Shigeo Masuda

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.

Mutations of the Notch1 gene in T-cell acute lymphoblastic leukemia: analysis in adults and children

Mutations of the Notch1 gene in T-cell acute lymphoblastic leukemia: analysis in adults and children

Notch1 oncoprotein antagonizes TGF‐β/Smad‐mediated cell growth suppression via sequestration of coactivator p300

The Notch proteins constitute a family of transmembrane receptors that play a pivotal role in cellular differentiation, proliferation and apoptosis. Although it has been recognized that excess Notch signaling is potentially tumorigenic, little is known about precise mechanisms through which dysregulated Notch signaling induces neoplastic transformation. Here we demonstrate that Notch signaling has a transcriptional cross‐talk with transforming growth factor‐β (TGF‐β) signaling, which is well characterized by its antiproliferative effects. TGF‐β‐mediated transcriptional responses are suppressed by constitutively active Notch1, and this inhibitory effect is canceled by introduction of transcriptional coactivator p300. We further show that this blockade of TGF‐β signaling is executed by the sequestration of p300 from Smad3. Moreover, in a human cervical carcinoma cell line, CaSki, in which Notch1 is spontaneously activated, suppression of Notch1 expression with small interfering RNA significantly restores the responsiveness to TGF‐β. Taken together, we propose that Notch oncoproteins promote cell growth and cancer development partly by suppressing the growth inhibitory effects of TGF‐β through sequestrating p300 from Smad3. (Cancer Sci 2005; 96: 274 –283)

Both Notch1 and Notch2 contribute to the regulation of melanocyte homeostasis.

Notch signaling affects a variety of mammalian stem cells, but there has been limited evidence that a specific Notch molecule regulates adult stem cells. Recently, it was reported that the reduced Notch signaling initiated at the embryonic stage results in a gradual hair graying phenotype after birth. Here we demonstrate that the oral administration of a γ‐secretase inhibitor (GSI) to wild‐type adult C57/Bl6 mice led to a gradual increase in gray spots, which remained unchanged for at least 20 weeks after discontinuing the GSI. In GSI‐treated mice, there was a severe decrease in unpigmented melanocytes in the bulge/subbulge region where melanocyte stem cells are located. While we confirmed that Notch1+/−Notch2+/− double heterozygous mice with a C57/Bl6 background were born with a normal hair color phenotype and gradually turned gray after the second hair cycle, in the c‐kit mutant Wv background, Notch1+/− and Notch2+/− mice had larger white spots on the first appearance of hair than did the Wv/+ mice, which did not change throughout life. Notch1+/−Notch2+/−Wv/+ mice had white hair virtually all over the body at the first appearance of hair and the depigmentation continued to progress thereafter. Using a neural crest organ culture system, GSI blocked the generation of pigmented melanocytes when added to the culture during the period of melanoblast proliferation, but not during the period of differentiation. These observations imply roles of Notch signaling in both development of melanocyte during embryogenesis and maintenance of melanocyte stem cells in adulthood, while the degree of requirement is distinct in these settings: the latter is more sensitive than the former to the reduced Notch signaling. Furthermore, Notch1 and Notch2 cooperates with c‐kit signaling during embryogenesis, and they cooperate with each other to regulate melanocyte homeostasis after birth.

Notch Activation Induces the Generation of Functional NK Cells from Human Cord Blood CD34-Positive Cells Devoid of IL-15

The development of NK cells from hematopoietic stem cells is thought to be dependent on IL-15. In this study, we demonstrate that stimulation of human cord blood CD34+ cells by a Notch ligand, Delta4, along with IL-7, stem cell factor, and Fms-like tyrosine kinase 3 ligand, but no IL-15, in a stroma-free culture induced the generation of cells with characteristics of functional NK cells, including CD56 and CD161 Ag expression, IFN-γ secretion, and cytotoxic activity against K562 and Jurkat cells. Addition of γ-secretase inhibitor and anti-human Notch1 Ab to the culture medium almost completely blocked NK cell emergence. Addition of anti-human IL-15-neutralizing Ab did not affect NK cell development in these culture conditions. The presence of IL-15, however, augmented cytotoxicity and was required for a more mature NK cell phenotype. CD56+ cells generated by culture with IL-15, but without Notch stimulation, were negative for CD7 and cytoplasmic CD3, whereas CD56+ cells generated by culture with both Delta4 and IL-15 were CD7+ and cytoplasmic CD3+ from the beginning and therefore more similar to in vivo human NK cell progenitors. Together, these results suggest that Notch signaling is important for the physiologic development of NK cells at differentiation stages beyond those previously postulated.

Homeoprotein DLX-1 interacts with Smad4 and blocks a signaling pathway from activin A in hematopoietic cells

In the transforming growth factor β (TGF-β) superfamily, activin A, TGF-β1, and bone morphogenic protein 4 (BMP-4) have various effects on hematopoiesis, including early mesodermo-hematogenesis. After these cytokines bind to their respective receptor, a regulatory Smad is phosphorylated and becomes associated with Smad4, the common Smad, and the resulting complex translocates to the nucleus to regulate transcription. DLX1 is the product of a member of the distal-less homeobox gene family, which is known to have important roles in embryogenesis, particularly in craniofacial development, and in GABAergic neurogenesis. DLX1 has been reported to be temporally and spatially coexpressed with BMP-4 during embryogenesis in selected contexts. We report here that, in addition to the previously reported regions/cells, DLX1 is expressed in hematopoietic cells in a lineage-dependent manner and that DLX1 interacts with Smad4 through its homeodomain. We show that it blocks multiple signals from TGF-β superfamily cytokines such as activin A, TGF-β1, and BMP-4, including differentiation of a hematopoietic cell line by activin A. Taken together, these data suggest that DLX1 may function as a regulator of multiple signals from TGF-β superfamily members in broad biological contexts during blood production.

Cotransplantation with MSCs improves engraftment of HSCs after autologous intra-bone marrow transplantation in nonhuman primates

OBJECTIVE Hematopoietic stem cells (HSCs) reside in the osteoblastic niche, which consists of osteoblasts. Mesenchymal stromal cells (MSCs) have an ability to differentiate into osteoblasts. Here, using nonhuman primates, we investigated the effects of cotransplantation with MSCs on the engraftment of HSCs after autologous intra-bone marrow transplantation. MATERIALS AND METHODS From three cynomolgus monkeys, CD34-positive cells (as HSCs) and MSCs were obtained. The former were divided into two equal aliquots and each aliquot was genetically marked with a distinctive retroviral vector to track the in vivo fate. Each HSC aliquot with or without MSCs was autologously injected into the bone marrow (BM) cavity of right or left side, enabling the comparison of in vivo fates of the two HSC grafts in the same body. RESULTS In the three monkeys, CD34(+) cells transplanted with MSCs engrafted 4.4, 6.0, and 1.6 times more efficiently than CD34(+) cells alone, as assessed by BM colony polymerase chain reaction. In addition, virtually all marked cells detected in the peripheral blood were derived from the cotransplantation aliquots. Notably, colony-forming units derived from the cotransplantation aliquots were frequently detected in BM distant sites from the injection site, implying that cotransplantation with MSCs also restored the ability of gene-marked HSCs to migrate and achieve homing in the distant BM. CONCLUSION Cotransplantation with MSCs would improve the efficacy of transplantation of gene-modified HSCs in primates, with enhanced engraftment in BM as well as increased chimerism in peripheral blood through migration and homing.

Dual antitumor mechanisms of Notch signaling inhibitor in a T-cell acute lymphoblastic leukemia xenograft model

Constitutive activation of Notch signaling is required for the proliferation of a subgroup of human T‐cell acute lymphoblastic leukemias (T‐ALL). Previous in vitro studies have demonstrated the therapeutic potential of Notch signaling inhibitors for treating T‐ALL. To further examine this possibility, we applied a γ‐secretase inhibitor (GSI) to T‐ALL xenograft models. Treatment of established subcutaneous tumors with GSI resulted in partial or complete regression of tumors arising from four T‐ALL cell lines that were also sensitive to GSI in vitro. To elucidate the mechanism of action, we transduced DND‐41 cells with the active form of Notch1 (aN1), which conferred resistance to in vitro GSI treatment. Nevertheless, in vivo treatment with GSI induced a partial but significant regression of subcutaneous tumors that developed from aN1‐transduced DND‐41 cells, whereas it induced complete regression of tumors that developed from mock‐transduced DND‐41 cells. These findings indicate that the remarkable efficacy of GSI might be attributable to dual mechanisms, directly via apoptosis of DND‐41 cells through the inhibition of cell‐autonomous Notch signaling, and indirectly via disturbance of tumor angiogenesis through the inhibition of non‐cell‐autonomous Notch signaling. (Cancer Sci 2009; 100: 2444–2450)

Variation in the incidence of teratomas after the transplantation of nonhuman primate ES cells into immunodeficient mice.

Embryonic stem (ES) cells have the ability to generate teratomas when transplanted into immunodeficient mice, but conditions affecting the generation remain to be elucidated. Nonhuman primate cynomolgus ES cells were transplanted into immunodeficient mice under different conditions; the number of transplanted cells, physical state (clumps or single dissociated cells), transplant site, differentiation state, and immunological state of recipient mice were all varied. The tumorigenicity was then evaluated. When cynomolgus ES cells were transplanted as clumps into the lower limb muscle in either nonobese diabetic/severe combined immunodeficiency (NOD/SCID) or NOD/SCID/?cnull (NOG) mice, teratomas developed in all the animals transplanted with 1 × 105 or more cells, but were not observed in any mouse transplanted with 1 × 103 cells. However, when the cells were transplanted as dissociated cells, the number of cells necessary for teratomas to form in all mice increased to 5 × 105. When the clump cells were injected subcutaneously (instead of intramuscularly), the number also increased to 5 × 105. When cynomolgus ES cell-derived progenitor cells (1 × 106), which included residual pluripotent cells, were transplanted into the lower limb muscle of NOG or NOD/SCID mice, the incidence of teratomas differed between the strains; teratomas developed in five of five NOG mice but in only two of five NOD/SCID mice. The incidence of teratomas varied substantially depending on the transplanted cells and recipient mice. Thus, considerable care must be taken as to tumorigenicity.

Generation of HLA‐DRB1*1501‐restricted p190 minor bcr–abl (e1a2)‐specific CD4+ T lymphocytes

A small population of cells in acute lymphoblastic leukaemia is characterized by a specific translocation of the c‐abl oncogene on chromosome 9 to the break point cluster lesion (bcr) on chromosome 22, t(9; 22)(q34; q11) (e1a2). Theoretically, the junction‐spanning sequences of oncogene fusion proteins might be ideal targets for immunotherapy because these are not present in normal cells. In this study, we show for the first time that in vitro immunization with a 17‐mer e1a2 peptide representing the p190 minor bcr–abl fusion protein resulted in HLA‐DRB1*1501‐restricted peptide‐specific proliferative CD4+ T lymphocytes, using peptide‐pulsed monocyte‐derived dendritic cells as the antigen‐presenting cells.