Engraftment of post 5-fluorouracil murine marrow into minimally myeloablated (100 cGy) murine hosts.

Minimal myeloablative approaches are now being widely applied in the treatment of different hematological malignancies. One hundred cGy whole-body irradiation is a stem-cell-toxic, relatively non-myelotoxic treatment that allows for relatively high levels of donor chimerism. 5-Fluorouracil (5-FU) treatment leads to a relative concentration of high proliferative potential-colony-forming cell (HPP-CFC) and is an approach that has been used to induce in vivo progenitor/stem cell cycling to facilitate retroviral integration in gene therapy approaches. We have now evaluated the capacity of marrow harvested 1, 2, 6, or 12 days after 5-FU treatment (150 mg/kg) to engraft in 100 cGy-treated female BALB/c mice. Engraftment was assessed at 3, 10, and 24 weeks. A rapid induction of an engraftment defect occurred 1 day post 5-FU and persisted through day 6 with a recovery by day 12. To evaluate cell cycle status of normal and 5-FU-treated marrow cells, male donors received hydroxyurea (900 mg/kg i.v.) or phosphate-buffered saline (PBS), 2 h prior to marrow harvest and transplantation into submyeloablated female recipients. Engraftment levels were similar for hydroxyurea-treated mice and controls. Thus, these studies show transiently defective engraftment of 5-FU-treated marrow into submyeloablated hosts, which may be related to the cell cycle status of the stem cells.

A method for clonal analysis of epidermal growth factor-responsive neural progenitors.

Epidermal growth factor (EGF) responsive neural progenitors are defined by clonal growth from single cells. In previous studies we were unable to obtain clones at single cell densities using trypsinized cells and trituration alone always gave cellular aggregates. Here we report on single cell derived clones using a technique involving trituration of EGF responsive neurospheres, cell filtration, and single cell sorting using a MoFlo high speed fluorescence activated cell sorter. Single cell deposition was confirmed by labeling cells with Hoechst 33342 and Flow-check Fluorospheres, and visualization by fluorescence microscopy. The cells were deposited into liquid medium and grown from single cells in 10-20 ng/ml EGF for 12-14 days. This gave a cloning efficiency of 2.12%+/-0.37. New colonies occurred as late as day 18 post-sort. Tritiated thymidine suicide indicates that a percentage of these cells are cycling. Immunohistochemical analysis for oligodendrocytes, astroglia, and neuronal lineages performed on colonies at 10-14 and 21-28 days gave 39% uni-lineage, 36% bi-lineage, and 25% tri-lineage colonies. A total of five different types of progenitor cells were observed. In individual colonies, oligodendrons predominated with a lesser presence of astroglial or neuronal cell types. This approach establishes a reliable and reproducible method for single cell cloning of neurosphere cells.