In Vitro Proliferation, Expansion, and Differentiation of a CD34+ Cell-Enriched Hematopoietic Cell Population from Human Umbilical Cord Blood in Response to Recombinant Cytokines

The conditions and mechanisms that control the in vitro growth of hematopoietic stem/progenitor cells (contained within the population of CD34+ cells) are still not completely understood.By using an immunomagnetic system, we have enriched for umbilical cord blood (UCB)-derived CD34+ cells (55% of total cells recovered vs. 0.8% of total cells prior to the enrichment procedure) and analyzed their in vitro growth (proliferation, expansion, and differentiation) in a liquid culture system in the absence or presence of different recombinant cytokine combinations.When the selected cells were cultured in the absence of recombinant cytokines, no proliferation or expansion was observed. In the presence of steel factor (SF) and interleukin-6 (IL-6), total cell number was increased nearly fourfold; however, no progenitor cell expansion took place. When cultures were supplemented with SF and IL-6 together with IL-3 and erythropoietin (EPO), a rapid proliferation of the CD34+ -enriched cell population was observed with a selective stimulation of erythropoiesis. However, this stimulation was only transient, suggesting that there was a rapid exhaustion of erythroid progenitor cells within the first 10 days. Significantly higher levels of proliferation and expansion of progenitor cells were observed in the presence of SF, IL-6, GM-CSF, and G-CSF with preferential stimulation of myelopoiesis. Interestingly, such stimulation of myelopoiesis was sustained for the entire culture period (>30 days). The highest levels of proliferation and expansion were observed in the presence of all six cytokines. Under these conditions, erythropoiesis was also sustained only transiently (10 days), whereas myelopoiesis was sustained for >30 days.This study indicates that significant proliferation and expansion of hematopoietic progenitors can be achieved in vitro when culturing a cell population in which CD34+ cells comprise only >50% of the total cells. Our results also suggest that myeloid progenitors (those responding to GM-CSF and G-CSF) possess higher expansion potentials in vitro than their erythroid counterparts. The methods described here for the enrichment and culture of CD34+ cells may be relevant in the development of protocols for the ex vivo proliferation and expansion of hematopoietic progenitors for transplantation.