Neural precursors derived from human embryonic stem cells maintain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons.

Human embryonic stem (hES) cells can provide a useful source of cells for basic developmental studies, cell-based therapies or drug screening for treatment of neurological disorders. Initial work with mouse embryonic stem (mES) cells provided the knowledge base for studies of hES cells. Several reports have demonstrated the capacity of mES cells to generate neural precursor (NP) cells that can differentiate into dopaminergic (DA) and motor neurons using optimal culture conditions and/or genetic manipulation (Kawasaki et al. 2000; Lee et al. 2000; Chung et al. 2002; Barberi et al. 2003; Kim et al. 2006). In particular, we recently showed that NP cells derived from mES cells (mES-NP) maintain the potential to differentiate into DA neurons after expansion in vitro. Therefore, it is reasonable to hypothesize that NP cells derived from hES cells would maintain their potential to differentiate into three cell types of neural lineage and DA neurons during in vitro differentiation after long-term mitogenic expansion. However, there are fundamental differences between mouse and human stem cell studies: dissociated single hES cells have poorer survival than mES cells (Amit et al. 2000), human leukemia inhibitor factor (LIF) is unable to maintain the pluripotent state of hES cells (Daheron et al. 2004; Sato et al. 2004), and human development is considerably longer than that of mouse as is reflected in longer cell cycle times. In addition, the maintenance of hES cells' ability for self-renewal will be critical to their large-scale growth and differential potential. It is important to note that preparation of feeder layers, such as fibroblasts for self-renewal and stromal cells or immortalized astrocytes for differentiation of hES cells is labor intensive. Furthermore, variations between batches of feeder layers and different passages of hES cells can cause undesirable experimental complexities. Recently, many laboratories have reported that hES cells can efficiently differentiate into DA neurons through an intermediate stage that has been known as hES-derived NP (hES-NP) cells (Reubinoff et al. 2001; Ben-Hur et al. 2004; Park et al. 2004; Perrier et al. 2004; Li et al. 2005; Park et al. 2005; Tabar et al. 2005; Yan et al. 2005; Sonntag et al. 2007). However, the proliferative and developmental potentials of hES-NP cells are still poorly understood although multipotent NP cells could be efficiently derived from ES cells. To address these, we expanded NP cells that were derived from H1 and HSF6 hES cell lines and investigated their developmental properties in vitro. Here, we report that hES-NP cells were able to proliferate approximately 380 000-fold after expansion for 12 weeks, and maintained the potential to generate Tuj1+ neurons, glial fibrillary acidic protein-positive (GFAP+) astrocytes and oligodendrocyte marker-positive (O4+) oligodendrocytes as well as functional DA neurons. In addition, hES-NP cells maintained their differentiation potential through long-term storage of over 2 years and multiple freeze-thaw cycles. We report that hESNP cells are fast, dependable, and unlimited source of neuronal cells for basic and applied research.