Caroline Brandl

Comparison of human dental follicle cells (DFCs) and stem cells from human exfoliated deciduous teeth (SHED) after neural differentiation in vitro

Dental stem cells from human exfoliated deciduous teeth (SHED) and dental follicle cells (DFCs) are neural crest-derived stem cells from human dental tissues. Interestingly, SHED and DFCs can successfully differentiate into neuron-like cells. We hypothesized that SHED and DFCs have the same neural cell differentiation potentials. To evaluate neural cell differentiation, we cultivated SHED and DFCs in four different serum-replacement media (SRMs) and analyzed cell morphology, cell proliferation, and gene expression patterns before and after differentiation. In a standard cell culture medium, SHED and DFCs have not only similar cell morphologies, but they also have similar gene expression patterns for known stem cell markers. However, only SHED expressed the neural stem cell marker Pax6. After cultivation in SRMs, cell proliferations of DFCs and SHED were reduced and the cell morphology was spindle-like with long processes. However, differentiated DFCs and SHED had different neural cell marker expression patterns. For example, gene expression of the late neural cell marker microtubule-associated protein 2 was upregulated in DFCs and downregulated in SHED in SRM with the B27 supplement. In contrast, SHED formed neurosphere-like cell clusters in SRM with the B27 supplement, epidermal growth factor, and fibroblast growth factor-2. Moreover, SHED differentially expressed the glial cell marker glial fibrillary acidic protein, which in contrast was weakly or not expressed in DFCs. In conclusion, SHED and DFCs have different neural differentiation potentials under the same cell culture conditions.

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells

Significance First insight into the molecular identity of volume-regulated anion channel (VRAC) emerged only recently by demonstrating a role for leucine-rich repeats containing 8A (LRRC8A) in channel activity. Our results now expand on VRAC biology, suggesting a model where VRAC subunit composition is cell type- or tissue-specific rather than a single ubiquitous channel formed solely by LRRC8A. Here, we show that bestrophin 1 (BEST1), but not LRCC8A, is crucial in cell volume regulation in retinal pigment epithelium (RPE) cells differentiated from human-induced pluripotent stem cells (hiPSCs). VRAC-mediated currents were strongly reduced in hiPSC-RPE from macular dystrophy patients with pathologic BEST1 mutations. Our model is further supported by in vivo effects of Best1 deficiency in the mouse that manifest as severe subfertility phenotype due to enhanced abnormal sperm morphology related to impaired volume regulation. In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1−/−) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1−/− mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex—that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies.

Identification of neural crest-derived stem cell-like cells from the corneal limbus of juvenile mice

The neural crest is a transient embryonic tissue alongside the lateral margins of the neural folds. It contains cells involved in the development of anterior eye segments such as the cornea. Previous studies have revealed the presence of neural crest-derived stem cells in the cornea of the adult murine eye. However, less is known about cell populations of the developing eye. In this study, we have identified neural crest-derived murine corneal cells (MCCs) with stem cell-like properties derived from the corneal limbus of mice between postnatal days 1 and 8. RT-PCR analysis and immunofluorescence staining demonstrate that MCCs express a unique profile of markers including typical neural crest-originated stem cell transcripts like Sca1. MCCs show a limited self-renewing capacity but possess multipotency under in vitro conditions after differentiation into cells with features resembling adipocytes, osteoblasts and neuronal cells. Interestingly, MCCs could not be isolated from corneas of adult animals. We conclude that MCCs are stem cell-like cells of an early postnatal period of murine eye growth, probably involved in the early development of the postnatal cornea.

In-Depth Characterisation of Retinal Pigment Epithelium (RPE) Cells Derived from Human Induced Pluripotent Stem Cells (hiPSC)

Induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) has widely been appreciated as a promising tool to model human ocular disease emanating from primary RPE pathology. Here, we describe the successful reprogramming of adult human dermal fibroblasts to iPSCs and their differentiation to pure expandable RPE cells with structural and functional features characteristic for native RPE. Fibroblast cultures were established from skin biopsy material and subsequently reprogrammed following polycistronic lentiviral transduction with OCT4, SOX2, KLF4 and L-Myc. Fibroblast-derived iPSCs showed typical morphology, chromosomal integrity and a distinctive stem cell marker profile. Subsequent differentiation resulted in expandable pigmented hexagonal RPE cells. The cells revealed stable RNA expression of mature RPE markers RPE65, RLBP and BEST1. Immunolabelling verified localisation of BEST1 at the basolateral plasma membrane, and scanning electron microscopy showed typical microvilli at the apical side of iPSC-derived RPE cells. Transepithelial resistance was maintained at high levels during cell culture indicating functional formation of tight junctions. Secretion capacity was demonstrated for VEGF-A. Feeding of porcine photoreceptor outer segments revealed the proper ability of these cells for phagocytosis. IPSC-derived RPE cells largely maintained these properties after cryopreservation. Together, our study underlines that adult dermal fibroblasts can serve as a valuable resource for iPSC-derived RPE with characteristics highly reminiscent of true RPE cells. This will allow its broad application to establish cellular models for RPE-related human diseases.

A Circulating MicroRNA Profile Is Associated with Late-Stage Neovascular Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of severe vision impairment in Western populations over 55 years. A growing number of gene variants have been identified which are strongly associated with an altered risk to develop AMD. Nevertheless, gene-based biomarkers which could be dysregulated at defined stages of AMD may point toward key processes in disease mechanism and thus may support efforts to design novel treatment regimens for this blinding disorder. Circulating microRNAs (cmiRNAs) which are carried by nanosized exosomes or microvesicles in blood plasma or serum, have been recognized as valuable indicators for various age-related diseases. We therefore aimed to elucidate the role of cmiRNAs in AMD by genome-wide miRNA expression profiling and replication analyses in 147 controls and 129 neovascular AMD patients. We identified three microRNAs differentially secreted in neovascular (NV) AMD (hsa-mir-301-3p, pcorrected = 5.6*10−5, hsa-mir-361-5p, pcorrected = 8.0*10−4 and hsa-mir-424-5p, pcorrected = 9.6*10−3). A combined profile of the three miRNAs revealed an area under the curve (AUC) value of 0.727 and was highly associated with NV AMD (p = 1.2*10−8). To evaluate subtype-specificity, an additional 59 AMD cases with pure unilateral or bilateral geographic atrophy (GA) were analyzed for microRNAs hsa-mir-301-3p, hsa-mir-361-5p, and hsa-mir-424-5p. While we found no significant differences between GA AMD and controls neither individually nor for a combined microRNAs profile, hsa-mir-424-5p levels remained significantly higher in GA AMD when compared to NV (pcorrected<0.005). Pathway enrichment analysis on genes predicted to be regulated by microRNAs hsa-mir-301-3p, hsa-mir-361-5p, and hsa-mir-424-5p, suggests canonical TGFβ, mTOR and related pathways to be involved in NV AMD. In addition, knockdown of hsa-mir-361-5p resulted in increased neovascularization in an in vitro angiogenesis assay.

Tapping Stem Cells to Target AMD: Challenges and Prospects

Human pluripotent stem cells (hPSCs) are increasingly gaining attention in biomedicine as valuable resources to establish patient-derived cell culture models of the cell type known to express the primary pathology. The idea of “a patient in a dish” aims at basic, but also clinical, applications with the promise to mimic individual genetic and metabolic complexities barely reflected in current invertebrate or vertebrate animal model systems. This may particularly be true for the inherited and complex diseases of the retina, as this tissue has anatomical and physiological aspects unique to the human eye. For example, the complex age-related macular degeneration (AMD), the leading cause of blindness in Western societies, can be attributed to a large number of genetic and individual factors with so far unclear modes of mutual interaction. Here, we review the current status and future prospects of utilizing hPSCs, specifically induced pluripotent stem cells (iPSCs), in basic and clinical AMD research, but also in assessing potential treatment options. We provide an outline of concepts for disease modelling and summarize ongoing and projected clinical trials for stem cell-based therapy in late-stage AMD.

Features of Age-Related Macular Degeneration in the General Adults and Their Dependency on Age, Sex, and Smoking: Results from the German KORA Study

Age-related macular degeneration (AMD) is a vision impairing disease of the central retina characterized by early and late forms in individuals older than 50 years of age. However, there is little knowledge to what extent also younger adults are affected. We have thus set out to estimate the prevalence of early AMD features and late AMD in a general adult population by acquiring color fundus images in 2,840 individuals aged 25 to 74 years of the Cooperative Health Research in the Region of Augsburg project (KORA) in South Germany. Among the 2,546 participants with gradable images for each eye, 10.9% (n = 277) had early AMD features (applying the 9-step Age-Related Eye Disease Study Severity Scale), 0.2% (n = 6) had late AMD. Prevalence increased with age, reaching 26.3% for early AMD features and 1.9% for late AMD at the age 70+. However, signs of early AMD were found in subjects as young as 25 years, with the risk for early AMD features increasing linearly by years of age in men, and, less consistent with a linear increase, in women. Risk for early AMD features increased linearly by pack years of smoking in men, not in women, nor was there any association with other lifestyle or metabolic factors. By providing much sought-after prevalence estimates for AMD from Central Europe, our data underscores a substantial proportion of the adult population with signs of early AMD, including individuals younger than 50 years. This supports the notion that early AMD features in the young might be under-acknowledged.

Spontaneous immortalization of neural crest‐derived corneal progenitor cells after chromosomal aberration

Objectives:  In a previous study, we have reported the existence of neural crest‐derived stem cell‐like cells originating from the corneal limbus of juvenile mice (termed murine corneal cells, MCCs). To yield a sufficient number of MCCs, for example, for cell‐therapy approaches, here we have investigated MCCs’ ability for extensive proliferation, and we have evaluated their stem cell qualities and genetic stability after large‐scale culture.

A Deep Learning Algorithm for Prediction of Age-Related Eye Disease Study Severity Scale for Age-Related Macular Degeneration from Color Fundus Photography

PURPOSE Age-related macular degeneration (AMD) is a common threat to vision. While classification of disease stages is critical to understanding disease risk and progression, several systems based on color fundus photographs are known. Most of these require in-depth and time-consuming analysis of fundus images. Herein, we present an automated computer-based classification algorithm. DESIGN Algorithm development for AMD classification based on a large collection of color fundus images. Validation is performed on a cross-sectional, population-based study. PARTICIPANTS We included 120 656 manually graded color fundus images from 3654 Age-Related Eye Disease Study (AREDS) participants. AREDS participants were >55 years of age, and non-AMD sight-threatening diseases were excluded at recruitment. In addition, performance of our algorithm was evaluated in 5555 fundus images from the population-based Kooperative Gesundheitsforschung in der Region Augsburg (KORA; Cooperative Health Research in the Region of Augsburg) study. METHODS We defined 13 classes (9 AREDS steps, 3 late AMD stages, and 1 for ungradable images) and trained several convolution deep learning architectures. An ensemble of network architectures improved prediction accuracy. An independent dataset was used to evaluate the performance of our algorithm in a population-based study. MAIN OUTCOME MEASURES κ Statistics and accuracy to evaluate the concordance between predicted and expert human grader classification. RESULTS A network ensemble of 6 different neural net architectures predicted the 13 classes in the AREDS test set with a quadratic weighted κ of 92% (95% confidence interval, 89%-92%) and an overall accuracy of 63.3%. In the independent KORA dataset, images wrongly classified as AMD were mainly the result of a macular reflex observed in young individuals. By restricting the KORA analysis to individuals >55 years of age and prior exclusion of other retinopathies, the weighted and unweighted κ increased to 50% and 63%, respectively. Importantly, the algorithm detected 84.2% of all fundus images with definite signs of early or late AMD. Overall, 94.3% of healthy fundus images were classified correctly. CONCLUSIONS Our deep learning algoritm revealed a weighted κ outperforming human graders in the AREDS study and is suitable to classify AMD fundus images in other datasets using individuals >55 years of age.

Investigating the modulation of genetic effects on late AMD by age and sex: Lessons learned and two additional loci

Late-stage age-related macular degeneration (AMD) is the leading cause of visual impairment in the elderly with a complex etiology. The most important non-modifiable risk factors for onset and progression of late AMD are age and genetic risk factors, however, little is known about the interplay between genetics and age or sex. Here, we conducted a large-scale age- and sex-stratified genome-wide association study (GWAS) using 1000 Genomes imputed genome-wide and ExomeChip data (>12 million variants). The data were established by the International Age-related Macular Degeneration Genomics Consortium (IAMDGC) from 16,144 late AMD cases and 17,832 controls. Our systematic search for interaction effects yielded significantly stronger effects among younger individuals at two known AMD loci (near CFH and ARMS2/HTRA1). Accounting for age and gene-age interaction using a joint test identified two additional AMD loci compared to the previous main effect scan. One of these two is a novel AMD GWAS locus, near the retinal clusterin-like protein (CLUL1) gene, and the other, near the retinaldehyde binding protein 1 (RLBP1), was recently identified in a joint analysis of nuclear and mitochondrial variants. Despite considerable power in our data, neither sex-dependent effects nor effects with opposite directions between younger and older individuals were observed. This is the first genome-wide interaction study to incorporate age, sex and their interaction with genetic effects for late AMD. Results diminish the potential for a role of sex in the etiology of late AMD yet highlight the importance and existence of age-dependent genetic effects.