RESUMO
Dental pulp stem cells (DPSCs) are an easily accessible, heterogenous source of mesenchymal stem cells (MSCs) that are derived from the neural crest. Evidence suggests that they have neurotrophic qualities in their undifferentiated state and can also be differentiated into neuronal and retinal cell types. There is growing interest in using DPSCs in cell-based therapies to treat glaucoma and blinding retinal diseases. However, careful characterization of these cells is necessary as direct intravitreal and subretinal MSC transplantation is known to lead to deleterious glial reaction and fibrosis. In this study, we provide evidence for the mesenchymal-predominant nature of DPSCs and show that DPSCs maintain their mesenchymal phenotype despite upregulating mature retinal markers under retinal differentiation conditions. CD56, which was previously thought to be a specific marker of neural crest lineage, is robustly co-expressed with mesenchymal markers and may not be adequate for isolating a subpopulation of neural crest cells in DPSCs. Therefore, identification of more specific markers is required to elucidate the heterogeneity of the population and to successfully isolate a putative neural stem cell population before DPSCs can be used for retinal therapy.
RESUMO
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
