Defined Culture of Human Embryonic Stem Cells and Xeno-Free Derivation of Retinal Pigmented Epithelial Cells on a Novel, Synthetic Substrate

Abstract

Age-related macular degeneration (AMD), a leading cause of blindness, is characterized by the death of the retinal pigmented epithelium (RPE), which is a monolayer posterior to the retina that supports the photoreceptors. Human embryonic stem cells (hESCs) can generate an unlimited source of RPE for cellular therapies, and clinical trials have been initiated. However, protocols for RPE derivation using defined conditions free of nonhuman derivatives (xeno-free) are preferred for clinical translation. This avoids exposing AMD patients to animal-derived products, which could incite an immune response. In this study, we investigated the maintenance of hESCs and their differentiation into RPE using Synthemax II-SC, which is a novel, synthetic animal-derived component-free, RGD peptide-containing copolymer compliant with good manufacturing practices designed for xenofree stem cell culture. Cells on Synthemax II-SC were compared with cultures grown with xenogeneic and xeno-free control substrates. This report demonstrates that Synthemax II-SC supports long-term culture of H9 and H14 hESC lines and permits efficient differentiation of hESCs into functional RPE. Expression of RPE-specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium-derived factor. Both hESCs and hESC-RPE maintained normal karyotypes after long-term culture on Synthemax II-SC. Furthermore, RPE generated on Synthemax II-SC are functional when seeded onto parylene-C scaffolds designed for clinical use. These experiments suggest that Synthemax II-SC is a suitable, defined substrate for hESC culture and the xeno-free derivation of RPE for cellular therapies. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:165-177

Neural commitment of human pluripotent stem cells under defined conditions recapitulates neural development and generates patient‐specific neural cells

“…Further differentiation into more mature cells was achieved within rosettes, which can be correlated with events leading to the neural tube formation in vivo [7,16]. As previously demonstrated in similar systems [19][20][21][22]38], peptide conjugates can support robust expansion and differentiation of human PSCs under defined conditions, and in our case VTN supported neural commitment of human PSCs in conjugation with N2B27 medium supplemented with SMAD inhibitors. …”

Robust Differentiation of mRNA-Reprogrammed Human Induced Pluripotent Stem Cells Toward a Retinal Lineage

“…In the near future, it will also be necessary to demonstrate additional levels of safety before the widespread application of hiPSCs for translational purposes. Recent efforts have focused on the elimination of xenogeneic components from media used to maintain and differentiate hiPSCs [16, 62, 76, 77], and these approaches will need to be combined with appropriate reprogramming strategies, such as mRNA reprogramming.…”

An Innovative Surgical Technique for Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigmented Epithelium in Yucatan Mini Pigs: Preliminary Results

“…The advantages of the surgically foldable MSPS carrier include customized macular shape in diameter and thickness [8], ultrathin parts with diffusion [7], and excellent RPE adherence [10,11] and survival [12], whereas its nondegradability and unknown long-term effects can be seen as disadvantages. The study criteria for successful implantation were appropriate placement of the implant in the subretinal space, as assessed by OCT and post-mortem histology.…”

“…Secondpassage hESC-RPE cells were cryopreserved as an intermediate cell bank. These cultures have been extensively characterized and are 99 % positive for RPE markers [10].…”