Expansion of cord blood CD34+ hematopoietic progenitor cells in coculture with autologous umbilical vein endothelial cells (HUVEC) is superior to cytokine-supplemented liquid culture.

Expansion of hematopoietic progenitor cells (HPC) in the presence of endothelium has been shown to result in grafts capable of restoring hematopoiesis in a myeloablated host. However, the use of xenogeneic endothelium or cell lines may carry risks in a clinical transplantation setting. We explored the feasibility of cord blood progenitor cell expansion in vitro in an autologous coculture system using umbilical vein endothelial cells (HUVEC). CD34+ HPC and HUVEC were isolated from the same umbilical cord. For 3 days, HPC were maintained in serum-free medium supplemented with a single cytokine (SCF) or a cytokine combination (SCF, Flt3-ligand, IL-6). Meanwhile, adherent HUVEC cultures were established. After addition of VEGF and IL-1 at day 3, the cells were either added to HUVEC cultures or grown without endothelial cells for further 7 days. Total cells, CD34+ and clonogenic progenitors were significantly increased when coculture was compared to liquid culture. Long-term culture-initiating cells (LTC-IC) and cobble stone area-forming cells (CAFC, limiting dilution analysis) were detected more frequently after coculture with endothelial cells. Also precursors and mature myeloid cells were observed after expansion. We conclude that coculture with autologous HUVEC represents a feasable approach for ex vivo expansion of cord blood HPC.

Killing of Schistosoma mansoni sporocysts by Biomphalaria glabrata hemolymph in vitro: alteration of hemocyte behavior after poly-L-lysine treatment of plastic, and the kinetics of killing by different host strains.

Behavior of hemocytes of the gastropod mollusc Biomphalaria glabrata was markedly changed on plastic by treatment of the substrate with 0.1 mg/ml poly-L-lysine compared to behavior on untreated plastic. On lysine, the cells showed minimal spreading, moved significantly faster, and formed aggregates. Cell-mediated cytotoxicity (CMC) assays were set up on the modified and untreated substrates to compare the killing capacities of B. glabrata hemocytes against Schistosoma mansoni sporocysts. Hemolymph from 1316-R1 (resistant) snails showed higher killing in lysine-treated wells; no significant difference in sporocyst mortality was observed in MO (susceptible) hemolymph between treated and untreated wells. The CMC assays on poly-L-lysine-treated plastic were used to compare the kinetics of parasite killing in hemolymph from 2 susceptible (MO, MRLc) and 2 resistant (1316-R1, 10R2) host strains. Marked differences could be observed between the two resistant snail strains, suggesting different mechanisms of parasite recognition, killing, or both.

Identification of a subpopulation of merozoites of Sarcocystis singaporensis that invades and partially develops inside muscle cells in vitro.

The affinity of merozoites of Sarcocystis singaporensis obtained from the lungs of acutely infected rats to muscle cells and other cell lines grown in vitro was examined. Two distinct types of mature schizonts developed in the lungs 11-13 days p.i. with sporocysts: those containing PAS- merozoites (type 1) which mainly reacted with antibodies prepared against sporozoites, and others containing PAS+ merozoites (type 2) which were antigenically close to bradyzoites. When inoculated onto cell cultures, type 1-merozoites induced schizogonic development in brain capillary endothelial cells of the rat. In contrast, type 2-merozoites invaded L6 myoblasts. In long-term cultures (50 days) of L6 cells, zoites transformed to a 8-15 microns long uninucleate stage which, tentatively, could be unizoite sarcocysts. Although the observed dichotomy in merozoite development is unprecedented in this form, evidence from previous work suggests that these observations are relevant to other Sarcocystis species. The presented cell culture system could be a first step towards successful growth of sarcocysts in vitro.