Douglas C. Dooley

Expression of Flt3 and c-kit during growth and maturation of human CD34+CD38- cells.

Studies of murine stem cells suggest that the cytokine receptors Flt3 and c-kit are expressed differentially on the earliest reconstitutional cells, such that Flt3 is not expressed until after stem cell activation. Much less is known about the expression of Flt3 and c-kit on primitive human cells, especially those mobilized into circulation for transplantation. In this study, early circulating precursors were analyzed for expression of Flt3 at the gene and protein levels. Flow cytometric studies showed that >90% of CD34+CD38- cells expressed Flt3 antigen (CD135). The proportion of fresh CD34+ cells expressing Flt3 decreased as CD38 staining increased. These results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) analyses, which showed that Flt3 gene expression generally was limited to the CD34+CD38- population. Because Flt3 ligand (FL) enhances the growth and/or maintenance of primitive cells, it was important to know how long early cells retain Flt3 receptor expression in expansion culture. Both RT-PCR analyses and functional tests demonstrated that primitive cells are capable of expressing Flt3 for as long as 2 weeks in liquid medium. During the first week of culture, FL enhanced the generation of cells and progenitors without causing a loss of primitive CD34+CD38-Flt3+ cells. Flt3 expression in cell cultures was limited to precursors retaining a CD34+CD38(-/lo) phenotype. Because the most primitive human precursors are believed to express c-kit at a low level, we examined the FL responsiveness of CD34+CD38-c-kit(-/lo) cells and CD34+CD38-c-kit+ cells. CD34+CD38-c-kit(-/lo), cells constituted a small fraction (12%) of the CD34+CD38- population. Whereas both c-kit(-/lo) and c-kit+ subsets were stimulated by FL, cell expansion (p < 0.01) and colony formation (p < 0.01) were greater and maintained longer with CD34+CD38-c-kit(-/lo) cells. Furthermore, the rapid response to FL suggests that primitive CD34+CD38-c-kit(-/lo) cells express Flt3 at the time of isolation or shortly thereafter. These results demonstrate the presence of Flt3 on CD34+CD38 blood cells and suggests that Flt3 also may be present on a c-kit(-/lo) subset, among the most primitive in circulation. Flt3 is lost during maturation to committed (CD34+CD38+) lineages. Addition of FL to primitive cell cultures stimulates cell expansion while maintaining early CD34+CD38-Flt3+ precursors for at least 7 days. The possible existence of a more primitive CD34+CD38-c-kit(-/lo) Flt3(-/lo) precursor remains to be determined.

Transforming growth factor-β1 induces apoptosis in CD34+CD38−/low cells that express Bcl-2 at a low level

OBJECTIVE Transforming growth factor-beta(1) (TGF-beta(1)) strongly inhibits the proliferation and differentiation of primitive CD34(+)CD38(-) hematopoietic cells. In contrast, Flt3 ligand (FL) is a positive effector of CD34(+)CD38(-/low) cell proliferation. Because apoptosis plays a critical role in hematopoietic development, TGF-beta(1) and FL were analyzed as possible modulators of apoptosis. Specifically, this report examined expression of apoptotic promoters Bax and Bad and apoptotic inhibitors Bcl-2 and Bcl-x (all members of the Bcl-2 protein family). Protein levels were determined in fresh and cultured CD34(+)CD38(+) cells and CD34(+)CD38(-/low) cells with and without treatment with TGF-beta(1) and FL. MATERIALS AND METHODS Cells fractions were purified by sorting CD34(+)-enriched mononuclear cells from mobilized peripheral blood. Expression of Bcl-2, Bcl-x, Bax, and Bad and the extent of apoptosis were determined by flow cytometric analysis of freshly isolated cells and cells cultured with TGF-beta(1) and FL effectors. RESULTS TGF-beta(1) reduced CD34(+)CD38(+) cell expansion and arrested cell division. Inhibition of growth was not accompanied by an increase in apoptosis. In CD34(+)CD38(-)(/low) cells, serum TGF-beta(1) and added TGF-beta(1) inhibited cell growth and significantly increased apoptotic cell death. Freshly isolated CD34(+)CD38(+) and CD34(+)CD38(-/low) cells expressed Bcl-2 at similar low levels. However, after 3 days, Bcl-2 expression was markedly higher in cultured CD34(+)CD38(+) cells. TGF-beta(1) significantly increased Bax expression in both fractions after 3 days cultivation (p = 0.0034). Thus, addition of TGF beta-1 further reduced the already low Bcl-2:Bax ratio in CD34(+)CD38(-/low) cells. CONCLUSIONS Compared to CD34(+)CD38(+) cells, CD34(+)CD38(-/low) cells were slow to up-regulate expression of Bcl-2 during ex vivo culture. TGF-beta(1) up-regulated Bax expression by both CD34(+)CD38(+) and CD34(+)CD38(-)(/low) cells and promoted apoptosis in the latter fraction. This suggests that the preferential induction of apoptosis in primitive cells by TGF-beta(1) may be due to its further reduction of the Bcl-2:Bax ratio.

Density distribution of hematopoietic progenitor cells and T lymphocytes from peripheral blood: heterogeneity of the human population

Abstract. Density gradient centrifugation has been used in animal systems to purify stem cells and eliminate T lymphocytes prior to allogeneic transplantation. There is substantial disagreement whether the same approach can be used to purify hematopoietic stem cells obtained from human peripheral blood. The purpose of the present study was to resolve that issue by determining the density distribution of 4 classes of human leucocytes: total mononuclear cells, T lymphocytes, CFUc, and BFUe. To ensure a representative sampling, a large number of randomly selected donors were analyzed. The results show that most T lymphocytes band between 1.068 and 1.071 g/ml, with relatively little variation from individual to individual. In contrast, the density distributions of both CFUc and BFUe fluctuated markedly from donor to donor. As a consequence, there was significant variability in the degree of progenitor‐T cell separation. The implications of these results for clinical application of the density separation technique are discussed.

Glycerolization of Chinese hamster ovary cells in monolayer culture

Abstract CHO monolayer cultures were used as a model system to examine the kinetics of cell glycerolization and deglycerolization. Both influx and efflux of [ 3 H]glycerol appear to be first-order processes, the rate of flux being proportional to glycerol concentration. When net flux ceases, the intracellular and extracellular concentrations of glycerol appear to be identical. Influx can be described by a single first-order curve, whereas efflux requires at least two such functions. Flux rates are sensitive to changes in osmolality, the fast efflux component accelerating and the slow component decelerating with increased application of osmotic stress. The rate of influx slows as the temperature drops and as the viscosity of the medium increases. Cells can tolerate a much steeper deglycerolizing gradient at 37 °C than at 0 °C.

Culture of colony-forming hematopoietic progenitor cells from human peripheral blood

Methods for culturing hematopoietic colonies from human peripheral blood are described in this report. Granulocyte-macrophage progenitor cells (CFU-GM: colony forming units-granulocyte/macrophage) are grown in 35-mm dishes containing 4 ml of 0.35% agarose in Iscoves modified Dulbeccos medium (IMDM) with 20% prescreened fetal bovine serum (FBS). Colony-stimulating activity is provided by leukocyte-conditioned medium, and nonactivated autologous T lymphocytes or recombinant granulocyte- and granulocyte/macrophage-colony stimulating factor or both. This procedure minimize the growth inhibition caused by monocytes. Erythroid progenitor cells (BFUe: burst forming units-erythroid) are cultured in 1 ml of 0.35% agarose in IMDM with 30% FBS and conditioned medium from human bladder carcinoma cell line 5637 or recombinant interleukin 3. Both assays have proved useful in studying the enrichment of peripheral blood hematopoietic cells.