Abstract
In pursuit of treating Parkinson’s disease with cell replacement therapy, differentiated induced pluripotent stem cells (iPSC) are an ideal source of midbrain dopaminergic (mDA) cells. We previously established a protocol for differentiating iPSC-derived post-mitotic mDA neurons capable of reversing 6-hydroxydopamine-induced hemiparkinsonism in rats. In the present study, we transitioned the iPSC starting material and defined an adapted differentiation protocol for further translation into a clinical cell transplantation therapy. We examined the effects of cellular maturity on survival and efficacy of the transplants by engrafting mDA progenitors (cryopreserved at 17 days of differentiation, D17), immature neurons (D24), and post-mitotic neurons (D37) into immunocompromised hemiparkinsonian rats. We found that D17 progenitors were markedly superior to immature D24 or mature D37 neurons in terms of survival, fiber outgrowth and effects on motor deficits. Intranigral engraftment to the ventral midbrain demonstrated that D17 cells had a greater capacity than D24 cells to innervate over long distance to forebrain structures, including the striatum. When D17 cells were assessed across a wide dose range (7,500-450,000 injected cells per striatum), there was a clear dose response with regards to numbers of surviving neurons, innervation, and functional recovery. Importantly, although these grafts were derived from iPSCs, we did not observe teratoma formation or significant outgrowth of other cells in any animal. These data support the concept that human iPSC-derived D17 mDA progenitors are suitable for clinical development with the aim of transplantation trials in patients with Parkinson’s disease.
Introduction
As the population continues to age, a pandemic of Parkinson’s disease (PD) is emerging, with conservative estimates of over 14 million victims globally by 20401. While PD patients display a wide range of non-motor features, the defining symptoms are progressive motor deficits due to striatal dopaminergic insufficiency secondary to loss of dopaminergic nigral neurons. Current therapies are symptomatic, mostly focused on ameliorating motor deficits. Since no current therapy arrests or reverses the disease process, there is a major unmet need for new and effective PD treatments. The principal pharmacologic therapies for PD are oral L-DOPA or dopaminergic agonists, which initially provide potent relief from motor symptoms, but after 5–10 years most patients experience debilitating motor fluctuations and dyskinesias2. Deep brain stimulation of the subthalamic nucleus (STN) or internal segment of the globus pallidus provides an alternate and effective approach to treat PD motor symptoms, but is primarily indicated for younger patients who do not display cognitive decline and requires periodic battery changes. An alternative approach is the transplantation of midbrain dopaminergic (mDA) cells which could offer a more physiologically relevant delivery of dopamine and confer functional benefits similar to L-DOPA, but without the adverse effects3,4.
A large body of work has demonstrated that rodent and human fetal ventral mesencephalic (hfVM) dopamine neurons survive well, innervate the host and form synapses, release dopamine, and alleviate motor deficits when grafted to the dopamine-depleted striatum of experimental animals5,6. Some patients in open-label hfVM trials7,8 exhibited clinical improvement. However, randomized double blinded, placebo-controlled, clinical trials indicated that these benefits were too variable to meet the trials’ primary endpoints, although predefined secondary endpoints (Unified Parkinson’s Disease Rating Scale, UPDRS) showed statistically significant benefits in younger (<60 years of age; ref. 9) or less impaired (UPDRS in off <49; ref. 10) subjects. Additionally, some patients developed graft-induced dyskinesias (GID)9,11,12, possibly related to pre-existing L-DOPA-induced dyskinesias and the transplants containing serotonergic cells alongside the desired dopaminergic neurons13,14. These findings prompted a re-evaluation of the approach. More recently, the European collaborative consortium, TRANSEURO, revisited fetal transplantation in an open-label trial (NCT01898390) with 11 patients at relatively early disease stages who had not developed significant L-DOPA-induced dyskinesias prior to grafting15.
The isolation of human embryonic stem cells (hESCs)16 and subsequent development of protocols to generate iPSCs17 allowed for the manufacturing of various adult cell types for therapeutic applications, unconstrained by the practical and ethical complexities of sourcing aborted fetal tissue. Studies utilizing hESCs18,19,20 or iPSCs21,22,23,24 have shown that PSCs can be differentiated into mDA neurons and reverse motor deficits in animal models of PD. As a renewable tissue source with the potential for “off-the-shelf” dosing and better batch-to-batch consistency, PSCs are a promising cellular substrate for mDA neurons as an alternative to hfVM tissue.
We have previously shown that iPSC-derived mDA neurons, differentiated via a floor plate intermediate, engraft, survive long-term, and reverse drug-induced motor asymmetry in athymic rats with unilateral 6-hydroxydopamine (6-OHDA) lesions21,22. However, innervation of the striatum by these cells was modest compared to published studies using fetal tissue25,26,27. Across many studies, cells in various stages of mDA development have been transplanted, and the most robust engraftment and functional recovery has been observed when using progenitors or immature neurons18,28,29,30.
Therefore, in developing our robust, clinically-compliant, mDA differentiation process, the present paper examined the engraftment, innervation, and functional efficacy in hemiparkinsonian rats using cells from different developmental stages. Specifically, we compared cells considered mDA progenitors (cryopreserved on Day 17 (D17)), immature mDA neurons (D24), and purified mDA neurons (D37), comparing them to R&D grade purified mDA neurons (D38, G418) that have been characterized previously and are available commercially21,22. Additionally, we explored whether D17 or D24 cells can provide long-distance innervation by grafting them into the substantia nigra (SN). Finally, to further characterize the performance of D17 mDA progenitors, which we had found to have the most robust survival and fiber outgrowth, we conducted a dose-ranging study and determined the lowest dose that exerted an early onset of functional recovery in hemiparkinsonian rats.
Results
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