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Hideki Kobayashi,
Jason M. Butler,
Rebekah, Mariko Kobayashi,
Bi-Sen Ding,
Bryant Bonner,
Vi K. Chiu,
Daniel J. Nolan,
Koji Shido,
Laura Benjamin,
Shahin Rafii
Nature Cell Biology 10/2010; 12(11):1046-1056. · 19.49 Impact Factor
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Hideki Kobayashi,
Jason M Butler,
Rebekah O'Donnell, Mariko Kobayashi,
Bi-Sen Ding,
Bryant Bonner,
Vi K Chiu,
Daniel J Nolan,
Koji Shido,
Laura Benjamin,
Shahin Rafii
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ABSTRACT: Endothelial cells establish an instructive vascular niche that reconstitutes haematopoietic stem and progenitor cells (HSPCs) through release of specific paracrine growth factors, known as angiocrine factors. However, the mechanism by which endothelial cells balance the rate of proliferation and lineage-specific differentiation of HSPCs is unknown. Here, we demonstrate that Akt activation in endothelial cells, through recruitment of mTOR, but not the FoxO pathway, upregulates specific angiocrine factors that support expansion of CD34(-)Flt3(-) KLS HSPCs with long-term haematopoietic stem cell (LT-HSC) repopulation capacity. Conversely, co-activation of Akt-stimulated endothelial cells with p42/44 MAPK shifts the balance towards maintenance and differentiation of the HSPCs. Selective activation of Akt1 in the endothelial cells of adult mice increased the number of colony forming units in the spleen and CD34(-)Flt3(-) KLS HSPCs with LT-HSC activity in the bone marrow, accelerating haematopoietic recovery. Therefore, the activation state of endothelial cells modulates reconstitution of HSPCs through the modulation of angiocrine factors, with Akt-mTOR-activated endothelial cells supporting the self-renewal of LT-HSCs and expansion of HSPCs, whereas MAPK co-activation favours maintenance and lineage-specific differentiation of HSPCs.
Nature Cell Biology 10/2010; 12(11):1046-56. · 19.49 Impact Factor
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Jason M Butler,
Daniel J Nolan,
Eva L Vertes,
Barbara Varnum-Finney,
Hideki Kobayashi,
Andrea T Hooper,
Marco Seandel,
Koji Shido,
Ian A White, Mariko Kobayashi,
Larry Witte,
Chad May,
Carrie Shawber,
Yuki Kimura,
Jan Kitajewski,
Zev Rosenwaks,
Irwin D Bernstein,
Shahin Rafii
[show abstract]
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ABSTRACT: Bone marrow endothelial cells (ECs) are essential for reconstitution of hematopoiesis, but their role in self-renewal of long-term hematopoietic stem cells (LT-HSCs) is unknown. We have developed angiogenic models to demonstrate that EC-derived angiocrine growth factors support in vitro self-renewal and in vivo repopulation of authentic LT-HSCs. In serum/cytokine-free cocultures, ECs, through direct cellular contact, stimulated incremental expansion of repopulating CD34(-)Flt3(-)cKit(+)Lineage(-)Sca1(+) LT-HSCs, which retained their self-renewal ability, as determined by single-cell and serial transplantation assays. Angiocrine expression of Notch ligands by ECs promoted proliferation and prevented exhaustion of LT-HSCs derived from wild-type, but not Notch1/Notch2-deficient, mice. In transgenic notch-reporter (TNR.Gfp) mice, regenerating TNR.Gfp(+) LT-HSCs were detected in cellular contact with sinusoidal ECs. Interference with angiocrine, but not perfusion, function of SECs impaired repopulation of TNR.Gfp(+) LT-HSCs. ECs establish an instructive vascular niche for clinical-scale expansion of LT-HSCs and a cellular platform to identify stem cell-active trophogens.
Cell stem cell 03/2010; 6(3):251-64. · 23.56 Impact Factor
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Andrea T Hooper,
Jason M Butler,
Daniel J Nolan,
Andrea Kranz,
Kaoruko Iida, Mariko Kobayashi,
Hans-Georg Kopp,
Koji Shido,
Isabelle Petit,
Kilangsungla Yanger,
Daylon James,
Larry Witte,
Zhenping Zhu,
Yan Wu,
Bronislaw Pytowski,
Zev Rosenwaks,
Vivek Mittal,
Thomas N Sato,
Shahin Rafii
[show abstract]
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ABSTRACT: Myelosuppression damages the bone marrow (BM) vascular niche, but it is unclear how regeneration of bone marrow vessels contributes to engraftment of transplanted hematopoietic stem and progenitor cells (HSPCs) and restoration of hematopoiesis. We found that chemotherapy and sublethal irradiation induced minor regression of BM sinusoidal endothelial cells (SECs), while lethal irradiation induced severe regression of SECs and required BM transplantation (BMT) for regeneration. Within the BM, VEGFR2 expression specifically demarcated a continuous network of arterioles and SECs, with arterioles uniquely expressing Sca1 and SECs uniquely expressing VEGFR3. Conditional deletion of VEGFR2 in adult mice blocked regeneration of SECs in sublethally irradiated animals and prevented hematopoietic reconstitution. Similarly, inhibition of VEGFR2 signaling in lethally irradiated wild-type mice rescued with BMT severely impaired SEC reconstruction and prevented engraftment and reconstitution of HSPCs. Therefore, regeneration of SECs via VEGFR2 signaling is essential for engraftment of HSPCs and restoration of hematopoiesis.
Cell stem cell 04/2009; 4(3):263-74. · 23.56 Impact Factor
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Marco Seandel,
Jason M Butler,
Hideki Kobayashi,
Andrea T Hooper,
Ian A White,
Fan Zhang,
Eva L Vertes, Mariko Kobayashi,
Yan Zhang,
Sergey V Shmelkov,
Neil R Hackett,
Sina Rabbany,
Julie L Boyer,
Shahin Rafii
[show abstract]
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ABSTRACT: Vascular cells contribute to organogenesis and tumorigenesis by producing unknown factors. Primary endothelial cells (PECs) provide an instructive platform for identifying factors that support stem cell and tumor homeostasis. However, long-term maintenance of PECs requires stimulation with cytokines and serum, resulting in loss of their angiogenic properties. To circumvent this hurdle, we have discovered that the adenoviral E4ORF1 gene product maintains long-term survival and facilitates organ-specific purification of PECs, while preserving their vascular repertoire for months, in serum/cytokine-free cultures. Lentiviral introduction of E4ORF1 into human PECs (E4ORF1(+) ECs) increased the long-term survival of these cells in serum/cytokine-free conditions, while preserving their in vivo angiogenic potential for tubulogenesis and sprouting. Although E4ORF1, in the absence of mitogenic signals, does not induce proliferation of ECs, stimulation with VEGF-A and/or FGF-2 induced expansion of E4ORF1(+) ECs in a contact-inhibited manner. Indeed, VEGF-A-induced phospho MAPK activation of E4ORF1(+) ECs is comparable with that of naive PECs, suggesting that the VEGF receptors remain functional upon E4ORF1 introduction. E4ORF1(+) ECs inoculated in implanted Matrigel plugs formed functional, patent, humanized microvessels that connected to the murine circulation. E4ORF1(+) ECs also incorporated into neo-vessels of human tumor xenotransplants and supported serum/cytokine-free expansion of leukemic and embryonal carcinoma cells. E4ORF1 augments survival of PECs in part by maintaining FGF-2/FGF-R1 signaling and through tonic Ser-473 phosphorylation of Akt, thereby activating the mTOR and NF-kappaB pathways. Therefore, E4ORF1(+) ECs establish an Akt-dependent durable vascular niche not only for expanding stem and tumor cells but also for interrogating the roles of vascular cells in regulating organ-specific vascularization and tumor neo-angiogenesis.
Proceedings of the National Academy of Sciences 12/2008; 105(49):19288-93. · 9.68 Impact Factor
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Yasuji Arase,
Hiromitsu Kumada,
Kazuaki Chayama,
Murashima Naoya,
Akihito Tsubota,
Isao Koida,
Masahiro Kobayashi,
Yoshiyuki Suzuki,
Kenji Ikeda,
Satoshi Saitoh, Mariko Kobayashi
[show abstract]
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ABSTRACT: The objective of this study was to determine how HCV-RNA counts before interferon (IFN) retreatment influence patient response. We retrospectively studied 60 consecutive Japanese patients with chronic hepatitis C. HCV-RNA counts were determined by a new assay; HCV-branched DNA (HCV-bDNA) probe. In this study, 60 patients were retreated with alpha interferon. Two patients were withdrawn because of side effects. In regard to the HCV-RNA level between before the first IFN treatment and before IFN retreatment, HCV-RNA level was not significantly different in 74.1% () of patients. Out of 58 patients, nine (15.5%) had complete response with normalization of alanine aminotransferase (ALT) levels and undetectable HCV-RNA more than 6 months after the completion of retreatment. Out of seven patients with a HCV-RNA level less than 0.5 Meq/ml before IFN retreatment, three (42.9%) had a complete response. On the other hand, out of 17 patients with HCV-RNA level more than 10 Meq/ml before IFN retreatment, there were none with a complete response. These results indicate that a complete response to readministration of interferon is related to HCV-RNA level before interferon retreatment.
International Hepatology Communications.
