Singapore Eye Research Institute, Singapore National Eye Centre, Singapore, Singapore.
Duke-NUS, Medical School, Singapore, Singapore.
Stem Cell Res Ther. 2020 Feb 3;11(1):47. doi: 10.1186/s13287-020-1568-3.
Despite increasing demand, current protocols for human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) remain time, labor, and cost intensive. Additionally, absence of robust methods for selective RPE purification and removal of non-RPE cell impurities prevents upscaling of clinical quality RPE production. We aimed to address these challenges by developing a simplified hPSC-derived RPE production and purification system that yields high-quality RPE monolayers within 90 days.
Human pluripotent stem cells were differentiated into RPE using an innovative time and cost-effective protocol relying entirely on 2D cultures and minimal use of cytokines. Once RPE identity was obtained, cells were transferred onto permeable membranes to acquire mature RPE morphology. RPE differentiation was verified by electron microscopy, polarized VEGF expression, establishment of high transepithelial electrical resistance and photoreceptor phagocytosis assay. After 4 weeks on permeable membranes, RPE cell cultures were incubated with Dil-AcLDL (DiI-conjugated acetylated low-density lipoproteins) and subjected to fluorescence-activated cell sorting (FACS) for purification and subculture.
Using our 2D cytokine scarce protocol, hPSC-derived functional RPE cells can be obtained within 2 months. Nevertheless, at this stage, most samples contain a percentage of non-RPE/early RPE progenitor cells that make them unsuitable for clinical application. We demonstrate that functional RPE cells express high levels of lipoprotein receptors and that this correlates with their ability to uptake lipoproteins. Combining photoreceptor uptake assay with lipoprotein uptake assay further confirms that only functional RPE cells uptake AcLDL. Incubation of mixed RPE/non-RPE cell cultures with fluorophore conjugated AcLDL and subsequent FACS-based isolation of labeled cells allows selective purification of mature functional RPE. When subcultured, DiI-AcLDL-labeled cells rapidly form pure homogenous high-quality RPE monolayers.
Pure functional RPE monolayers can be derived from hPSC within 90 days using simplified 2D cultures in conjunction with our RPE PLUS protocol (RPE Purification by Lipoprotein Uptake-based Sorting). The simplicity of this protocol makes it scalable, and the rapidity of production and purification allows for high-quality RPE to be produced in a short span of time making them ideally suited for downstream clinical and in vitro applications.
尽管需求不断增加，但目前用于人类多能干细胞（hPSC）衍生的视网膜色素上皮（RPE）的方案仍然耗时、费力且成本高昂。此外，缺乏有效的 RPE 选择性纯化方法和去除非 RPE 细胞杂质的方法，阻碍了临床质量 RPE 生产的扩大。我们旨在通过开发一种简化的 hPSC 衍生的 RPE 生产和纯化系统来解决这些挑战，该系统可在 90 天内产生高质量的 RPE 单层。
使用一种创新的、省时且经济高效的方案，完全依赖于 2D 培养和最小化细胞因子的使用，将人类多能干细胞分化为 RPE。一旦获得 RPE 特性，就将细胞转移到可渗透的膜上以获得成熟的 RPE 形态。通过电子显微镜、极化 VEGF 表达、建立高跨上皮电阻和光受体吞噬测定来验证 RPE 分化。在可渗透膜上培养 4 周后，用 Dil-AcLDL（DiI 缀合的乙酰化低密度脂蛋白）孵育 RPE 细胞培养物，并进行荧光激活细胞分选（FACS）以进行纯化和亚培养。
使用我们的 2D 细胞因子稀少方案，可在 2 个月内获得 hPSC 衍生的功能性 RPE 细胞。然而，在这个阶段，大多数样本中含有一定比例的非 RPE/早期 RPE 祖细胞，这使得它们不适合临床应用。我们证明功能性 RPE 细胞表达高水平的脂蛋白受体，这与其摄取脂蛋白的能力相关。将光受体摄取测定与脂蛋白摄取测定相结合，进一步证实只有功能性 RPE 细胞摄取 AcLDL。用荧光染料标记的 AcLDL 孵育混合的 RPE/非 RPE 细胞培养物，并随后进行基于 FACS 的标记细胞分离，可选择性地纯化成熟的功能性 RPE。当进行亚培养时，DiI-AcLDL 标记的细胞迅速形成纯同质的高质量 RPE 单层。
使用简化的 2D 培养并结合我们的 RPE PLUS 方案（基于脂蛋白摄取的分选的 RPE 纯化），可在 90 天内从 hPSC 中获得纯功能性 RPE 单层。该方案的简单性使其具有可扩展性，并且生产和纯化的快速性允许在短时间内产生高质量的 RPE，使其非常适合下游临床和体外应用。
