This relatively large primate transplant study was designed to examine parameters of tissue preparation and graft location relative to obtaining maximum functional benefit following striatal fetal cell transplantation. The study demonstrates that striatal fetal VM transplants both as cell suspensions and solid pieces, in the Cd and Put locations, are effective in improving parkinsonian signs and activities of normal daily living in a bilateral MPTP primate model of PD. We have also shown that, similarly to human tissue, long post-transplantation periods may be necessary to observe a meaningful functional recovery (at least 9 months post-transplantation in monkeys). This recovery is associated with the time fetal primate DA cells need for maturation, outgrowth and functional connectivity (Isacson et al., 2003
; Isacson and Deacon, 1997
) and is proportional to the reported outcomes of the clinical trials using human tissue, where substantial clinical benefits appear 12-24 months post-transplantation, but are not complete until about 3-4 years after surgery (Isacson et al., 2003
; Lindvall et al., 1994
; Piccini et al., 1999
Functional benefits in our study were particularly convincing in the most severely parkinsonian monkeys (equivalent to Hoehn-Yahr V, PD patients). Previous studies from our group show that these severe MPTP treated monkeys usually do not survive for a extended period of time, despite medical treatment (D.E. Redmond, unpublished observations). In contrast, all these animals in the present study survived and showed functional improvements both in general health and in motor parameters. This highlights the fact that striatal cell transplantation can offer a substantial clinical benefit also in moderate to severe PD patients, as has been recently reported in an open label study (Mendez et al., 2005
). It is also worth noting that these functional benefits were achieved with only unilateral striatal transplantation, in a bilateral PD primate model, similar to cases in clinical studies of fetal transplantation (Defer et al., 1996
; Lindvall et al., 1994
; Mendez et al., 2005
; Piccini et al., 1999
), unilateral striatal GDNF infusion (Slevin et al., 2007
; Slevin et al., 2005
), and DBS stimulation (Slowinski et al., 2007
). In these previous reports, bilateral functional benefit included mild to moderate ipsilateral and axial improvements and a pronounced contralateral improvements (Bastian et al., 2003
; Germano et al., 2004
), although the magnitude of ipsilateral and axial changes was not studied in detail. In the current primate study, contralateral improvement may have been the first signs of graft function. To objectively determine ipsilateral and contralateral function in non-human primates would have required the use of tests specifically designed to analyze motor performance of both sides independently (Gash et al., 1999
; Jenkins et al., 2004
). The PRS and ADL behavioral scales used in the present study were not designed to measure side bias. However, although the scales used did not specifically address the amount of unilateral improvement achieved, we would have detected major asymetries in normal motor function. Such major asymmetries were not detected post-transplantation, indicating that while contralateral improvements may have dominated, there must also have been considerable ipsilateral improvements. Such bilateral changes after unilateral basal ganglia manipulations have been described, originally reported by Nieoullon et al (Nieoullon et al., 1977
). The neural substrate responsible for these bilateral responses to unilateral basal ganglia manipulations appears to involve glutamatergic thalamocortical and corticostriatal circuits with extensive bilateral connections, as well as descending projections from the basal ganglia to the brainstem and spinal cord (Barbeito et al., 1989
No differences in behavioral recovery were seen between the Cd and Put locations. Nevertheless, there was a significant correlation between reinnervation and functional recovery in the Put, but not in the Cd graft location. The functional improvement in these grafted animals clearly correlated with the net increase in DA innervation. This functional-histological correlation for animals grafted in the Put support the concept that these animals experienced graft-mediated functional benefits. It is also worth noting that, before transplantation, despite this similar recovery rate, monkeys grafted in the Put were somewhat more severe (although not significantly; see ) than monkeys grafted in the Cd.
A factor that complicates the analysis, and could account for this lack of differences in behavioral improvement between target locations, is the presence of a considerable reinnervation of the non-grafted striatal nuclei ipsilateral to the transplant, regardless of the primary graft target. That is, when grafts were placed into the caudate nucleus they innervated they partially innervated the putamen as well as the caudate. Similarly, when the grafts were placed into the putamen, they innervated the caudate nucleus as well as the putamen. Extensive work both in animal models and in post-mortem evaluation studies of grafted PD patients have shown that immature DA cells preserve the capacity to grow to their natural target when placed in a denervated adult striatum (Haque et al., 1997
; Isacson and Deacon, 1996
; Isacson et al., 1995
; Mendez et al., 2005
; Thompson et al., 2005
). The ventral mesencephalon, at this embryonic developmental stage, is normally composed of a mixture of SNc (A9) and VTA (A10) immature DA cells that normally project to motor (including Cd and post-commissural Put) or non-motor (precommissural putamen and Cd) striatal areas, respectively. Thus, this innervation of both striatal nuclei is, most likely, due to the presence of these 2 different DA cell types in the grafted fetal VM tissue.
Although there are anatomical and clinical reasons to favor the post-commissural putamen as the best target for cell transplantation in PD, there are also observations that support the possibility that Cd nucleus reinnervation could provide an additional improvement of motor function. The role of the Cd nucleus in motor planning (Schultz and Romo, 1992
), as well as reports of clinical improvements after Cd transplantation in non-human primates and PD patients (Lopez-Lozano et al., 1997a
; Taylor et al., 1991
), supports this concept. This possibility is also supported by the results reported in an open trial where both Cd and Put nuclei received DA innervation from fetal cells (Kordower et al., 1996
; Mendez et al., 2005
No differences were found in the survival of grafted TH-ir neurons between the VMsolid and VMsusp transplantation groups. Among several factors, donor gestational age at the time of dissection plays an important role in this outcome. In order to have the optimal cell survival post-transplantation, fetal VM needs to be dissected at a very specific time of embryonic development (Sladek et al., 1993
). This optimal period is limited by the beginning of DA neuron neurogenesis and the time these DA neurons differentiate and extend neurites. In the AFG, this period corresponds to embryonic days 36 to 44 (Sladek et al., 1993
; Tarantal and Hendrickx, 1988a
; Tarantal and Hendrickx, 1988b
Postmortem analyses of PD patients grafted with cell suspensions, showed SNc-like (A9) DA neurons located along the graft-host border, and VTA-like (A10) DA neurons preferentially distributed towards central areas of the graft (Mendez et al., 2005
). Here, analysis of graft structure and integration revealed that this typical distribution of DA cell types of VM suspension grafts, was not seen in VM solid grafts. Additionally, there were large areas in the center of solid grafts without any TH-ir neurons or fibers, especially in grafts located in the Cd, in close proximity to the lateral ventricle. Nevertheless overall, both graft types had similar distribution and cell composition, with TH-ir cells preferentially distributed along the periphery of the graft and good graft-host integration.
Our data, to some extent, supports the general notion that fetal tissue grafted as solid pieces elicits a stronger host tissue reaction than tissue grafted as a cell suspension. We observed a stronger astroglial reaction by the host to solid pieces even though exactly the same dissection techniques were used. In contrast, we could not confirm previous observations of increased microglial reaction after transplantation of solid VM pieces versus cell suspensions in PD patients grafted with fetal VM tissue (Freed et al., 2001
; Kordower et al., 1997
; Mendez et al., 2005
; Olanow et al., 2003
). These and other studies in animal models of PD, show that the presence of donor blood vessels in grafted solid VM pieces caused a more pronounced inflammatory response than the response elicited by cell suspension grafts (Lindsay and Raisman, 1984
). The pristine condition and easy access to fetal material in this study, in contrast with fetal tissue fragments obtained from elective abortions, allowed us to perform a more thorough dissection in which all the remaining meninges and other membranes were completely peeled from the VM during the preparation of both solid pieces and cell suspensions. This thorough dissection may have almost eliminated the presence of donor blood vessels in the grafted solid pieces, and thus the formation of the typical microglial response to this tissue preparation.
Inflammatory responses may interfere with the host-graft interaction and with the normal process of striatal DA reinnervation, thus affecting graft-mediated functional recovery. This role of inflammation is supported by the findings in one of the double labeled clinical trials, in which the presence of graft-induced dyskinesias (in particular off-state dyskinesias) occurred in 50% of the subjects following discontinuation of immunosuppression (Olanow et al., 2003
). The interruption of cyclosporine treatment also correlated with loss of transplant-induced clinical benefit, despite continued presence of healthy-appearing grafted neurons (Olanow et al., 2003
). This is consistent with findings from xenograft studies, where functional loss occurs slowly and prior to complete elimination of xenogenic transplanted cells, due to a rejection process (Galpern et al., 1996
; Widner, 1999
). Finally, there are other factors influencing survival and integration of the grafts, such as tissue storage method, number of grafted embryos, and distribution of grafted tissue (Isacson et al., 2003
). In our study, all these factors were kept constant and even dissection procedures were performed in the exact same way for VMsolid and VMsusp.
The hope that stem cells may be the most practical donor material for use in cell replacement is largely based on potential advantages in the supply and quality control of the donor material, not based on the assumption that stem cell derived midbrain DA neurons would be functionally superior to DA neurons from dissected fetal VM. Stem cell-derived midbrain DA neurons will be at, or go through, the same developmental fetal stage as we have used in this study, prior to or after transplantation, as well as experience similar tissue and guidance cues for integration into the recipient brain(Isacson et al., 2003
). It is therefore likely that stem cell derived DA neurons of VM phenotype will function in a manner similar to dissected fetal DA neurons, and studies would encounter similar problems to those observed using fetal DA neuron grafts(Isacson, 2003
). Consequently, it is imperative to better understand the mechanisms behind functional improvements, and in particular the molecular and neuronal substrates underlying the side effects observed in some clinical cell replacement trials (Freed et al., 2001
; Olanow et al., 2003
In summary, we show that grafted VM tissue survives and provides a significant striatal dopaminergic reinnervation, resulting in improvements of motor and general health parameters, with a high success rate in a primate model of PD. Based on our data, there are no major differences between implantation sites in the Cd or the Put, although the putaminal location seems to have a better correlation with the functional outcome. The (1) lack of benefits of solid tissue over cell suspensions, (2) increased glial response induced by solid grafts, and (3) the control over cell numbers using dissociated cells and (4) easier translation of methods and results to stem cell derived neural transplantation, all support the use of cell suspensions in future studies of fetal cell transplantation in PD.
In the exploration of DA neuron transplantation for future rational clinical use, the PD primate model studies presented here provide a basis for additional studies of efficacy and safety. Work in progress involves the transplantation of fetal DA neurons in this primate model, in animals with prior L-dopa induced dyskinesia (Youngerman BE, 2005
) to better model the clinical reality and therapeutic trials.