Cell Replacement Therapy for Parkinson's Disease: Potential for Circuitry Repair

Abstract: The derivation of dopamine neurons from human embryonic stem cells (hESCs) now offers a promising alternative to fetal tissue for cell replacement therapy (CRT) in Parkinson´s disease (PD). Using the appropriate chemical cues in vitro, hESCs can be patterned towards bona-fide ventral midbrain (VM) DA neurons that survive, reinnervate, release DA and provide functional recovery when transplanted into rodent and non-human primate models of PD. However, the extent to which transplanted neurons integrate into the damaged host circuitry, which is necessary for regulated DA release, and hence to elicit a more complete circuitry repair, remains unknown.In this thesis, the potential for transplanted hESC-derived neurons to repair damaged circuitry in parkinsonian rats, as assessed by synaptic integration and targeted axonal outgrowth, was investigated. The role of graft- and host-dependent variables on synaptic integration and innervation were investigated in view of a better understanding of transplant biology, with potential to optimize the functionality of the graft. We established a modified rabies-based tracing methodology that allows for the identification of monosynaptic inputs to a defined starter population in order to assess host-to-graft and graft-to-host synaptic integration in a cell transplantation model. In Paper I, we investigated the integration of hESC-derived neurons, and in Paper II during in vivo reprogramming, to investigate the integration of in situ converted neurons into pre-existing circuitry. Subsequently in Paper III, this methodology was utilized to reveal that transplanted hESC-derived VM neurons receive the correct set of presynaptic inputs from the host circuitry when placed in their homotopic location within the substantia nigra, while the profile of axonal outgrowth showed that transplanted neurons innervate across long-distances in a target-specific manner towards appropriate forebrain targets. Moreover, functional recovery as assessed by amphetamine-induced rotations matched the presence and timing of the arrival of graft-derived dopaminergic innervation in the dorsolateral striatum. In Paper IV, the role of graft neuronal phenotype and host environment on synaptic integration and graft-derived axonal outgrowth was investigated. These results show that graft derived innervation is determined by cell intrinsic factors, as only the correct hESC-derived VM neurons innervate the appropriate forebrain DA target regions. On the other hand, monosynaptic tracing showed that the pattern of integration is dependent on graft placement, as monosynaptic inputs to intrastriatal and intranigral grafts differed. Nonetheless, a certain level of anatomical and phenotypic overlap in presynaptic inputs to both ectopic and homotopic hESC-derived VM grafts was detected, suggesting that ectopically placed grafts may be modulated by functionally relevant structures involved in motor control, supporting the validity of this grafting paradigm in the clinic. Finally, transplantation of hESC-derived neurons in intact or 6-OHDA-lesioned animals revealed that the globus pallidus is differentially connected to transplanted neurons, identifying this structure as a possible important modulator of graft function in the DA-depleted parkinsonian brain.Overall, the results of this thesis suggest that transplanted hESC-derived VM DA neurons have the capacity to achieve a more complete repair of the damaged host circuitry beyond simple DA neuron replacement. As such, they support the validity of ectopic grafting into the lesioned brain as a valid strategy for CRT in PD. Moreover, this work identifies host nuclei that may play an important role in graft modulation, hence prompting further functional experimentation.