Functional and phenotypic analysis of human CD34⁺CD38⁻Lin⁻ cells cultured in serum-free conditions containing soluble forms of hDelta-1 and hDelta-4.

Highly purified CD34⁺CD38⁻Lin⁻cells (purity greater than 99%; data not shown) were seeded in 96-well culture plates containing serum-free BIT medium and growth factors as described in “Materials and methods.” Cells were harvested at the indicated times for proliferative and differentiative analysis by fluorescence-activated cell sorting (FACS) or assayed for functional progenitors in methylcellulose containing human cytokines. (A) The fold increase in total cell number relative to cells seeded on Day 0. Cells from individual wells were counted, and the mean fold increase in total cell number was calculated. (B) Changes in the total number of primitive CD34⁺CD38⁻ cells in culture. Total number of primitive CD34⁺CD38⁻ cells was determined for each well from the frequencies of CD34⁺CD38⁻ cells examined by FACS analysis. Mean frequencies of CD34⁺CD38⁻ cells expressed as a percentage of the total cells in the culture is indicated for each treatment. (C) Effect of hDelta-1 and hDelta-4 treatment on the total number of clonogenic progenitors. Aliquots of cells were plated in methylcellulose cultures as described in “Materials and methods.” Hematopoietic progenitors (colony-forming units [CFUs]) were scored, and the frequency of progenitors was determined from the input cell number. The frequency of CFUs and the total number of cells harvested from each well were used to calculate the total number of clonogenic progenitors. Values shown are the mean ± SEM (n = 6). *Significant difference; P < .01.