The availability of in vitro models of the human retina in which to perform pharmacological and toxicological studies is an urgent and unmet need. An essential step for developing in vitro models of human retina is the ability to generate laminated, physiologically functional, and light-responsive retinal organoids from renewable and patient specific sources. We investigated five different human-induced pluripotent stem cell (iPSC) lines and showed a significant variability in their efficiency to generate retinal organoids. Despite this variability, by month 5 of differentiation, all iPSC-derived retinal organoids were able to generate light responses, albeit immature, comparable to the earliest light responses recorded from the neonatal mouse retina, close to the period of eye opening. All iPSC-derived retinal organoids exhibited at this time a well-formed outer nuclear like layer containing photoreceptors with inner segments, connecting cilium, and outer like segments. The differentiation process was highly dependent on seeding cell density and nutrient availability determined by factorial experimental design. We adopted the differentiation protocol to a multiwell plate format, which enhanced generation of retinal organoids with retinal-pigmented epithelium (RPE) and improved ganglion cell development and the response to physiological stimuli. We tested the response of iPSC-derived retinal organoids to Moxifloxacin and showed that similarly to in vivo adult mouse retina, the primary affected cell types were photoreceptors. Together our data indicate that light responsive retinal organoids derived from carefully selected and differentiation efficient iPSC lines can be generated at the scale needed for pharmacology and drug screening purposes. Stem Cells 2018;36:1535-1551.

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Large-scale manufacture of human embryonic stem cells (hESCs) is prerequisite to their widespread use in biomedical applications. However, current hESC culture strategies are labor-intensive and employ highly variable processes, presenting challenges for scaled production and commercial development. Here we demonstrate that passaging of the hESC lines, HUES7, and NOTT1, with trypsin in feeder-free conditions, is compatible with complete automation on the CompacT SelecT, a commercially available and industrially relevant robotic platform. Pluripotency was successfully retained, as evidenced by consistent proliferation during serial passage, expression of stem cell markers (OCT4, NANOG, TRA1-81, and SSEA-4), stable karyotype, and multi-germlayer differentiation in vitro, including to pharmacologically responsive cardiomyocytes. Automation of hESC culture will expedite cell-use in clinical, scientific, and industrial applications.

In this study, we demonstrate the ability of a three-dimensional co-culture model to preserve some key aspects of differentiated hepatocyte function in vitro. Freshly isolated rat hepatocytes in co-culture with activated stellate cells rapidly aggregate to form well-defined viable spheroids. After 5 days in culture, the spheroids have a complex extracellular matrix support and hepatic ultrastructure including bile canaliculi, tight junctions, desmosomes and lipid storage. Co-culture spheroids have superior cytochrome P₄₅₀ (CYP₄₅₀) 3A and 2B function, and increased inducibility of 2B function, relative to a range of hepatocyte monoculture techniques (high-performance liquid chromatography of testosterone metabolites). Increased function in co-culture is supported by greater expression of CYP₄₅₀ 3A23, 1A2, and 2E1 mRNA relative to monoculture (reverse transcriptase quantitative polymerase chain reaction). Also, high hepatocyte growth factor mRNA expression in co-culture suggests a post-traumatic, or possibly regenerative, environment. A preliminary study of human hepatocytes co-cultured with rat stellate cells demonstrated prolonged function of CYP₄₅₀ 3A4, 2C19 and 2C9. This study shows that stellate cells facilitate spheroid formation, influence spheroid architecture, and are an effective method of preserving some aspects of hepatocyte function in the early stage of culture.