The role of Notch in Hematopoiesis
In the early 1990’s Nohla Scientific Founder Dr. Irwin Bernstein at the Fred Hutchinson Cancer Research Center postulated that, as in other developing systems, hematopoietic stem cell-fate specification resulted from intercellular interactions with adjacent cells and was modulated by several families of molecules, including the Notch gene family. At that time, the Notch pathway was particularly well-studied in invertebrate systems with clear evidence that Notch played an important role in mediating intercellular interactions affecting cell-fate decisions within the central nervous system, eye, mesoderm, and ovaries.
A role for Notch in hematopoiesis was then further suggested by Dr. Bernstein’s detection of the human Notch1 gene in CD34+ or CD34+lin− human hematopoietic precursors. Subsequently, primary murine hematopoietic stem cells (HSC) were transduced with a retrovirus leading to constitutively active expression of the intracellular domain (ICD) of Notch1 and led to the emergence of an immortalized pluripotent cytokine-dependent cell line capable of both lymphoid and myeloid repopulation in vivo, thereby demonstrating a role for Notch in hematopoietic stem progenitor cell (HSPC) self-renewal. Although at the time a biological function for Notch in HSPC was not determined, this work suggested that manipulation of the Notch signaling pathway ex vivo in primary HSC could prove to be a novel approach for expanding HSPC.
To avoid the potential safety concerns of retroviral transduction, it was decided to activate endogenous Notch signaling in primary HSPC during culture. To that end, engineered Notch ligands were developed consisting of the extracellular domain (ECD) of the Notch ligands Jagged1 and Delta1. Notch ligands activate their receptors by physically pulling on the ECD in vivo; thus immobilization of the ligand proved necessary to activate endogenous Notch signaling in vitro. Using an immobilized form of the Notch ligand Delta1, Dr. Bernstein’s group was able to sufficiently activate endogenous receptors and induce expansion of murine stem progenitor cells capable of in vivo reconstitution similar to that seen in the retroviral-mediated Notch overexpression experiments. In fact, culture of murine hematopoietic precursors with the immobilized ligand Delta1 and cytokines resulted in a several-log increase in progenitors capable of short-term lymphoid and myeloid repopulation.
Cord Blood Expansion
Given the clinical need for large numbers of hematopoietic progenitor cells capable of providing rapid myeloid recovery in vivo, especially in patients undergoing cord blood transplant (CBT), Nohla Scientific Founder and Chief Medical Officer Dr. Colleen Delaney extended the ex vivo expansion platform from murine to human HSPC where she noted a unique response of cord blood (CB) HSPC to Notch ligands as compared to CD34+ bone marrow cells or mobilized peripheral blood stem cells. She found that culture of isolated CB CD34+ HSPC in the presence of an engineered form of Delta1 ligand in serum free media supplemented with cytokines led to a greater than 2-log increase in the number of CD34+ cells and nearly a 16-fold increase in NOD/SCID mouse repopulating cell frequency compared to control.
The ability of these human cells to rapidly reconstitute (as early as 10 days post infusion) the myeloid compartment in immunodeficient mice indicated their potential clinical utility. In contrast to the studies using primary murine HSPCs, in vivo persistence of transplanted cells at 9 weeks and secondary transplantation studies suggested the presence of both long-term and short-term repopulating cells following culture of human CB progenitor cells on Delta ligand.
A key aspect of these studies was determination of whether the magnitude of Notch signaling played a role in the optimal generation of repopulating cells. Murine studies showed that the relatively lower amount of Notch signaling induced in cells cultured with lower densities of Delta-1 led to self-renewal of progenitors with primarily B-lymphoid and myeloid potential, whereas higher amounts of Notch signaling inhibited B cell differentiation and promoted differentiation towards the T cell lineage.
Concurrent studies performed with human HSPC also revealed important ligand dose-dependent effects whereby relatively lower densities of immobilized ligand substantially enhanced generation of NOD/SCID repopulating cells, with higher ligand densities promoting differentiation towards the T-cell lineage at the expense of repopulating cells.