In vitro expansion of long-term repopulating hematopoietic stem cells in the presence of immobilized Jagged-1 and early acting cytokines

Abstract

There is an increasing body of evidence that suggests that genes involved in cell fate decisions and pattern formation during development also play a key role in the continuous cell fate decisions made by adult tissue stem cells. Here we show that prolonged in vitro culture (14 days) of murine bone marrow lineage negative cells in medium supplemented with three early acting cytokines (stem cell factor, Flk-2/Flt-3 ligand, thrombopoietin) and with immobilized Notch ligand, Jagged-1, resulted in robust expansion of serially transplantable hematopoietic stem cells with long-term repopulating ability. We found that the absolute number of marrow cells was increased approximately 8 to 14-fold in all cultures containing recombinant growth factors. However, the frequency of high quality stem cells was markedly reduced at the same time, except in cultures containing growth factors and Jagged-1-coated Sepharose-4B beads. The absolute number of hematopoietic cells with long-term repopulating ability was increased approximately 10 to 20-fold in the presence of multivalent Notch ligand. These results support a role for combinatorial effects by Notch and cytokine-induced signaling pathways in regulating hematopoietic stem cell fate and to a potential role for Notch ligand in increasing cell numbers in clinical stem cell transplantation.

Mimicking stem cell niches to increase stem cell expansion

“…Culture on Delta1-Fc doubled the number of cells able to reconstitute multi-lineage human hematopoiesis in immunodeficient mice, and bone marrow cells collected from primary recipients supported engraftment of human cells in two of three secondary transplant recipients [*11]. In a similar manner, Jagged1 immobilized on Sepharose beads had little effect on the ex vivo expansion of lineage negative (Lin − ) mouse bone marrow cells during two weeks in culture, but greatly increased the production of male Lin − cells able to generate hematopoietic cell chimerism in secondary female transplant recipients [12]. In the presence of epithelial differentiation-inducing growth factors, immobilized rat Jagged1-Fc (via adsorbed protein G or bound anti-Fc antibody), but not soluble Jagged1, greatly enhanced Notch/CBF-1 signaling and differentiation of rat primary esophageal epithelial stem cells [13].…”

Designing and Engineering Stem Cell Niches

“…63 A separate study reported the conjugation of Jagged1 to sephadex beads and similarly obtained no clear effect on the expansion of HSCs, but it had an obvious effect on the efficacy of the expanded cells shown in the hematopoietic cell chimerism in secondary transplant recipients. 64 Another example concerns the immobilization of stem cell factor (SCF) using low-molecular-weight heparin/protamine microparticles, which can be stably adsorbed onto both plastic surfaces and cytokines. The SCF-loaded microparticle-coatings were shown to produce about a six-fold increase in the expansion of HSC.…”

Hematopoietic Stem Cells

“…The quantitative aspects of Notch signaling in determining hematopoietic precursor fate has been demonstrated by Delaney et al [223] who showed in CD34þ CB cultures that low densities of the Notch ligand Delta1 enhanced in vitro generation of CD34þ cells as well as CD14 and CD7 cells consistent with myeloid and lymphoid differentiation whereas higher concentrations induced apoptosis of CD34þ cells but not CD7 T cell precursors. A role for combinatorial effects of Jagged stimulation of Notch and cytokine-induced signaling pathways (Kit, Flt3, Mpl) was reported in murine HSC cultures, with a 10e20 fold expansion of HSC with long-term repopulating potential [224]. Inhibition of Notch signaling leads to accelerated differentiation of HSC in vitro and depletion of HSC in vivo [225].…”