Precision spinal gene delivery-induced functional switch in nociceptive neurons reverses neuropathic pain.

Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.

AAV5-miHTT Gene Therapy Demonstrates Broad Distribution and Strong Human Mutant Huntingtin Lowering in a Huntington's Disease Minipig Model.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Previously, we showed strong huntingtin reduction and prevention of neuronal dysfunction in HD rodents using an engineered microRNA targeting human huntingtin, delivered via adeno-associated virus (AAV) serotype 5 vector with a transgene encoding an engineered miRNA against HTT mRNA (AAV5-miHTT). 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 particularly relevant for gene therapy approaches, where distribution achieved upon local administration into the parenchyma is likely dependent on brain size and structure. Here, we aimed to demonstrate the translation of huntingtin-lowering gene therapy to a large-animal brain. We investigated the feasibility, efficacy, and tolerability of one-time intracranial administration of AAV5-miHTT in the transgenic HD (tgHD) minipig model. We 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. The 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.

Age-Related Oxidative Changes in Primary Porcine Fibroblasts Expressing Mutated Huntingtin.

BACKGROUND: Huntington's disease (HD) is a devastating neurodegenerative disorder caused by CAG triplet expansions in the huntingtin gene. Oxidative stress is linked to HD pathology, although it is not clear whether this is an effect or a mediator of disease. The transgenic (TgHD) minipig expresses the N-terminal part of human-mutated huntingtin and represents a unique model to investigate therapeutic strategies towards HD. A more detailed characterization of this model is needed to fully utilize its potential. METHODS: In this study, we focused on the molecular and cellular features of fibroblasts isolated from TgHD minipigs and the wild-type (WT) siblings at different ages, pre-symptomatic at the age of 24-36 months and with the onset of behavioural symptoms at the age of 48 months. We measured oxidative stress, the expression of oxidative stress-related genes, proliferation capacity along with the expression of cyclin B1 and D1 proteins, cellular permeability, and the integrity of the nuclear DNA (nDNA) and mitochondrial DNA in these cells. RESULTS: TgHD fibroblasts isolated from 48-month-old animals showed increased oxidative stress, which correlated with the overexpression of SOD2 encoding mitochondrial superoxide dismutase 2, and the NEIL3 gene encoding DNA glycosylase involved in replication-associated repair of oxidized DNA. TgHD cells displayed an abnormal proliferation capacity and permeability. We further demonstrated increased nDNA damage in pre-symptomatic TgHD fibroblasts (isolated from animals aged 24-36 months). CONCLUSIONS: Our results unravel phenotypic alterations in primary fibroblasts isolated from the TgHD minipig model at the age of 48 months. Importantly, nDNA damage appears to precede these phenotypic alterations. Our results highlight the impact of fibroblasts from TgHD minipigs in studying the molecular mechanisms of HD pathophysiology that gradually occur with age.

Gradual Phenotype Development in Huntington Disease Transgenic Minipig Model at 24 Months of Age.

BACKGROUND: Huntington disease (HD) is an incurable neurodegenerative disease caused by the expansion of a polyglutamine sequence in a gene encoding the huntingtin (Htt) protein, which is expressed in almost all cells of the body. In addition to small animal models, new therapeutic approaches (including gene therapy) require large animal models as their large brains are a more realistic model for translational research. OBJECTIVE: In this study, we describe phenotype development in transgenic minipigs (TgHD) expressing the N-terminal part of mutated human Htt at the age of 24 months. METHODS: TgHD and wild-type littermates were compared. Western blot analysis and subcellular fractionation of different tissues was used to determine the fragmentation of Htt. Immunohistochemistry and optical analysis of coronal sections measuring aggregates, Htt expression, neuroinflammation, and myelination was applied. Furthermore, the expression of Golgi protein acyl-CoA binding domain containing 3 (ACBD3) was analyzed. RESULTS: We found age-correlated Htt fragmentation in the brain. Among various tissues studied, the testes displayed the highest fragmentation, with Htt fragments detectable even in cell nuclei. Also, Golgi protein ACBD3 was upregulated in testes, which is in agreement with previously reported testicular degeneration in TgHD minipigs. Nevertheless, the TgHD-specific mutated Htt fragments were also present in the cytoplasm of striatum and cortex cells. Moreover, microglial cells were activated and myelination was slightly decreased, suggesting the development of a premanifest stage of neurodegeneration in TgHD minipigs. CONCLUSIONS: The gradual development of a neurodegenerative phenotype, ac-companied with testicular degeneration, is observed in 24- month-old TgHD minipigs.

Mitochondrial Metabolism in a Large-Animal Model of Huntington Disease: The Hunt for Biomarkers in the Spermatozoa of Presymptomatic Minipigs.

BACKGROUND: Huntington disease (HD) is a fatal neurodegenerative disorder involving reduced muscle coordination, mental and behavioral changes, and testicular degeneration. In order to further clarify the decreased fertility and penetration ability of the spermatozoa of transgenic HD minipig boars (TgHD), we applied a set of mitochondrial metabolism (MM) parameter measurements to this promising biological material, which can be collected noninvasively in longitudinal studies. OBJECTIVE: We aimed to optimize methods for MM measurements in spermatozoa and to establish possible biomarkers of HD in TgHD spermatozoa expressing the N-terminal part of mutated human huntingtin. METHODS: Semen samples from 12 TgHD and wild-type animals, aged 12-65 months, were obtained repeatedly during the study. Respiration was measured by polarography, MM was assessed by the detection of oxidation of radiolabeled substrates (mitochondrial energy-generating system; MEGS), and the content of the oxidative phosphorylation system subunits was detected by Western blot. Three possibly interfering factors were statistically analyzed: the effect of HD, generation and aging. RESULTS: We found 5 MM parameters which were significantly diminished in TgHD spermatozoa and propose 3 specific MEGS incubations and complex I-dependent respiration as potential biomarkers of HD in TgHD spermatozoa. CONCLUSIONS: Our results suggest a link between the gain of toxic function of mutated huntingtin in TgHD spermatozoa and the observed MM and/or glycolytic impairment. We determined 4 biomarkers useful for HD phenotyping and experimental therapy monitoring studies in TgHD minipigs.

Mutated Huntingtin Causes Testicular Pathology in Transgenic Minipig Boars.

BACKGROUND: Huntington's disease is induced by CAG expansion in a single gene coding the huntingtin protein. The mutated huntingtin (mtHtt) primarily causes degeneration of neurons in the brain, but it also affects peripheral tissues, including testes. OBJECTIVE: We studied sperm and testes of transgenic boars expressing the N-terminal region of human mtHtt. METHODS: In this study, measures of reproductive parameters and electron microscopy (EM) images of spermatozoa and testes of transgenic (TgHD) and wild-type (WT) boars of F1 (24-48 months old) and F2 (12-36 months old) generations were compared. In addition, immunofluorescence, immunohistochemistry, Western blot, hormonal analysis and whole-genome sequencing were done in order to elucidate the effects of mtHtt. RESULTS: Evidence for fertility failure of both TgHD generations was observed at the age of 13 months. Reproductive parameters declined and progressively worsened with age. EM revealed numerous pathological features in sperm tails and in testicular epithelium from 24- and 36-month-old TgHD boars. Moreover, immunohistochemistry confirmed significantly lower proliferation activity of spermatogonia in transgenic testes. mtHtt was highly expressed in spermatozoa and testes of TgHD boars and localized in all cells of seminiferous tubules. Levels of fertility-related hormones did not differ in TgHD and WT siblings. Genome analysis confirmed that insertion of the lentiviral construct did not interrupt any coding sequence in the pig genome. CONCLUSIONS: The sperm and testicular degeneration of TgHD boars is caused by gain-of-function of the highly expressed mtHtt.

Revelation of fibroblast protein commonalities and differences and their possible roles in wound healing and tumourigenesis using co-culture models of cells.

BACKGROUND INFORMATION: The in vitro co-culture models of communication between normal fibroblasts and epithelial cells, such as keratinocytes or squamous cell carcinoma cells of FaDu line representing wound healing or cancer development, were established by non-direct contact between the cells and utilised in this study to examine epithelia-induced changes in overall fibroblast proteome patterns. RESULTS: We were able to select the proteins co-regulated in both models in order to evaluate possible molecular commonalities between wound healing and tumour development. Amongst the most pronounced were the proteins implemented in contractile activity and formation of actin cytoskeleton such as caldesmon, calponin-2, myosin regulatory light-chain 12A and cofilin-1, which were expressed independently of the presence of α-smooth muscle actin. Additionally, proteins altered differently highlighted functional and cellular phenotypes during transition of fibroblasts towards myofibroblasts or cancer-associated fibroblasts. Results showed coordinated regulation of cytoskeleton proteins selective for wound healing which were lost in tumourigenesis model. Vimentin bridged this group of proteins with other regulated proteins in human fibroblasts involved in protein or RNA processing and metabolic regulation. CONCLUSIONS: The findings provide strong support for crucial role of stromal microenvironment in wound healing and tumourigenesis. In particular, epithelia-induced protein changes in fibroblasts offer new potential targets which may lead to novel tailored cancer therapeutic strategies.

Feasibility of Direct Vitrectomy-Sparing Subretinal Injection for Gene Delivery in Large Animals.

PURPOSE: To assess the safety and feasibility of direct vitrectomy-sparing subretinal injection for gene delivery in a large animal model. METHODS: The experimental Libechov minipigs were used for subretinal delivery of a plasmid DNA vector (pS/MAR-CMV-copGFP) with cytomegalovirus (CMV) promoter, green fluorescent protein (GFP) reporter (copGFP) and a scaffold/matrix attachment region (S/MAR) sequence. The eyes were randomized to subretinal injection of the vector following pars plana vitrectomy (control group) or a direct injection without prior vitrectomy surgery (experimental group). Intra- and post-operative observations up to 30 days after surgery were compared. RESULTS: Six eyes of three mini-pigs underwent surgery for delivery into the subretinal space. Two eyes in the control group were operated with a classical approach (lens-sparing vitrectomy and posterior hyaloid detachment). The other four eyes in the experimental group were injected directly with a subretinal cannula without vitrectomy surgery. No adverse events, such as endophthalmitis, retinal detachment and intraocular pressure elevation were observed post-operatively. The eyes in the experimental group had both shorter surgical time and recovery while achieving the same surgical goal. CONCLUSIONS: This pilot study demonstrates that successful subretinal delivery of gene therapy vectors is achievable using a direct injection without prior vitrectomy surgery.

Upregulation of IL-6, IL-8 and CXCL-1 production in dermal fibroblasts by normal/malignant epithelial cells in vitro: Immunohistochemical and transcriptomic analyses.

BACKGROUND INFORMATION: Considering an analogy between wound healing and tumour progression, we studied chemokine and cytokine transcription and expression in normal fibroblasts by co-culture and in situ. RESULTS: Whole-genome transcriptome profiling revealed strong upregulation for the interleukin (IL)-6, IL-8 and the chemokine CXCL-1 in in vitro co-cultures of normal fibroblasts with either normal or malignant epithelial cells compared to fibroblast cultures. The same ILs/chemokines were distinctly upregulated in clinical samples of squamous cell carcinoma when compared with paired normal mucosae. Analysis of culture supernatants showed that during the course of co-culture of the fibroblasts with the epithelial cells, IL-6, IL-8 and CXCL-1 were secreted to the culture medium. Experiments with addition of any of the proteins to the culture medium supported the notion that these ILs/chemokines strongly contributed to maintenance of a low-differentiation phenotype of epithelial cells, evaluated by the detection of keratin-8. Simultaneous addition of all factors increased the extent of the effect. These studies were extended by experiments with epithelial cells, either cultured in medium conditioned by preceding use for malignant keratinocytes without and in the presence of normal or cancer-associated fibroblasts or medium containing antibodies against IL-6, IL-8 and CXCL-1. CONCLUSIONS: Our results indicate an analogy between wound healing and tumour growth, support the importance of epithelial-mesenchymal interaction in this model system and establish a potential bio-inspired anticancer therapy.

Rapid and Reliable Quantification of Prime Editing Targeting Within the Porcine ABCA4 Gene Using a BRET-Based Sensor.

Stargardt disease (STGD) leads to blindness in children and young adults. So far, no curative therapy is available and gene augmentation therapies have not yet advanced to the clinics, in part, due to the limited packaging capacity of adeno-associated viruses used to transfer genes into photoreceptor cells. Prime editing offers a new perspective to treat mutations on the genomic level. A nicking variant of Cas9 fused to a reverse transcriptase complex with an elongated guideRNA force intracellular mismatch repair to correct the targeted mutation even in postmitotic cells such as photoreceptors in the eye. Using a custom-made bioluminescence resonance energy transfer (BRET)-based editing sensor in HEK293 cells, we tested 27 different prime editing guide RNAs (pegRNAs) and additional 4 nicking guide RNAs (ngRNAs) with regard to their efficiency to induce sequences changes in exon 43 of the porcine ATP binding cassette subfamily A member 4 (ABCA4) gene that eliminate a mutagenic adenine frameshift insertion, which has been associated with STGD in humans. We identified nine working pegRNAs, and in combination with ngRNAs, we achieved a correction rate of up to ≈92% measured with the BRET-based reporter system. Our data prove the high efficiency of prime editors to correct mutations and highlight the importance of optimal ngRNA design, thus offering a promising editing tool to correct ABCA4 mutations in the disease context.

Retinal Pigment Epithelium Cell Development: Extrapolating Basic Biology to Stem Cell Research.

The retinal pigment epithelium (RPE) forms an important cellular monolayer, which contributes to the normal physiology of the eye. Damage to the RPE leads to the development of degenerative diseases, such as age-related macular degeneration (AMD). Apart from acting as a physical barrier between the retina and choroidal blood vessels, the RPE is crucial in maintaining photoreceptor (PR) and visual functions. Current clinical intervention to treat early stages of AMD includes stem cell-derived RPE transplantation, which is still in its early stages of evolution. Therefore, it becomes essential to derive RPEs which are functional and exhibit features as observed in native human RPE cells. The conventional strategy is to use the knowledge obtained from developmental studies using various animal models and stem cell-based exploratory studies to understand RPE biogenies and developmental trajectory. This article emphasises such studies and aims to present a comprehensive understanding of the basic biology, including the genetics and molecular pathways of RPE development. It encompasses basic developmental biology and stem cell-based developmental studies to uncover RPE differentiation. Knowledge of the in utero developmental cues provides an inclusive methodology required for deriving RPEs using stem cells.

Progress in Stem Cells-Based Replacement Therapy for Retinal Pigment Epithelium: In Vitro Differentiation to In Vivo Delivery.

Retinal pigment epithelium (RPE) is a critical cell monolayer forming the blood-retina-barrier (BRB) and a permeable bridge between the choriocapillaris and the retina. RPE is also crucial in maintaining photoreceptor function and for completing the visual cycle. Loss of the RPE is associated with the development of degenerative diseases like age-related macular degeneration (AMD). To treat diseases like AMD, pluripotent stem cell-derived RPE (pRPE) has been recently explored extensively as a regenerative module. pRPE like other ectodermal tissues requires specific lineage differentiation and long-term in vitro culturing for maturation. Therefore, understanding the differentiation process of RPE could be useful for stem cell-based RPE derivation. Developing pRPE-based transplants and delivering them into the subretinal space is another aspect that has garnered interest in the last decade. In this review, we discuss the basic strategies currently employed for stem cell-based RPE derivation, their delivery, and recent clinical studies related to pRPE transplantation in patients. We have also discussed a few limitations with in vitro RPE culture and potential solutions to overcome such problems which can be helpful in developing functional RPE tissue.

Aurora kinase A drives MTOC biogenesis but does not trigger resumption of meiosis in mouse oocytes matured in vivo.

Aurora kinase A (AURKA) is an important mitotic kinase involved in the G2/M transition, centrosome maturation and separation, and spindle formation in somatic cells. We used transgenic models that specifically overexpress in mouse oocytes either wild-type (WT-AURKA) or a catalytically inactive (kinase-dead) (KD-AURKA) AURKA to gain new insights regarding the role of AURKA during oocyte maturation. AURKA activation occurs shortly after hCG administration that initiates maturation in vivo. Although AURKA activity is increased in WT-AURKA oocytes, resumption of meiosis is not observed in the absence of hCG administration. Control oocytes contain one to three microtubule organizing centers (MTOCs; centrosome equivalent) at prophase I. At the time of germinal vesicle breakdown (GVBD), the first visible marker of resumption of meiosis, the MTOC number increases. In WT-AURKA oocytes, the increase in MTOC number occurs prematurely but transiently without GVBD, whereas the increase in MTOC number does not occur in control and KD-AURKA oocytes. AURKA activation is biphasic with the initial activation not requiring CDC25B-CDK1 activity, whereas full activation, which is essential for the increase in MTOCs number, depends on CDK1 activity. AURKA activity also influences spindle length and regulates, independent of its protein kinase activity, the amount of MTOC associated with gamma-tubulin. Both WT-AURKA and KD-AURKA transgenic mice have normal fertility during first 6 mo of life. These results suggest that although AURKA is not a trigger kinase for G2/M transition in mouse oocytes, it regulates MTOC number and spindle length, and, independent of its protein kinase activity, gamma-tubulin recruitment to MTOCs.

Advantages of nanofibrous membranes for culturing of primary RPE cells compared to commercial scaffolds.

PURPOSE: Dysfunction of the retinal pigment epithelium (RPE) causes numerous forms of retinal degeneration. RPE replacement is a modern option to save vision. We aimed to test the results of transplanting cultured RPEs on biocompatible membranes. METHODS: We cultivated porcine primary RPE cells isolated from cadaver eyes from the slaughterhouse on two types of membranes: commercial polyester scaffolds Transwell (Corning Inc., Kenneburg, ME, USA) with 0.4 µm pore size and prepared Poly (L-lactide-co-DL-lactide) (PDLLA) nanofibrous membranes with an average pore size of 0.4 µm. RESULTS: Five types of assays were used for the analysis: immunocytochemistry (ICC), phagocytosis assay, Western blotting, real-time qPCR (RT-qPCR) and electron microscopy. RT-qPCR demonstrated that RPEs cultured on nanofibrous membranes have higher expressions of BEST1 (bestrophin 1), RLBP1 (retinaldehyde-binding protein 1), RPE65 (retinal pigment epithelium-specific 65 kDa protein), PAX6 (transcription factor PAX6), SOX9 (transcription factor SOX9), DCT (dopachrome tautomerase) and MITF (microphthalmia-associated transcription factor). ICC of the RPEs cultured on nanofibrous membranes showed more intensive staining of markers such as BEST1, MCT1 (monocarboxylate transporter 1), Na+ /K+ ATPase, RPE65 and acetylated tubulin in comparison with commercial ones. Additionally, the absence of α-SMA proved the stability of the RPE polarization state and the absence of epithelial-to-mesenchymal transition. RPE possessed high phagocytic activity. Electron microscopy of both membranes confirmed a confluent layer of RPE cells and their genuine morphological structure, which was comparable to native RPEs. CONCLUSIONS: Retinal pigment epitheliums cultured on polylactide nanofibrous membranes improved the final quality of the cell product by having better maturation and long-term survival of the RPE monolayer compared to those cultured on commercial polyester scaffolds. PDLLA-cultured RPEs are a plausible source for the replacement of non-functioning RPEs during cell therapy.

Mitochondrial Dysfunction in a High Intraocular Pressure-Induced Retinal Ischemia Minipig Model.

Purpose: Retinal ischemia (RI) and progressive neuronal death are sight-threatening conditions. Mitochondrial (mt) dysfunction and fusion/fission processes have been suggested to play a role in the pathophysiology of RI. This study focuses on changes in the mt parameters of the neuroretina, retinal pigment epithelium (RPE) and choroid in a porcine high intraocular pressure (IOP)-induced RI minipig model. Methods: In one eye, an acute IOP elevation was induced in minipigs and compared to the other control eye. Activity and amount of respiratory chain complexes (RCC) were analyzed by spectrophotometry and Western blot, respectively. The coenzyme Q10 (CoQ10) content was measured using HPLC, and the ultrastructure of the mt was studied via transmission electron microscopy. The expression of selected mt-pathway genes was determined by RT-PCR. Results: At a functional level, increased RCC I activity and decreased total CoQ10 content were found in RPE cells. At a protein level, CORE2, a subunit of RCC III, and DRP1, was significantly decreased in the neuroretina. Drp1 and Opa1, protein-encoding genes responsible for mt quality control, were decreased in most of the samples from the RPE and neuroretina. Conclusions: The eyes of the minipig can be considered a potential RI model to study mt dysfunction in this disease. Strategies targeting mt protection may provide a promising way to delay the acute damage and onset of RI.

Subretinal Implantation of Human Primary RPE Cells Cultured on Nanofibrous Membranes in Minipigs.

PURPOSE: The development of primary human retinal pigmented epithelium (hRPE) for clinical transplantation purposes on biodegradable scaffolds is indispensable. We hereby report the results of the subretinal implantation of hRPE cells on nanofibrous membranes in minipigs. METHODS: The hRPEs were collected from human cadaver donor eyes and cultivated on ultrathin nanofibrous carriers prepared via the electrospinning of poly(L-lactide-co-DL-lactide) (PDLLA). "Libechov" minipigs (12-36 months old) were used in the study, supported by preoperative tacrolimus immunosuppressive therapy. The subretinal implantation of the hRPE-nanofibrous carrier was conducted using general anesthesia via a custom-made injector during standard three-port 23-gauge vitrectomy, followed by silicone oil endotamponade. The observational period lasted 1, 2, 6 and 8 weeks, and included in vivo optical coherence tomography (OCT) of the retina, as well as post mortem immunohistochemistry using the following antibodies: HNAA and STEM121 (human cell markers); Bestrophin and CRALBP (hRPE cell markers); peanut agglutining (PNA) (cone photoreceptor marker); PKCα (rod bipolar marker); Vimentin, GFAP (macroglial markers); and Iba1 (microglial marker). RESULTS: The hRPEs assumed cobblestone morphology, persistent pigmentation and measurable trans-epithelial electrical resistance on the nanofibrous PDLLA carrier. The surgical delivery of the implants in the subretinal space of the immunosuppressed minipigs was successfully achieved and monitored by fundus imaging and OCT. The implanted hRPEs were positive for HNAA and STEM121 and were located between the minipig's neuroretina and RPE layers at week 2 post-implantation, which was gradually attenuated until week 8. The neuroretina over the implants showed rosette or hypertrophic reaction at week 6. The implanted cells expressed the typical RPE marker bestrophin throughout the whole observation period, and a gradual diminishing of the CRALBP expression in the area of implantation at week 8 post-implantation was observed. The transplanted hRPEs appeared not to form a confluent layer and were less capable of keeping the inner and outer retinal segments intact. The cone photoreceptors adjacent to the implant scaffold were unchanged initially, but underwent a gradual change in structure after hRPE implantation; the retina above and below the implant appeared relatively healthy. The glial reaction of the transplanted and host retina showed Vimentin and GFAP positivity from week 1 onward. Microglial activation appeared in the retinal area of the transplant early after the surgery, which seemed to move into the transplant area over time. CONCLUSIONS: The differentiated hRPEs can serve as an alternative cell source for RPE replacement in animal studies. These cells can be cultivated on nanofibrous PDLLA and implanted subretinally into minipigs using standard 23-gauge vitrectomy and implantation injector. The hRPE-laden scaffolds demonstrated relatively good incorporation into the host retina over an eight-week observation period, with some indication of a gliotic scar formation, and a likely neuroinflammatory response in the transplanted area despite the use of immunosuppression.

Subretinal Implantation of RPE on a Carrier in Minipigs: Guidelines for Preoperative Preparations, Surgical Techniques, and Postoperative Care.

Degenerative disorders of the retina (including age-related macular degeneration), which originate primarily at or within the retinal pigmented epithelial (RPE) layer, lead to a progressive disorganization of the retinal anatomy and the deterioration of visual function. The substitution of damaged RPE cells (RPEs) with in vitro cultured RPE cells using a subretinal cell carrier has shown potential for re-establishing the anatomical structure of the outer retinal layers and is, therefore, being further studied. Here, we present the principles of a surgical technique that allows for the effective subretinal transplantation of a cell carrier with cultivated RPEs into minipigs. The surgeries were performed under general anesthesia and included a standard lens-sparing three-port pars plana vitrectomy (PPV), subretinal application of a balanced salt solution (BSS), a 2.7 mm retinotomy, implantation of a nanofibrous cell carrier into the subretinal space through an additional 3.0 mm sclerotomy, fluid-air exchange (FAX), silicone oil tamponade, and closure of all the sclerotomies. This surgical approach was used in 29 surgeries (18 animals) over the past 8 years with a success rate of 93.1%. Anatomic verification of the surgical placement was carried out using in vivo fundus imaging (fundus photography and optical coherence tomography). The recommended surgical steps for the subretinal implantation of RPEs on a carrier in minipig eyes can be used in future preclinical studies using large-eye animal models.

Early disruption of photoreceptor cell architecture and loss of vision in a humanized pig model of usher syndromes.

Usher syndrome (USH) is the most common form of monogenic deaf-blindness. Loss of vision is untreatable and there are no suitable animal models for testing therapeutic strategies of the ocular constituent of USH, so far. By introducing a human mutation into the harmonin-encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction, and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis, and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Fibroblasts from USH1C pigs or USH1C patients showed significantly elongated primary cilia, confirming USH as a true and general ciliopathy. Primary cells also proved their capacity for assessing the therapeutic potential of CRISPR/Cas-mediated gene repair or gene therapy in vitro. AAV-based delivery of harmonin into the eye of USH1C pigs indicated therapeutic efficacy in vivo.

Mapping of the secretome of primary isolates of mammalian cells, stem cells and derived cell lines.

Within a mammalian organism, the interaction among cells both at short and long distances is mediated by soluble factors released by cells into the extracellular environment. The secreted proteins may involve extracellular matrix proteins, proteinases, growth factors, protein hormones, immunoregulatory cytokines, chemokines or other bioactive molecules that have a direct impact on target cell phenotype. Stem cells of mesenchymal, adipose, neural and embryonic origin, fibroblast feeder cells as well as primary isolates of astrocytes, endothelial and muscle cells have recently become targets of intensive secretome profiling with the search for proteins regulating cell survival, proliferation, differentiation or inflammatory response. Recent advances and challenges of the stem cell and primary cell secretome analysis together with the most relevant results are discussed in this review.

Analysis of marker expression in porcine cell lines derived from blastocysts produced in vitro and in vivo.

The present study was designed to extensively characterize cell lines derived from porcine blastocysts by several methodical approaches, including morphological observation, cytogenetic analysis, estimation of alkaline phosphatase activity and detection of specific marker expression at the mRNA/protein level. A comparison was made between the properties of cell lines isolated from in vivo- and in vitro-obtained blastocysts. Our results showed that 57.1% of the in vivo-obtained blastocysts attached to the feeder layer and that 33.3% of them started to grow in a monolayer. The percentage of attached in vitro-produced blastocysts was lower (24.6%), and only 6.9% of them started to grow. Outgrowths from the in vitro-produced blastocysts formed mainly trophectoderm or epithelial-like monolayer, whereas the in vivo-obtained blastocysts formed heterogeneous outgrowths that also contained cells with embryonic stem (ES)-like morphology. Detailed analyses showed that the primary outgrowths with ES-like morphology expressed the pluripotency markers OCT-4 and NANOG and revealed intensive alkaline phosphatase staining, while they did not express markers of differentiation. The majority of passaged cells, including those with ES-like morphology, lacked OCT-4 protein and revealed expression of specific differentiation markers (cytokeratin 18, lamins A/C, transferrin, α-fetoprotein and GATA-4), although they still expressed NANOG and exhibited weak alkaline phosphatase activity. Moreover, these cells spontaneously differentiated into neural, fibroblast or epithelial-like cells, even in the presence of leukaemia inhibitory factor. Our results show that complex analysis of markers of pluripotency as well as differentiation markers is necessary for proper interpretation of data in porcine embryonic stem cell studies.