The functional effects of neurons derived from human and primate ES cells in vivo
have been inconsistent. Furthermore, no previous studies have shown that parthenogenetic cells function in vivo
. In this study we show for the first time a robust motor recovery in hemi-parkinsonian rats that was directly correlated with the number of parthenogenetic ES-derived DA neurons present in the grafts. Modifications of our in vitro
differentiation protocol led to an increase in the number of DA neurons and to an improved survival, which in turn resulted in a sufficient number of midbrain-like DA neurons in the grafts (Ferrari et al.
) to mediate a reversal of typical parkinsonian motor deficits. In addition to the remarkable effect on amphetamine-induced rotation, which is a useful indicator of the presence of mature DA neurons in the graft, because it requires localization of the DA transporter to the cell membrane and the synthesis and release of DA, we also observed a significant attenuation in apomorphine-induced rotation and, importantly, an improvement in spontaneous exploratory behaviour to the same extent reported for l
-DOPA in the cylinder test (Mela et al.
). These behaviours are indicative of a substantial degree of graft integration, because basal (spontaneous) release of DA by grafted neurons is necessary to attenuate the post-synaptic adaptations that mediate apomorphine-induced rotational response and to restore symmetric exploratory behaviour (Dunnett et al.
; Nikkhah et al.
). Axonal outgrowth and synaptic contacts between grafted DA neurons and host DARPP-32 striatal neurons was confirmed by the post-mortem analysis at the cellular level, using confocal imaging. Other factors, in particular compression, or lesion of striatofugal fibres, may indeed diminish the rotational response to apomorphine (Marshall and Ungerstedt, 1977
; Christophersen and Brundin, 2007
), but such complications were never observed in these animals. Analysis of graft cell composition revealed uniform expression of neuronal proteins such as neural cell adhesion molecule and Dcx, with little expression of nestin within the grafts at 16 weeks post-transplantation and no evidence of teratoma formation in any of the experimental groups. While nestin expression was low, the presence of uncommitted cells within the grafts is still a matter of great concern and emphasizes the need for selection procedures.
Another fundamental contribution of this study is the demonstration of the absence of BrdU incorporation by DA neurons after transplantation. Knowing whether DA neurons present in the grafts are the ones differentiated in vitro
, or are born in the transplants is relevant for implementing selection and transplantation methods, in particular for negative selection of proliferating cells and cell dose optimization. Indeed, the developmental stage is crucial in terms of predictability of graft composition and phenotypic stability based on in vitro
characterization of the cells (on which transplantation parameters are necessarily based). Because post-mitotic neurons are fragile, many in vivo
studies have been performed using minimally differentiated neural progenitors or spheres from human (Ben-Hur et al.
; Roy et al.
; Ko et al.
) or primate (Takagi et al.
) ES cells for transplantation into parkinsonian animals. In those studies, the TH+ neurons identified in the grafts were most likely born in vivo
(see Discussion in Brederlau et al.
) and the regional phenotype was not established. Moreover, while such an approach sometimes enhances grafted cell survival, the lack of control over proliferation and differentiation makes it unsuitable for therapeutic applications, because of the development of teratomas or graft overgrowth (Roy et al.
). In our study, administration of BrdU was designed specifically to detect DA neurons born in vivo
and our results demonstrate that there were none. The absence of BrdU labelling in TH+ neurons in the grafts indicates that these neurons were born in vitro
. It is very unlikely that there were proliferating DA precursors that died because of BrdU neurotoxicity, as we found doublecortin+ neurons that were BrdU+. The fact that very few cells were Ki67+ at the end of the study supports the idea that the cells that are proliferating in vivo
are not stem cells but committed precursor cells, close to their developmental cell-cycle exit. Consistently, many double-labelled HNA+/BrdU+ cells expressed Dcx. Importantly, our results demonstrate that it is feasible to harvest and transplant in vitro
born post-mitotic DA neurons that function in vivo
; this post-mitotic stage would be preferable in terms of safety and reproducibility for clinical applications. Notably, the low numbers of Ki67+ cells in the graft, together with the fact that some BrdU labelled cells were detectable 10 to 14 weeks post-oral administration emphasizes the low proliferation rate when ES cells are differentiated for long time periods in vitro
, like in the present study (6 weeks).
We found here that the addition of factors (Wnt5a/FGF20/FGF2), which are normally secreted by mesencephalic glia, resulted in a significant increase of TH+ neurons and enhanced survival. In vitro
(Murase and McKay, 2006
), FGF20 has been shown to promote survival of the CB– subpopulation of DA neurons when the cells were subjected to oxidative stress. Although we did not observe a effect on the TH+CB– population, the presence of FGF20 in the last stage of differentiation may have contributed to the enhanced survival to transplantation. In a recent publication (Correia et al.
) FGF20 appeared to promote DA differentiation from hES cells in vitro
, using PA6 co-culture inductive protocols. FGF2 trophic support appears to be critical for post-mitotic DA neurons while it maybe redundant (i.e. compensated by other trophic factors) during development (Timmer et al.
). Our data suggest that Wnt5a had a strong effect in vitro
and in vivo
. Wnt5a promotes the differentiation of Nurr-1+ precursors to DA neurons and has been shown to induce a significant increase in TH+ neurons in vitro
, in primary ventral midbrain DA cultures (Castelo-Branco et al.
; Castelo-Branco et al.
) and in locus coeruleus
noradrenergic cultures (Holm et al.
). Interestingly, in those in vitro
studies, the effect of Wnt5a (at late developmental stages that roughly correspond with the time of in vitro
exposure in our study) was hypothesized to be due to direct TH induction or phenotypic stabilization in post-mitotic progenitors (Holm et al.
). In addition, this group has recently shown that Wnt5a over-expression in foetal ventral midbrain neurons is related to better survival of transplanted DA neurons and to faster functional recovery in vivo
(Parish et al.
), like in our study, without having a direct impact on either proliferation or cell death. While Wnt5a promotes maturation, transcriptional stabilization and cell-cycle exit of DA precursors (Castelo-Branco et al.
) and probably enhanced the generation and/or maturation of DA neurons in our paradigm in vitro
, it is likely that FGFs acted synergistically to improve overall survival to transplantation. It is possible that this particular trophic factor combination enhanced not only survival, but also proliferation of non-DA cells in vivo
, early after transplantation, yet additional labelling studies would be required to identify such effects.
Our results here demonstrate that parthenogenetic stem cell lines could be utilized to derive functional DA neurons for cell therapy in Parkinson's disease patients. Recent studies have pointed to substantial differences between hES cell lines upon differentiation (Wu et al.
). Whether a propensity to generate neurons, or, more specifically, ventral neuronal phenotypes may be related to the parthenogenetic origin is currently under investigation (for example, there may be maternally imprinted genes involved in the patterning, differentiation or maturation of these neuronal populations or lack of paternally imprinted genes facilitating it). Nevertheless, while we acknowledge that further enrichment and purification of neurons derived from ES cells is required (Hedlund et al.
), this study conclusively demonstrates that is feasible to restore motor function in parkinsonian animals using DA neurons derived in vitro
from primate parthenogenetic stem cells.