Human embryonic stem cells (hESCs), initiated from the inner cell mass of a blastocyst embryo, can be expanded continuously in a stem cell state and, in response to appropriate signals, produce expected differentiated progenies [1, 2]. These two unique characteristics make hESCs a useful tool for exploring novel medical therapies, as well as for developmental studies. In the past several years, strategies have been developed for directing these naïve stem cells to neuroectodermal cells [3, –5] and more differentiated neuronal and glial subtypes [6, , , –10] (reviewed in ). In particular, tremendous efforts have been made in developing hESC-derived cells to replace dopamine (DA) neurons that are degenerated in Parkinson disease [7, 8, 12, , , , , , , , –21]. The earliest transplantation study involved the use of hESC-derived neural progenitors by Ben-Hur et al. . They showed that the grafted cells differentiated into DA neurons in vivo, although at a low prevalence, and that the grafted cells induced partial behavioral recovery. Their data suggest a requirement of optimal in vitro dopaminergic neural differentiation prior to transplantation to achieve a complete behavior recovery. Currently, the vast majority of the in vitro DA neuronal differentiation protocols involve the use of cell-cell contact coculture of hESCs with stromal cells, such as PA6 and MS5 cells [8, 13, –15, 18, 20, 21]. Most recently, Roy et al. have reported a robust protocol for differentiating hESCs to DA neurons by coculturing with telomerase-immortalized midbrain astrocytes . We have developed a chemically defined culture system that also allows efficient differentiation of DA neurons from hESCs . A similar approach has been reported recently . Although they have various degrees of efficiency, most of the hESC-derived DA neurons can release dopamine and exhibit some electrophysiological properties of a neuron [7, 8, 14, 16, 18]. However, transplantations of these in vitro-produced human DA neurons, without genetic modification, have not yet produced any meaningful functional contributions in the 6-hydroxydopamine (6-OHDA)-lesioned rodents [13, –15, 17, 21]. One exception is the recent demonstration by Roy et al., which is accompanied by graft overgrowth within 10 weeks . However, teratoma formation, overgrowth, and the presence of primitive neuroepithelia are commonly observed using hESC-derived DA cell preparations even after a relatively short time (8–13 weeks) of survival [15, 16, 20, 21]. In addition, current differentiation approaches produce a mixed population of DA neurons with forebrain and midbrain phenotypes (reviewed in ). It remains unknown which type of stem cell-derived DA neural cells contributes to functional recovery (if any) following transplantation into the brain of Parkinson animals. This leaves open the questions of whether hESC-produced DA neural cells can functionally engraft in a safe manner and how to accomplish this.
In an effort to better understand the cellular behaviors and functional contribution of hESC-derived DA neural cells in vivo during a longer term, we transplanted in vitro-generated human DA neural cells into the 6-OHDA-lesioned rat striatum and dynamically monitored cell survival, differentiation, and proliferation and functional behavior changes 1, 4, and 20 weeks later. We demonstrated the presence of human DA neurons in the grafts and functional contribution at 5 months. The majority of dopaminergic neurons were differentiated from the grafted progenitors and exhibited some midbrain phenotype. Furthermore, grafted cells exhibited a significantly diminished mitotic activity by 5 months and chiefly differentiated into neurons and glial cells.