Dopaminergic neurons (DAns), generated from human pluripotent stem cells (hPSCs), are capable of functionally integrating following transplantation and have recently advanced to clinical trials for ...Parkinson’s disease (PD). However, pre-clinical studies have highlighted the low proportion of DAns within hPSC-derived grafts and their inferior plasticity compared to fetal tissue. Here, we examined whether delivery of a developmentally critical protein, glial cell line-derived neurotrophic factor (GDNF), could improve graft outcomes. We tracked the response of DAns implanted into either a GDNF-rich environment or after a delay in exposure. Early GDNF promoted survival and plasticity of non-DAns, leading to enhanced motor recovery in PD rats. Delayed exposure to GDNF promoted functional recovery through increases in DAn specification, DAn plasticity, and DA metabolism. Transcriptional profiling revealed a role for mitogen-activated protein kinase (MAPK)-signaling downstream of GDNF. Collectively, these results demonstrate the potential of neurotrophic gene therapy strategies to improve hPSC graft outcomes.
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•DAn grafting into a GDNF-rich host aids pan-neuronal survival but impedes plasticity•Delaying graft exposure to GDNF increases DAn maturation, plasticity, and function•GDNF promotes functional integration of DAn grafts via MAPK signaling•Modulating the host environment can improve the integration of human neural grafts
Gantner et al. provide preclinical evidence that the timely overexpression of glial cell-derived neurotrophic factor (GDNF) within the host brain can promote the maturation, plasticity, and functional integration of human stem cell-derived dopamine grafts in a rodent model of Parkinson’s disease.
Midbrain dopamine (mDA) neurons can be replaced in patients with Parkinson’s disease (PD) in order to provide long-term improvement in motor functions. The limited capacity for long-distance axonal ...growth in the adult brain means that cells are transplanted ectopically, into the striatal target. As a consequence, several mDA pathways are not re-instated, which may underlie the incomplete restoration of motor function in patients. Here, we show that viral delivery of GDNF to the striatum, in conjunction with homotopic transplantation of human pluripotent stem-cell-derived mDA neurons, recapitulates brain-wide mDA target innervation. The grafts provided re-instatement of striatal dopamine levels and correction of motor function and also connectivity with additional mDA target nuclei not well innervated by ectopic grafts. These results demonstrate the remarkable capacity for achieving functional and anatomically precise reconstruction of long-distance circuitry in the adult brain by matching appropriate growth-factor signaling to grafting of specific cell types.
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•A PITX3-eGFP iPSC line reveals patterns of mDA neuron growth from stem cell grafts•mDA neurons in homotopic grafts target nuclei not well innervated by ectopic grafts•Forebrain GDNF facilitates robust striatal innervation by homotopic mDA grafts
Moriarty et al. show that dopamine neurons in homotopic grafts generated from pluripotent stem cells can provide long-distance, anatomically precise innervation of appropriate targets throughout the brain, including robust striatal innervation when combined with forebrain GDNF delivery, in order to restore motor function in a rodent model of Parkinson’s disease.
...derived biomaterials, such as collagen hydrogels, hold the advantage of being characteristically similar to the body’s native tissue, making them highly biocompatible and biodegradable, while also ...naturally supporting cell adhesion without the need for further chemical alterations which may disrupt the immunogenicity of the scaffold. ...we then sought to determine if the collagen hydrogel was capable of providing a growth factor reservoir in the brain by functionalising the gels with the dopaminergic neurotrophin, glial-derived neurotrophic factor (GDNF). An interesting addition to this scaffold was the tethering of GDNF to short nanofibers, alongside the presence of soluble GDNF throughout the hydrogel, thus providing long-term GDNF delivery at the graft site and sustained release from the hydrogel. ...since the tethering of GDNF to the short nanofibers alone did not result in improved cell survival or re-innervation, this highlights the importance of GDNF release from the graft core to the surrounding striatum, where it can guide and support neurite outgrowth. ...this study demonstrated the ability of the functionalised hydrogel to improve the efficacy of dopaminergic neuronal differentiation, with a higher fraction of dopaminergic cells obtained in vitro, and an increase in the number of surviving cells during enzymatic cell harvest, a step that is thought to be a major contributing factor to pre-transplantation cell death in stem cell therapies.
Abstract
Despite advancements in human pluripotent stem cells (hPSCs) differentiation protocols to generate appropriate neuronal progenitors suitable for transplantation in Parkinson’s disease, ...resultant grafts contain low proportions of dopamine neurons. Added to this is the tumorigenic risk associated with the potential presence of incompletely patterned, proliferative cells within grafts. Here, we utilised a hPSC line carrying a FailSafe
TM
suicide gene (thymidine kinase linked to cyclinD1) to selectively ablate proliferative cells in order to improve safety and purity of neural transplantation in a Parkinsonian model. The engineered FailSafe
TM
hPSCs demonstrated robust ventral midbrain specification in vitro, capable of forming neural grafts upon transplantation. Activation of the suicide gene within weeks after transplantation, by ganciclovir administration, resulted in significantly smaller grafts without affecting the total yield of dopamine neurons, their capacity to innervate the host brain or reverse motor deficits at six months in a rat Parkinsonian model. Within ganciclovir-treated grafts, other neuronal, glial and non-neural populations (including proliferative cells), were significantly reduced—cell types that may pose adverse or unknown influences on graft and host function. These findings demonstrate the capacity of a suicide gene-based system to improve both the standardisation and safety of hPSC-derived grafts in a rat model of Parkinsonism.
Clinical studies have provided evidence for dopamine (DA) cell replacement therapy in Parkinson's Disease. However, grafts derived from foetal tissue or pluripotent stem cells (PSCs) remain ...heterogeneous, with a high proportion of non-dopaminergic cells, and display subthreshold reinnervation of target tissues, thereby highlighting the need to identify new strategies to improve graft outcomes. In recent work, Stromal Cell-Derived Factor-1 (SDF1), secreted from meninges, has been shown to exert many roles during ventral midbrain DA development and DA-directed differentiation of PSCs. Related, co-implantation of meningeal cells has been shown to improve neural graft outcomes, however, no direct evidence for the role of SDF1 in neural grafting has been shown. Due to the rapid degradation of SDF1 protein, here, we utilised a hydrogel to entrap the protein and sustain its delivery at the transplant site to assess the impact on DA progenitor differentiation, survival and plasticity. Hydrogels were fabricated from self-assembling peptides (SAP), presenting an epitope for laminin, the brain's main extracellular matrix protein, thereby providing cell adhesive support for the grafts and additional laminin-integrin signalling to influence cell fate. We show that SDF1 functionalised SAP hydrogels resulted in larger grafts, containing more DA neurons, increased A9 DA specification (the subpopulation of DA neurons responsible for motor function) and enhanced innervation. These findings demonstrate the capacity for functionalised, tissue-specific hydrogels to improve the composition of grafts targeted for neural repair.
Poor graft survival limits the use of primary dopaminergic neurons for neural repair in Parkinson's disease. Injectable hydrogels have the potential to significantly improve the outcome of such ...reparative approaches by providing a physical matrix for cell encapsulation which can be further enriched with pro-survival factors. Therefore, this study sought to determine the survival and efficacy of primary dopaminergic grafts after intra-striatal delivery in a glial-derived neurotrophic factor (GDNF)-loaded collagen hydrogel in a rat model of Parkinson's disease. After intra-striatal transplantation into the lesioned striatum, the GDNF-enriched collagen hydrogel significantly improved the survival of dopaminergic neurons in the graft (5-fold), increased their capacity for striatal re-innervation (3-fold), and enhanced their functional efficacy. Additional studies suggested that this was due to the hydrogel's ability to retain GDNF in the microenvironment of the graft, and to protect the transplanted cells from the host immune response. In conclusion, the encapsulation of dopaminergic neurons in a GDNF-loaded hydrogel dramatically increased their survival and function, providing further evidence of the potential of biomaterials for neural transplantation and brain repair in neurodegenerative diseases such as Parkinson's disease.
Biomaterials have been shown to significantly improve the outcome of cellular reparative approaches for Parkinson's disease in experimental studies because of their ability to provide transplanted ...cells with a supportive microenvironment and shielding from the host immune system. However, given that the margin for improvement in such reparative therapies is considerable, further studies are required to fully investigate and harness the potential of biomaterials in this context. Given that several recent studies have demonstrated improved brain repair in Parkinsonian models when using dopaminergic grafts derived from younger foetal donors, we hypothesized that encapsulating these cells in a supportive biomaterial would further improve their reparative efficacy. Thus, this study aimed to determine the impact of a GDNF‐loaded collagen hydrogel on the survival, reinnervation, and functional efficacy of dopaminergic neurons derived from young donors. To do so, hemi‐Parkinsonian (6‐hydroxydopamine‐lesioned) rats received intrastriatal transplants of embryonic day 12 cells extracted from the rat ventral mesencephalon either alone, in a collagen hydrogel, with GDNF, or in a GDNF‐loaded collagen hydrogel. Methamphetamine‐induced rotational behaviour was assessed at three weekly intervals for a total of 12 weeks, after which rats were sacrificed for postmortem assessment of graft survival. We found that, following intrastriatal transplantation to the lesioned striatum, the GDNF‐loaded collagen hydrogel significantly increased the survival (4‐fold), reinnervation (5.4‐fold), and functional efficacy of the embryonic day 12 dopaminergic neurons. In conclusion, this study further demonstrates the significant potential of biomaterial hydrogel scaffolds for cellular brain repair approaches in neurodegenerative diseases such as Parkinson's disease.
Delivery of primary dopaminergic neurons for brain repair in Parkinson's disease is limited by poor survival, whereas, delivery in a growth factor‐loaded biomaterial scaffold improves the survival, striatal reinnervation, and functional efficacy of the transplanted cells.
The dopamine precursor, levodopa, remains the “gold standard” treatment for Parkinson's disease, and, although it provides superlative efficacy in the early stages of the disease, its long‐term use ...is limited by the development of severe motor side effects and a significant abating of therapeutic efficacy. Therefore, there remains a major unmet clinical need for the development of effective neuroprotective, neurorestorative or neuroreparatory therapies for this condition. The relatively selective loss of dopaminergic neurons from the nigrostriatal pathway makes Parkinson's disease an ideal candidate for reparative cell therapies, wherein the dopaminergic neurons that are lost in the condition are replaced through direct cell transplantation into the brain. To date, this approach has been developed, validated and clinically assessed using dopamine neuron‐rich foetal ventral mesencephalon grafts which have been shown to survive and reinnervate the denervated brain after transplantation, and to restore motor function. However, despite long‐term symptomatic relief in some patients, significant limitations, including poor graft survival and the impact this has on the number of foetal donors required, have prevented this therapy being more widely adopted as a restorative approach for Parkinson's disease. Injectable biomaterial scaffolds have the potential to improve the delivery, engraftment and survival of these grafts in the brain through provision of a supportive microenvironment for cell adhesion, growth and immune shielding. This article will briefly review the development of primary cell therapies for brain repair in Parkinson's disease and will consider the emerging literature which highlights the potential of using injectable biomaterial hydrogels in this context.
Conventional delivery of primary dopamine neurons for brain repair in Parkinson's disease is limited by poor survival, whereas, delivery in growth‐factor functionalised biomaterial scaffolds improves both survival and efficacy through the provision of: (a) a physical scaffold for cell adhesion during intracerebral delivery and engraftment; (b) a local reservoir for growth factors at the implantation site; and (c) a protective barrier against the host immune response.
•Soluble low-molecular-weight oligomers have been hypothesized as causative to neurodegeneration in Alzheimer’s disease.•The oligomers are challenging to characterize due to their polymorphic, ...heterogeneous and transient nature.•We performed the first synergic validation of the structure, dynamics and toxicity of full-length Aβ42 oligomers.•Aβ oligomers assumed nanosized β-barrels and impaired the cognitive function in a mouse model.•The occurrence of β-barrels supports their roles as the common toxic intermediates in Alzheimer’s and a therapeutic target.
Free-energy landscape of Aβ peptides in aggregation. Aβ monomers initially assemble into low β-sheet content oligomers, followed by conformational conversion into high β-sheet abundant oligomers (including β-barrels). Eventually, the β-sheet rich oligomers are converted into proto-fibrils by crossing a high free-energy barrier. Display omitted
Soluble low-molecular-weight oligomers formed during the early aggregation of amyloid peptides have been hypothesized as a major toxic species of amyloidogenesis. Herein, we performed the first synergic in silico, in vitro and in vivo validations of the structure, dynamics and toxicity of Aβ42 oligomers. Aβ peptides readily assembled into β-rich oligomers comprised of extended β-hairpins and β-strands. Nanosized β-barrels were observed with certainty with simulations, transmission electron microscopy and Fourier transform infrared spectroscopy, corroborated by immunohistochemistry, cell viability, apoptosis, inflammation, autophagy and animal behavior assays. Secondary and tertiary structural properties of these oligomers, such as the sequence regions with high β-sheet propensities and inter-residue contact frequency patterns, were similar to the properties known for Aβ fibrils. The unambiguous spontaneous formation of β-barrels in the early aggregation of Aβ42 supports their roles as the common toxic intermediates in Alzheimer’s pathobiology and a target for Alzheimer’s therapeutics.
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