transfer. These HAC-containing hematopoietic cells showed resistance to geneticin, expressed EGFP and retained the ability to differentiate into various lineages, and the EGFP-21ΔqHAC was successfully transduced into primary hematopoietic cells. Hematopoietic cells harboring the EGFP-21ΔqHAC could still be detected at two weeks post-transplantation in immunodeficient mice. We also showed effective expansion of hematopoietic cells by introducing the 21ΔqHAC containing ScFvg, a gp130-based chimeric receptor that transmits growth signals in response to specific-antigen of this receptor. All of these results demonstrate the usefulness of HAC in gene therapy.