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Cell therapies represent a promising approach to slow down the progression of currently untreatable neurodegenerative diseases (e.g., Alzheimer's and Parkinson's disease or amyotrophic lateral sclerosis), as well as to support the reconstruction of functional neural circuits after spinal cord injuries. In such therapies, the grafted cells could either functionally integrate into the damaged tissue, partially replacing dead or damaged cells, modulate inflammatory reaction, reduce tissue damage, or support neuronal survival by secretion of cytokines, growth, and trophic factors. Comprehensive characterization of cells and their proliferative potential, differentiation status, and population purity before transplantation is crucial to preventing safety risks, e.g., a tumorous growth due to the proliferation of undifferentiated stem cells. We characterized changes in the proteome and secretome of human neural stem cells (NSCs) during their spontaneous (EGF/FGF2 withdrawal) differentiation and differentiation with trophic support by BDNF/GDNF supplementation. We used LC-MS/MS in SWATH-MS mode for global cellular proteome profiling and quantified almost three thousand cellular proteins. Our analysis identified substantial protein differences in the early stages of NSC differentiation with more than a third of all the proteins regulated (including known neuronal and NSC multipotency markers) and revealed that the BDNF/GDNF support affected more the later stages of the NSC differentiation. Among the pathways identified as activated during both spontaneous and BDNF/GDNF differentiation were the HIF-1 signaling pathway, Wnt signaling pathway, and VEGF signaling pathway. Our follow-up secretome analysis using Luminex multiplex immunoassay revealed significant changes in the secretion of VEGF and IL-6 during NSC differentiation. Our results further demonstrated an increased expression of neuropilin-1 as well as catenin β-1, both known to participate in the regulation of VEGF signaling, and showed that VEGF-A isoform 121 (VEGF121), in particular, induces proliferation and supports survival of differentiating cells.

In late stages of inherited and acquired retinal diseases such as Stargardt disease (STGD) or dry age‐related macular degeneration (AMD), loss of retinal pigment epithelia (RPE) cells and subsequently photoreceptors in the macular area result in a dramatic decline of central visual function. Repopulating this area with functional RPE cells may prevent or decline the progression of photoreceptor loss. In the present study, the viability, survival, and integration of human induced pluripotent stem cell (hiPSC)‐derived RPE cells (hiPSC‐RPE) is assessed generated using clinical‐grade protocol and cultured on a clinically relevant scaffold (poly‐L‐lactide‐co‐D, L‐lactide, PDLLA) after subretinal implantation in immunosuppressed minipigs for up to 6 weeks. It is shown that transplanted hiPSC‐RPE cells maintain the RPE cell features such as cell polarity, hexagonal shape, and cell–cell contacts, and interact closely with photoreceptor outer segments without signs of gliosis or neuroinflammation throughout the entire period of examination. In addition, an efficient immunosuppressing strategy with a continuous supply of tacrolimus is applied. Continuous verification and improvement of existing protocols are crucial for its translation to the clinic. The results support the use of hiPSC‐RPE on PDLLA scaffold as a cell replacement therapeutic approach for RPE degenerative diseases. Human induced pluripotent stem cell‐derived retinal pigment epithelial (hiPSC‐RPE) cells, cultured on a clinically relevant PDLLA scaffold, are transplanted into immunosuppressed minipigs. The study demonstrates the viability, integration, and preservation of RPE‐specific features without gliosis or neuroinflammation, supporting their potential as a cell replacement therapy for retinal degenerative diseases.

BackgroundGiven evidence that mutated huntingtin is expressed in peripheral immune cells, it is possible that inflammatory changes detected in peripheral tissues may reflect the inflammatory process in CNS. Peripheral immune cells are collected by non-invasive approach which is easily feasible in patients. AimsThe objective of this observation was to identify alterations in molecules produced by peripheral immune cells and in serum in transgenic HD minipig model. MethodsA multiplex immunoassay (bead-based Luminex xMAP technology) was applied for the simultaneous determination of 7 cytokines in serum and in secretomes of myeloid cells (monocytes and macrophages). Additionally, an activity of different complement pathways was measured in serum by Wieslab®Complement system Screen kit. Age-matched TgHD and WT minipigs with similar genetic background and before the onset of clinical symptoms were used in this study. ResultsSerum level of IL-8 was found to be significantly increased in TgHD animals. It did not correlate with findings in monocyte secretomes where the IL-8 level oscillated. Measurements of cytokine level in macrophage secretomes showed raised production of IL-1β, IFNα and TNFα after cell stimulation in TgHD animals at the age of 48–56 months. Besides that, the complement test revealed a significantly increased activity of the classical pathway in serum from TgHD animals at the age of 24 and 36 months.ConclusionsThis study identified molecules animals which were altered in the periphery of TgHD. These molecules could serve as biomarkers for monitoring of organism response to treatment against mHTT effect in transgenic HD minipigs. SupportCHDI (A-8248, A-5378), EXAM (CZ.1.05/2.1.00/03.0124), National Sustainability Programme (LO1609), RVO67985904, GAUK No 378215

BackgroundHuntington disease (HD) is a fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Great effort has been put in proof-of-concept studies of therapeutic agents in HD rodent models. One of the challenges of rodents as a model of neurodegenerative diseases is their relatively small brain, making successful translation to the HD patient difficult. This is particular relevant for gene therapy approaches, where distribution achieved upon local administration into the parenchyma is likely dependent on brain size and structure.AimsHere, we investigated the feasibility, efficacy, and tolerability of huntingtin-lowering gene therapy in a large animal brain.MethodsTransgenic HD (tgHD) minipigs were injected with an engineered microRNA targeting human huntingtin, delivered via adeno-associated viral vector serotype 5 (AAV5-miHTT) or AAV5-GFP as control. The viruses were intracranially administered into the striatum and thalamus.ResultsWe detected widespread dose-dependent distribution of AAV5-miHTT throughout the tgHD minipig brain that correlated with the engineered microRNA expression. Both human mutant huntingtin mRNA and protein were significantly reduced in all brain regions transduced by AAV5-miHTT.ConclusionThe combination of widespread vector distribution and extensive huntingtin lowering observed with AAV5-miHTT supports the translation of a huntingtin-lowering gene therapy for HD from preclinical studies into the clinic.