Yingsha Zhang

Specification of distinct cell types from human embryonic stem cells (hESCs) is key to the potential application of these naïve pluripotent cells in regenerative medicine. Determination of the nontarget differentiated populations, which is lacking in the field, is also crucial. Here, we show an efficient differentiation of motor neurons ( approximately 50%) by a simple sequential application of retinoid acid and sonic hedgehog (SHH) in a chemically defined suspension culture. We also discovered that purmorphamine, a small molecule that activates the SHH pathway, could replace SHH for the generation of motor neurons. Immunocytochemical characterization indicated that cells differentiated from hESCs were nearly completely restricted to the ventral spinal progenitor fate (NKX2.2+, Irx3+, and Pax7-), with the exception of motor neurons (HB9+) and their progenitors (Olig2+). Thus, the directed neural differentiation system with small molecules, even without further purification, will facilitate basic and translational studies using human motoneurons at a minimal cost.

Lentiviruses have been increasingly used for genetic modification of human cells including embryonic stem (ES) cells. Using four ubiquitous promoters--cytomegalovirus (CMV), cytomegalovirus immediate-early enhancer/chicken beta-actin hybrid (CAG), phosphoglycerate kinase (PGK), and human elongation factor-1alpha (EF1alpha)--in a lentiviral vector to drive the expression of the enhanced green fluorescent protein (EGFP) gene in human ES cells and mouse ES cells, we determined the extent of EGFP suppression by assessing the percentage of cells that were transduced with the EGFP gene but did not fluoresce green. A much higher level of transgene suppression was observed in human ES cells as compared to mouse ES cells. The suppression was also highly promoter dependent, leading to inactivation of more than 95% of the EGFP genes under the CMV or CAG promoter while only 55% under the PGK promoter. No promoter-dependent suppression was observed in transient transfection of human ES cells. Thus, the common phenomenon of poor transgene expression in human ES cells may be caused mainly by suppression of the transgene right after transduction and integration. Cautions should be taken to choose the optimal promoter when lentiviruses are used for genetic modification of human ES cells.