Lanning Emily P, Branch Matthew J, Harding Philippa, Margari Miriam, Smith Alexander J, Ali Robin R, Pearson Rachael A
Ocular Cell and Gene Therapy Group, Centre for Gene Therapy and Regenerative Medicine, King's College London, Tower Wing, Guy's Hospital, London, United Kingdom.
Invest Ophthalmol Vis Sci. 2025 Jun 2;66(6):40. doi: 10.1167/iovs.66.6.40.
Little is known about the development of Bruch's membrane (BrM), the structure separating and supporting the retina and choroid, nor whether differentiation of human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) accurately replicates BrM. This has relevance for tissue engineering strategies, both in the development of accurate in vitro models, and effective RPE transplant strategies. Here, we investigated BrM-associated protein production in human fetal tissue and hPSC-derived RPE.
The presence of laminin, elastin, fibronectin, and types I/III/IV collagen was examined in human fetal eyes at 6 to 21 post-conception weeks (PCWs) and hPSC-derived RPE cultures at 1 to 6 weeks in culture using immunohistochemistry/immunocytochemistry and quantitative PCR (qPCR).
In human fetal retina, laminin and fibronectin were present from 6 PCW, type IV collagen from 8 PCW, elastin from 12 PCW, type I collagen by 17 PCW, and type III collagen from 21 PCW. BrM layering was discernible from 12 PCW, becoming distinct by 17 PCW. In hPSC-derived RPE cultures, basement membranes containing laminin and fibronectin were present from week 1, type IV collagen from week 2, and type I collagen from week 4. Type III collagen was present at all timepoints, although not localized as a basement membrane. Elastin was absent at all timepoints.
BrM-like membrane synthesis in hPSC-derived RPE largely recapitulates the temporal sequence seen in human development, excluding elastin. These support the utility of hPSC-derived RPE in in vitro systems to model RPE/retina interactions in health and disease, and inform cell therapy approaches, as de novo BrM-like membrane has the potential to support transplanted donor RPE.
关于布鲁赫膜（BrM）的发育情况所知甚少，布鲁赫膜是分隔并支撑视网膜和脉络膜的结构，也不清楚人类多能干细胞（hPSC）衍生的视网膜色素上皮（RPE）的分化是否能准确复制布鲁赫膜。这对于组织工程策略具有重要意义，无论是在开发精确的体外模型还是有效的RPE移植策略方面。在此，我们研究了人胎儿组织和hPSC衍生的RPE中与布鲁赫膜相关的蛋白质产生情况。
使用免疫组织化学/免疫细胞化学和定量PCR（qPCR）检测了孕后6至21周（PCW）的人胎儿眼睛以及培养1至6周的hPSC衍生的RPE培养物中纤连蛋白、弹性蛋白、纤连蛋白以及I/III/IV型胶原蛋白的存在情况。
在人胎儿视网膜中，6 PCW时存在纤连蛋白和纤连蛋白，8 PCW时存在IV型胶原蛋白，12 PCW时存在弹性蛋白，17 PCW时存在I型胶原蛋白，21 PCW时存在III型胶原蛋白。12 PCW时可辨别出布鲁赫膜分层，17 PCW时变得明显。在hPSC衍生的RPE培养物中，第1周时存在含有纤连蛋白和纤连蛋白的基底膜，第2周时存在IV型胶原蛋白，第4周时存在I型胶原蛋白。III型胶原蛋白在所有时间点均存在，尽管未定位为基底膜。所有时间点均不存在弹性蛋白。
hPSC衍生的RPE中类布鲁赫膜的合成在很大程度上重现了人类发育中所见的时间顺序，但不包括弹性蛋白。这些结果支持了hPSC衍生的RPE在体外系统中用于模拟健康和疾病状态下RPE/视网膜相互作用的实用性，并为细胞治疗方法提供了信息，因为新生的类布鲁赫膜有可能支持移植的供体RPE。
