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Peptide-Based Scaffolds for the Culture and Transplantation of Human Dopaminergic Neurons.

ABSTRACT: Cell replacement therapy is a promising treatment strategy for Parkinson's disease (PD); however, the poor survival rate of transplanted neurons is a critical barrier to functional recovery. In this study, we used self-assembling peptide nanofiber scaffolds (SAPNS) based on the peptide RADA16-I to support the in vitro maturation and in vivo post-transplantation survival of encapsulated human dopaminergic (DA) neurons derived from induced pluripotent stem cells. Neurons encapsulated within the SAPNS expressed mature neuronal and midbrain DA markers and demonstrated in vitro functional activity similar to neurons cultured in two dimensions. A microfluidic droplet generation method was used to encapsulate cells within monodisperse SAPNS microspheres, which were subsequently used to transplant adherent, functional networks of DA neurons into the striatum of a 6-hydroxydopamine-lesioned PD mouse model. SAPNS microspheres significantly increased the in vivo survival of encapsulated neurons compared with neurons transplanted in suspension, and they enabled significant recovery in motor function compared with control lesioned mice using approximately an order of magnitude fewer neurons than have been previously needed to demonstrate behavioral recovery. These results indicate that such biomaterial scaffolds can be used as neuronal transplantation vehicles to successfully improve the outcome of cell replacement therapies for PD. Impact Statement Transplantation of dopaminergic (DA) neurons holds potential as a treatment for Parkinson's disease (PD), but low survival rates of transplanted neurons is a barrier to successfully improving motor function. In this study, we used hydrogel scaffolds to transplant DA neurons into PD model mice. The hydrogel scaffolds enhanced survival of the transplanted neurons compared with neurons that were transplanted in a conventional manner, and they also improved recovery of motor function by using significantly fewer neurons than have typically been transplanted to see functional benefits. This cell transplantation technology has the capability to improve the outcome of neuron transplantation therapies.

SUBMITTER: Francis NL

PROVIDER: S-EPMC7044800 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Peptide-Based Scaffolds for the Culture and Transplantation of Human Dopaminergic Neurons.

Francis Nicola L NL Zhao Nanxia N Calvelli Hannah R HR Saini Astha A Gifford Janace J JJ Wagner George C GC Cohen Rick I RI Pang Zhiping P ZP Moghe Prabhas V PV

Tissue engineering. Part A 20191017 3-4

Cell replacement therapy is a promising treatment strategy for Parkinson's disease (PD); however, the poor survival rate of transplanted neurons is a critical barrier to functional recovery. In this study, we used self-assembling peptide nanofiber scaffolds (SAPNS) based on the peptide RADA16-I to support the <i>in vitro</i> maturation and <i>in vivo</i> post-transplantation survival of encapsulated human dopaminergic (DA) neurons derived from induced pluripotent stem cells. Neurons encapsulated ...[more]

PMID: 31537172

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Project description:Macroautophagy/autophagy cytoplasmic quality control pathways are required during neural development and are critical for the maintenance of functional neuronal populations in the adult brain. Robust evidence now exists that declining neuronal autophagy pathways contribute to human neurodegenerative diseases, including Parkinson disease (PD). Reliable and relevant human neuronal model systems are therefore needed to understand the biology of disease-vulnerable neural populations, to decipher the underlying causes of neurodegenerative disease, and to develop assays to test therapeutic interventions in vitro. Human induced pluripotent stem cell (hiPSC) neural model systems can meet this demand: they provide a renewable source of material for differentiation into regional neuronal sub-types for functional assays; they can be expanded to provide a platform for screening, and they can potentially be optimized for transplantation/neurorestorative therapy. So far, however, hiPSC differentiation protocols for the generation of ventral midbrain dopaminergic neurons (mDANs) - the predominant neuronal sub-type afflicted in PD - have been somewhat restricted by poor efficiency and/or suitability for functional and/or imaging-based in vitro assays. Here, we describe a reliable, monolayer differentiation protocol for the rapid and reproducible production of high numbers of mDANs from hiPSC in a format that is amenable for autophagy/mitophagy research. We characterize these cells with respect to neuronal differentiation and macroautophagy capability and describe qualitative and quantitative assays for the study of autophagy and mitophagy in these important cells.Abbreviations: AA: ascorbic acid; ATG: autophagy-related; BDNF: brain derived neurotrophic factor; CCCP: carbonyl cyanide m-chlorophenylhydrazone; dbcAMP: dibutyryl cAMP; DAN: dopaminergic neuron; DAPI: 4',6-diamidino-2-phenylindole; DAPT: N-[N-(3,5-difluorophenacetyl)-L-alanyl]-sphenylglycine; DLG4/PSD95: discs large MAGUK scaffold protein 4; DMEM: Dulbecco's modified eagle's medium; EB: embryoid body; ECAR: extracellular acidification rate; EGF: epidermal growth factor; FACS: fluorescence-activated cell sorting; FCCP: arbonyl cyanide p-triflouromethoxyphenylhydrazone; FGF: fibroblast growth factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GDNF: glia cell derived neurotrophic factor; hiPSC: human induced pluripotent stem cell; LAMP2A: lysosomal associated membrane protein 2A; LT-R: LysoTracker Red; MAP1LC3: microtubule associated protein 1 light chain 3; mDAN: midbrain dopaminergic neuron; MEF: mouse embryonic fibroblast; MT-GR: MitoTracker Green; MT-R: MitoTracker Red; NAS2: normal SNCA2; NEM: neuroprogenitor expansion media; NR4A2/NURR1: nuclear receptor subfamily group A member 2; OA: oligomycin and antimycin A; OCR: oxygen consumption rate; PD: Parkinson disease; SHH: sonic hedgehog signaling molecule; SNCA/α-synuclein: synuclein alpha; TH: tyrosine hydroxylase; VTN: vitronectin.

Project description:Human induced pluripotent stem cells (hiPSCs) represent an unlimited cell source for the generation of in vitro models of patient-specific dopaminergic (DA) neurons, posing a possibility to overcome the restricted accessibility to disease-affected tissue for mechanistic studies on Parkinson’s disease (PD). However, the complexity of the human brain is not fully recapitulated by existing monolayer culture methods. Consequently, neurons differentiated in a three dimensional (3D) in vitro culture system might better mimic the in vivo cellular environment for basic mechanistic studies and represent better predictors of drug responses in vivo. Thus, in this work we established a new in vitro cell culture model based on the microencapsulation of hiPSCs in small alginate/fibronectin beads and their differentiation to DA neurons. Optimisation of hydrogel matrix concentrations and composition resulted in high viability of embedded iPSCs. Neural differentiation capacity and DA neuronal yield, analyzed by qRT-PCR, immunofluorescence, and western blot analyses, were increased in the 3D neuronal cultures compared to neurons generated using the conventional 2D culture system. Additionally, electrophysiological parameters and metabolic switching profile supported the increased functionality and an anticipated metabolic resetting of neurons grown in alginate scaffolds with respect to the 2D neurons. We also report long-term maintenance of neuronal cultures and mature functional properties. Furthermore, our findings indicate that our 3D model system can recapitulate mitochondrial superoxide production as an important mitochondrial phenotype observed in PD patients’-derived neurons and that this phenotype might be detectable earlier during neuronal differentiation. Taken together, these results indicate that our alginate-based 3D culture system offers an advantageous strategy for the reliable and rapid derivation of mature and functional DA neurons from iPSCs.

Dataset Information

Peptide-Based Scaffolds for the Culture and Transplantation of Human Dopaminergic Neurons.

Publications

Peptide-Based Scaffolds for the Culture and Transplantation of Human Dopaminergic Neurons.

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