Notch-induced hIL-6 production facilitates the maintenance of self-renewal of hCD34+ cord blood cells through the activation of Jak-PI3K-STAT3 pathway.
ABSTRACT Ex vivo expansion of CD34(+) stem cells in contact culture between hCD34(+)CD38(-)Lin(-) cord blood stem cells and human delta-like-expressing AFT024 feeder cells revealed increased amounts of stemness-related proteins such as HoxB4, GATA2, Bmi-1, and p21 and anti-apoptotic proteins such as Bcl-2, Bcl-xL, Mcl-1, and phospho-Bad, when compared with control or noncontact culture. Production of human IL-6 (hIL-6) was markedly elevated in the culture, but was profoundly inhibited by treatment with γ-secretase inhibitor. In addition, Notch-induced activation of STAT3 was directly involved in gene expression of hIL-6 and soluble hIL-6Rα, indicating the close linkage between Notch signaling and hIL-6 production. Furthermore, depletion of soluble hIL-6 (with hIL-6-specific antibodies) and inhibition of IL-6-mediated signals (with a Jak1 inhibitor and wortmannin) severely affected the maintenance of self-renewal of hCD34(+) cord blood cells. It was also observed that the ex vivo expanded CD34(+) cord blood cells were induced to reconstitute human immune cells in nonobese diabetic mice with severe combined immunodeficiency when compared with freshly isolated CD34(+) cord blood cells. Together, these results strongly demonstrate that Notch signaling in the "cell-to-cell contact" between hCD34(+) cord blood and delta-like-expressing AFT024 feeder cells facilitates maintenance of self-renewal of hCD34(+) cord blood cells through direct regulation of hIL-6 production.
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ABSTRACT: Constitutive activity of kinases has been reported in many types of cancers, so that inhibition of "onco-kinases" became a validated anti-cancer strategy. We found that the polyphenol 13c, a tri-vanillate derivative, inhibited kinase phosphorylation in leukemia cells. P-JAK2, P-Src and P-PI3Kp85 inhibition occurred independently of phosphatase involvement in JAK2V617F expressing HEL cells while 13c inhibited Bcr-Abl expression without inhibition of phosphorylation in chronic myelogenous cell lines (K562, MEG-01). In correlation with kinase inhibition, 13c abolished constitutive P-STAT3/P-STAT5 expression, down-regulated Mcl-1 and c-Myc gene expression and induced apoptosis. Altogether, polyphenol 13c displays potential antitumor activities by affecting onco-kinases and STAT activities.Cancer letters 06/2013; · 4.86 Impact Factor
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ABSTRACT: Increasing evidence supports the importance of cell extrinsic regulation in stem cell fate control. Hematopoietic stem cells (HSC) are responsive to local signals from their niche and to systemic feedback from progenitors and mature cells. The Notch ligand Delta-1 (DL1), a key component of the stem cell niche, regulates human hematopoietic lineage development in a dose-dependent manner and has been used clinically for primitive progenitor expansion. How DL1 acts to regulate HSC fate, and whether these actions are related to its lineage skewing effects, is poorly understood. Herein we demonstrate that although DL1 activates STAT3 signaling similarly to the gp130-activating cytokine IL-6, it has opposite effects on myeloid cell production. Mechanistically, these different outcomes are attributable to a DL1-mediated reduction in membrane (m) bound IL-6R expression, converting progenitor cells from being directly IL-6 responsive, to requiring both IL-6 and the soluble (s) IL-6R for activation. Concomitant reduction of both mIL-6R (by DL1 supplementation) and sIL6R (using dynamically fed cultures) reduced myeloid cell production and led to enhanced outputs of human HSCs. This work describes a new mode of cytokine action wherein DL1 changes cytokine receptor distributions on hematopoietic cells, altering feedback networks and their impact on stem cell fate.Blood 11/2013; · 9.06 Impact Factor
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ABSTRACT: Notch signalling has been implicated in haematopoietic stem cell self-renewal. Although several studies have tested the effect of activating or inhibiting the Notch signalling pathway in stem cells, no study has yet determined the functional differences associated with expressing Notch1. The aims of this study were to characterise the expression of human cell surface Notch1 in cord blood (CB) CD34+ cells and to study the function of Notch in CD34+ cells in vitro. A monoclonal antibody against the extracellular domain of Notch1 was developed, and Notch1 expression in CB CD34+ cells was assessed by flow cytometry. CB CD34+ cells were sorted on the basis of their Notch1 expression and cultured in serum-free media. Single sorted CD34+CD38- Notch1+/- cells were cultured for 8 weeks on murine stroma monolayers and assayed for stem cell activity and lineage potential using a cobblestone area-forming cell (CAFC) assay. Cell surface Notch1 expression was characterised in various primitive CD34+ cell compartments including a small subpopulation of CD34+CD38-cells. We found the CD34+CD38-Notch1+ population to be enriched for stem cell activity. Moreover, CD34+CD38-Notch1+ but not Notch1- cells demonstrated multi-lineage potential. These data show that Notch1 is expressed on a functionally distinct sub-population of CD34+ cells that is highly enriched for stem cell activity and multi-lineage potential and could suggest that Notch1 could be used as a novel stem cell marker. This article is protected by copyright. All rights reserved.European Journal Of Haematology 09/2013; · 2.55 Impact Factor