Our data indicate that the in vitro-produced human dopaminergic neurons contribute to partial locomotive functional recovery in a hemi-Parkinsonian rat. Although some grafted dopamine neurons survive the transplant procedure, the majority of the DA neurons in the 5-month-old graft appears to differentiate from the grafted progenitors. The majority of DA neurons in the graft exhibited a midbrain phenotype despite the mixed population of midbrain and forebrain donor DA neurons. Although the cell preparation contained large numbers of dividing cells, cells in the graft exhibited greatly diminished proliferation and differentiated to neurons and glia by 5 months. In contrast to tumor formation by hESC-derived dopaminergic cultures within 8–13 weeks reported to date, our extensive analyses showed no obvious tumor formation after 5 months of survival, the longest time so far achieved for hESC-derived dopaminergic cell transplants in the Parkinson rat model. Therefore, hESC-produced dopamine neurons can functionally engraft in the brain.
Several groups have reported that hESC-produced DA neurons are present in the graft in the 6-OHDA-lesioned rodents in the short term (2–13 weeks), sometimes in impressive numbers [12
]. However, functional contribution has not been reported, with a few exceptions. In a report by Roy et al., recovery of rotation behavior is observed as early as 4 weeks post-transplantation [16
]. In our present study, functional recovery did not occur until approximately 3 months following transplant and began to exhibit a statistically significant difference, compared with the control transplants, by the 5th month. This is generally consistent with reports of behavioral recovery 2–3 months after transplantation with fetal human mesencephalic tissues as a donor [41
]. Sonntag et al. also reported that 1 of the 20 grafted animals exhibited significant reduction in amphetamine-induced rotation at 12 weeks postgraft, and this rat had more than 500 TH+ cells [20
]. The delayed functional effect in our present study does not appear to be due to the lack of DA neurons. DA neurons are present in the graft shortly after transplantation (1st week), and there are significant numbers of them (>300 per graft) in the 4th week of transplantation. This exceeds the minimum numbers that are thought to be sufficient to produce locomotive behavioral changes in the 6-OHDA-lesioned rat [43
]. This implies that a simple secretion of DA is not sufficient and that the integration of DA neurons into the striatal tissue may be necessary to achieve a stable functional recovery [46
]. There are signs of potential integration of hESC-derived cells into the host striatum in our present study, including the extension of TH+ fibers into the striatum and synaptic-like connections between grafted human neurons and the host DARPP32-expressing GABAergic neurons. Synaptic-like connections between grafted primate ESC-derived TH- and DARPP32-positive cells have also been reported lately [48
], although the DARPP32-positive cells in our present study are always endogenous. The requirement of an extended period for human DA neurons to integrate into the host animal brain may be related to the intrinsic property of human neurons that take at least 10 weeks of hESC differentiation to exhibit functional synaptic transduction [49
]. This is also consistent with previous observations that hESC-derived neural progenitors take months after transplantation into the rodent brain to become mature neurons [50
], fire action potentials [51
], and form synapses [40
]. The relatively low degree of DA neuron fiber output to the striatum may be another factor. These factors may explain why the cylinder test and the stepping test had not begun to show obvious improvement. The initial contralateral rotation following amphetamine challenge in the cell-grafted but not the medium-injected rats suggests that the reduction in amphetamine-induced rotation is caused by functional implantation, even in the absence of improvement for the stepping test.
It has been proposed that midbrain dopamine neurons are necessary for functional engraftment [52
]. In our grafts, very few DA neurons were present in the 1st week. Most of the DA neurons in the 5-month graft were presumably differentiated from their progenitors, although re-expression of TH was not excluded. Differentiation of DA neurons from grafted progenitors has been reported by Ben-Hur et al., in a study in which they transplanted hESC-derived neural progenitors instead of DA neurons [12
]. The facts that the DA neurons in our grafts did not colabel with GABA and the forebrain transcription factor Foxg1, whereas GABA- and Foxg1-expressing cells were present in the grafts, indicate that the DA neurons in the grafts do not resemble forebrain DA neurons. This could be due to a selective loss of the forebrain DA neurons that normally do not form synapses with striatal GABA neurons. However, the midbrain transcription factor En1 was not detected in the TH+ cells by immunohistochemistry either. Since many DA neurons and neural progenitors carry a midbrain phenotype before transplantation, it is likely that the expression of developmentally regulated transcription factor En1 is suppressed in the striatal environment, thus making it undetectable. Developmentally, mid/hindbrain and forebrain transcription factors reciprocally antagonize each other [53
]. Phenotypic instability of in vitro-generated human neurons might be another possibility. The expression of Girk2 in DA neurons in the graft supports the likelihood of a midbrain phenotype, especially the nigra phenotype, for the functionally grafted DA neurons.
Tumorigenic potential is a major concern if hESC-derived cells are going to be applied clinically, especially in light of the reports that hESC-derived dopaminergic transplants result in overgrowth or even teratoma formation within a short period (8–13 weeks) of survival [16
]. The lack of rosette structures in the graft, the presence of scant Ki67+ cells in the 5-month-old graft, and the lack of expression of primitive stem cell markers, such as Oct4 and Pax6, indicate the absence of tumors. We believe that the lack of tumorigenic potential of our dopaminergic cultures may be related to the following factors. First, our neuroepithelial differentiation protocol yields a nearly pure population of synchronized neuroectodermal cells, with undetectable expression of mesodermal and endodermal markers [22
]. This step essentially eliminates undifferentiated ESCs and cells of a non-neural lineage. Most other dopaminergic differentiation cultures to date use stromal cell coculture or suspension aggregation cultures [8
], which either carry over tumorigenic stromal cells or inefficiently exclude undifferentiated cells in the aggregates. Second, we differentiated the hESCs to DA neurons for a total of 7 weeks. This period is equivalent to 8 weeks of human gestation, in which most fetal mesencephalic cells are chosen for clinical transplant therapy [54
]. Reports to date, including our own unpublished experiments, indicate that cells differentiated for a shorter period often result in large grafts in the rodent brain. These cultures usually contain more progenitors, which explains why most TH+ DA neurons are present in the graft periphery [21
]. In our present transplants, DA neurons are distributed throughout the graft, similar to the distribution pattern seen with the primary mesencephalic transplant [41
]. Third, our cells are largely restricted to the midbrain fate. However, large grafts were observed in two of six rats by 5 months after transplantation, indicating neural overgrowth. This may be attributed to the injection of more cells in these two animals, as our cell preparation is not completely dissociated into individual cells. It may also be attributed to the presence of neural progenitors with forebrain phenotypes in our cell preparation. Forebrain progenitors intrinsically undergo many more cell divisions than midbrain progenitors before exiting the cell cycle. We have recently shown that hESC-derived neuroepithelial cells (without FGF8 and SHH treatment and displaying forebrain phenotypes), following transplantation to postnatal immune-deficient mice, divided in the first 2–3 months before differentiating to postmitotic neurons and glia [40
]. Indeed, we have observed a large population of Foxg1-expressing cells in the larger grafts. Minimizing the proportion of forebrain progenitors by more completely patterning the cells to the midbrain phenotype will likely reduce the initial cell division, which will be a critical area to be further explored.
Although our transplant work is performed mainly using the H9 cell line, we and others have previously shown that many different hESC lines exhibit a similar behavior in dopaminergic differentiation with similar phenotypes [7
]. The demonstration of functional engraftment of the hESC-generated dopaminergic cells for 5 months without tumor formation, similar to what has been observed using human fetal tissue transplant [41
], raises hopes for potential uses of stem cell-produced progenies for therapy.