The marrow cell continuum: stochastic determinism.

Traditional models of hematopoiesis have been hierarchical in nature. Over the past 10 years, we have developed data indicating that hematopoiesis is regulated in a continuum with deterministic and stochastic components. We have shown that the most primitive stem cells, as represented by lineage negative rhodamine(low) Hoechst(low) murine marrow cells are continuously or intermittently cycling as determined by in vivo BrdU labeling. When marrow stem cells are induced to transit cell cycle by in vitro exposure to cytokines, either IL-3, IL-6, IL-11, and steel factor or thrombopoietin, FLT3 ligand, and steel factor, they progress through cycle in a highly synchronized fashion. We have determined that when the stem cells progress through a cytokine stimulated cell cycle the homing, engraftment, adhesion protein, global gene expression, and hematopoietic differentiation phenotypes all change in a reversible fashion. This has led to the continuum model, in which, with cycle transit, chromatin is continually changing altering open transcription areas and providing a continually changing landscape of transcriptional opportunity. More recently, we have extended the changing differentiation profiles to differentiation into lung cells and found that non-hematopoietic differentiation also shows cycle related reversibly modulation. These observations all together support a continuum model of stem cell regulation in which the phenotype of the marrow stem cells is continually and reversibly changing over time.

The stem cell continuum: considerations on the heterogeneity and plasticity of marrow stem cells.

Traditional models of hematopoiesis have been hierarchical. Recent evidence showing that marrow stem cells are a cycling population and that the hematopoietic phenotype of these cells reversibly changes with cycle transit have suggested a continuum model of stem cell regulators. Studies on marrow cell conversion to lung cells have extended this continuum to cycle-related differentiation into nonhematopoietic stem cells. We postulate that stem cells transiting cell cycle continually change their chromatin structure, thus providing different windows of transcriptional opportunity and a continually changing phenotype. Final outcomes with this continuum model would be determined by the specific chromatin state of the cell and the presence of specific differentiation inducers.

The molecular basis for the cytokine-induced defect in homing and engraftment of hematopoietic stem cells.

OBJECTIVE: Hematopoietic stem cell homing and engraftment is dramatically altered by cytokine exposure. These studies address the molecular mechanisms responsible for the observed changes in transplantation biology. METHODS: Primitive murine hematopoietic stem cells were isolated by fluorescence-activated cell sorting of lineage depleted (Lin(-)) cells exhibiting low staining of Hoechst 33342 and rhodamine 123 dyes or Lin(-) cells bearing Sca. Adhesion receptor expression was examined by immunofluorescence and reverse transcriptase polymerase chain reaction. In vitro adhesion assays were employed to define binding interactions between stem cells and stroma or extracellular matrix proteins. RESULTS: Adhesion of Lin(-)Sca+ cells to Dexter stroma could be blocked by about 90% with antibodies to PECAM-1, alphaa(4), or beta(1), and partially blocked by antibodies to alpha(5), CD44, or L-selectin. By immunofluorescence, about 30% of purified Lin(-)Ho(lo)Rho(lo) cells expressed alpha(4), alpha(5), beta(1), and L-selectin, about 15% expressed alpha(L) and alpha(6), half expressed PECAM-1, and none expressed alpha(1) or alpha(2). After 48 hours in expansion cytokines, only 9% of the cells expressed alpha(4) and none expressed beta(1), whereas alpha(L) expression was fully restored, PECAM-1 and L-selectin partially restored, CD44 expression was newly induced, and adhesion to both fibronectin and laminin was reduced. Adhesion to purified collagen, fibronectin, or laminin enhanced expression of beta(1) integrins. CONCLUSION: Expansion cytokines that move quiescent primitive hematopoietic stem cells into S phase markedly altered adhesion receptor expression and reduced their functional binding to extracellular matrix, which could reduce engraftment after transplant.

Gene transfer to ankyrin-deficient bone marrow corrects spherocytosis in vitro.

OBJECTIVE: The goal of this study was to transfer by retroviral vector the cDNA for ankyrin to progenitors from normal bone marrow and from the nb/nb spherocytosis mutant deficient in expression of full-length ankyrin to achieve erythroid expression of functional ankyrin protein. MATERIALS AND METHODS: A minigene composed of the human ankyrin promoter, murine ankyrin cDNA, and the 3' human domain corresponding to the ankyrin 2.2 isoform was assembled in the retroviral vector, pG1. Murine erythroleukemia (MEL) cells, normal murine bone marrow cells, 3T3 fibroblasts, and nb/nb mutant bone marrow and spleen cells were transduced with the retroviral supernatant. Transduced mutant cells were induced to differentiate in liquid culture. Gene transfer was assessed by colony polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR, immunofluorescence, and Southern, Northern, and Western blot analysis. RESULTS: MEL cells, normal bone marrow progenitors, and nb/nb cells were all successfully transduced and expressed ankyrin by RT-PCR and Western blot. Transduced murine 3T3 fibroblasts and MEL cells exhibited cell membrane staining by immunofluorescence. Colony RT-PCR demonstrated dependence of expression on erythropoietin. In vitro, the transduced nb/nb cells matured to polychromatophils, whereas nontransduced nb/nb cells matured to microspherocytes. CONCLUSION: Retroviral transfer of ankyrin corrected the defect leading to formation of microspherocytes in erythroid differentiation cultures from the nb/nb mutant. The human ankyrin promoter conferred erythropoietin-dependent expression in normal and mutant erythroid progenitors, which could have implications for the gene therapy of human hemolytic anemias.

Effect of ex vivo cytokine treatment on human cord blood engraftment in NOD-scid mice.

Umbilical cord blood transplantation is considered an alternative to traditional bone marrow transplantation for patients who do not have matched sibling donors. In this study, we examined the effects of ex vivo treatment of human cord blood cells with cytokine mixtures and assessed the ability of treated cells to engraft in NOD-scid mice. We incubated the cord blood with a four-factor cytokine mixture of interleukin (IL)-3, IL-6, IL-11 and stem cell factor, or with a two-factor cytokine mixture of thrombopoietin and flt-3. Incubation of cord blood for 48 h with either cytokine mixture did not affect progenitor cell number or proliferative potential as measured by the high proliferative potential (HPP) assay. Cytokine-treated cord blood injected into irradiated NOD-scid mice resulted in multilineage human engraftment. Overall, incubation with cytokines resulted in variable levels of engraftment with different cord blood samples. Incubation of cord blood with the four-factor cytokine mixture resulted in increased survival of irradiated NOD-scid recipients. These results demonstrate that short-term ex vivo treatment of human progenitor cells gives variable results on in vivo multipotential capabilities.