Dose-dependent effects of the Notch ligand Delta1 on ex vivo differentiation and in vivo marrow repopulating ability of cord blood cells

Abstract

IntroductionThe widespread expression of Notch family members by hematopoietic cells, including stem cells, has led to speculation about their role in hematopoiesis. A critical role for Notch signaling in T versus B cell-fate decisions has been established in vivo with gain-and loss-of-function studies. 1-3 Despite profound effects on lymphoid cell fates, these studies have failed to identify significant effects on myeloid differentiation. However, in contrast to these in vivo studies, in vitro studies in which the constitutively active intracellular domain of Notch1 was overexpressed did reveal inhibition of myeloid differentiation and enhanced generation of precursor cells in addition to promotion of early T-cell differentiation, indicating a potential role of Notch signaling on multipotent precursor cells. [4][5][6][7] To exploit Notch signaling as a means of directing desired cell-fate outcomes or generating precursor cells from nontransduced stem cells, soluble or cell-expressed Notch ligands have been used by a number of laboratories. Initial studies met with only modest success, resulting in only a few-fold increase in progenitor cell number, 4,5,[7][8][9][10][11][12] but our more recent studies using an engineered Notch ligand Delta1 in immobilized form demonstrated profound effects on murine precursors with a multilog increase in the number of Sca-1 ϩ c-kit ϩ precursors with short-term lymphoid and myeloid repopulating ability. 7 Based on these findings, we examined Notch signaling in human cord blood precursors because inadequate stem cell numbers in cord blood grafts have been associated with significantly delayed engraftment and, as a result, increased early transplant-related mortality from infection, thereby limiting the use of cord blood for hematopoietic cell transplantation (HCT) in adults and larger children. Our studies demonstrated an increase in early human hematopoietic reconstitution in NOD/SCID mice, indicating potential clinical importance in overcoming the delayed engraftment in umbilical cord blood transplants. 5 We investigated whether quantitative differences in ligandinduced activation of Notch signaling could be the basis for the reported variability with Notch activation and cell fate outcomes of hematopoietic precursors and whether such quantitative differences might determine whether precursors self-renew or adopt a lymphoid cell fate. The importance of quantitative aspects of Notch signaling has been shown in Drosophila where different functions of Notch can require different thresholds of signaling. For example, Notch haploinsufficiency suffices to perform most functions of Notch indistinguishably from wild type, but causes improper specification of the dorsoventral margin of the wing, giving rise to the eponymous "notched wing" phenotype. 13,14 In mammals, a reduction in Notch1 gene dosage in developing T cells favors the ␥␦ T-cell fate over the ␣␤ T-cell fate, 15 and a dose-dependent effect of Delta1 on the determination of type 1 helper T cell (Th1) versus Th2 cell-fate dec...

NOTCH1 directly regulates c-MYC and activates a feed-forward-loop transcriptional network promoting leukemic cell growth

“…Oligo sequences for shRNAs targeting NOTCH1 (9) or the luciferase gene were cloned in the pLKO-puro and pGK-GFP lentiviral vectors (gift from William Hahn of the Dana-Farber Cancer Institute). Lentivirus production and infections were performed as described (24).…”

Notch ligands potentiate IL‐7‐driven proliferation and survival of human thymocyte precursors

“…These results demonstrate that Notch signaling increases IL-7-driven thymocyte proliferation. A relationship between the density of Notch ligands, the intensity of Notch signaling, and cell proliferation has been described by several authors [18,19]. Therefore, we performed experiments in which CD34 1 TN thymocytes were plated with growing numbers of OP9-DL1 or OP9-DL4 cells.…”

Characterization of the growth modulatory activities of osteoblast conditioned media on cord blood progenitor cells

“…One strategy has been focused on the ex vivo expansion of HSPC prior to transplantation which has now been tested in several clinical trials (Pineault and Abu-Khader 2015;Norkin et al 2013;Delaney et al 2005;Cairo et al 2016;de Lima et al 2012). Indeed, decreased graft failure and accelerated neutrophil recovery have been reported in patients undergoing double CB transplantation with an expanded and unmanipulated units, each unit providing short-and long-term engraftment, respectively (de Lima et al 2012;Delaney et al 2010;Horwitz et al 2014).…”