Abstract
The ability to generate purified neural progenitors is critical to the development of embryonic stem cell-based therapies to alleviate human neurological disorders. While many cell culture protocols for directed differentiation of human embryonic stem (hES) cells into neural cells have been described, most yield mixed populations, some containing cells of different embryonic germ layer lineages, or even undifferentiated embryonic stem cells. In this study, we describe a method for single-cell dissociation, isolation by flow cytometry, and subsequent culture of neural progenitors from hES cells. As reported earlier, hES cells treated with the bone morphogenetic protein (BMP) antagonist noggin gave rise to neurospheres at a relatively high frequency. However, these noggin-treated embryonic stem cell cultures were heterogeneous, with cells expressing embryonic stem markers still detectable even following 14 days of differentiation. In order to isolate pure human neural progenitors, we fractionated noggin-treated ES cells on the basis of their expression of the putative neural stem cell marker, CD133, and the GCTM-2, and SSEA-1 antigens, which mark pluripotent stem cells and differentiated cells respectively from hES cell culture. CD133+ cells formed larger spheres compared to CD133− cells. CD133+SSEA1+ cells and CD133+SSEA-1− cells expressed similar levels of neural genes and formed neurospheres at similar frequencies. By contrast, CD133+GCTM-2+ cells expressed high levels of OCT4 but not neural lineage genes and failed to form neurospheres. CD133+GCTM-2− cells formed neurospheres at the relative highest frequency. Thus, negative selection with GCTM-2 may be useful for the purification of specific cell types differentiated from hES cells.