Delayed myeloid engraftment following cord blood transplantation (CBT) is thought to result from inadequate numbers of progenitor cells in the graft and is associated with increased early transplant related morbidity and mortality. Novel culture strategies that increase the number of cord blood (CB) progenitors capable of rapid myeloid engraftment following CBT would allow more widespread use of this stem cell source for transplantation. Here we report development of a clinically relevant Notchmediated ex vivo expansion system for human CD34 + CB progenitors that results in a >100 fold increase in the absolute number of stem/progenitor cells, including those capable of enhanced repopulation in the marrow of immunodeficient NOD/SCID mice. Furthermore, when Notchmediated ex vivo expanded CB progenitors were infused in a clinical setting, rapid recovery of myeloid cells was achieved, demonstrating the first observation of rapid engraftment derived from ex vivo expanded stem/progenitor cells in humans.

Hematopoietic stem cells give rise to progeny that either self-renew in an undifferentiated state or lose self-renewal capabilities and commit to lymphoid or myeloid lineages. Here we evaluated whether hematopoietic stem cell self-renewal is affected by the Notch pathway. Notch signaling controls cell fate choices in both invertebrates and vertebrates by inhibiting certain differentiation pathways, thereby permitting cells to either differentiate along an alternative pathway or to self-renew. Notch receptors are present in hematopoietic precursors and Notch signaling enhances the in vitro generation of human and mouse hematopoietic precursors, determines T- or B-cell lineage specification from a common lymphoid precursor and promotes expansion of CD8(+) cells. Here, we demonstrate that constitutive Notch1 signaling in hematopoietic cells established immortalized, cytokine-dependent cell lines that generated progeny with either lymphoid or myeloid characteristics both in vitro and in vivo. These data support a role for Notch signaling in regulating hematopoietic stem cell self-renewal. Furthermore, the establishment of clonal, pluripotent cell lines provides the opportunity to assess mechanisms regulating stem cell commitment and demonstrates a general method for immortalizing stem cell populations for further analysis.

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...

We investigated whether combined signaling induced by engineered Notch ligands and hematopoietic growth factors influences hematopoietic stem-cell differentiation. We show that incubation of murine marrow precursors with Delta1 ext-IgG , a Notch ligand consisting of the Delta1 extracellular domain fused to the Fc portion of human immunoglobulin G1 (IgG1), and growth factors stem cell factor (SCF), interleukin 6 (IL-6), IL-11, and Flt3-l inhibited myeloid differentiation and promoted a several-log increase in the number of precursors capable of short-term lymphoid and myeloid repopulation. Addition of IL7 promoted early T-cell development, whereas addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) led to terminal myeloid differentiation. These results support a role for combinatorial effects by Notch and cytokine-induced signaling pathways in regulating hematopoietic cell fate and suggest the usefulness of Notch ligand in increasing hematopoietic precursor numbers for clinical stem-cell transplantation. (Blood.