Quantification of Retinogenesis in 3D Cultures Reveals Epigenetic Memory and Higher Efficiency in iPSCs Derived from Rod Photoreceptors

Abstract

Summary Cell-based therapies to treat retinal degeneration are now being tested in clinical trials. However, it is not known if the source of stem cells is important for the production of differentiated cells suitable for transplantation. To test this, we generated iPSCs murine rod photoreceptors (r-iPSCs) and scored their ability to make retina using a standardized quantitative protocol called STEM-RET. We discovered that r-iPSCs were more efficient at producing differentiated retina than embryonic stem cells (ESCs) or fibroblast-derived iPSCs (f-iPSCs). Retinae derived from f-iPSCs had a reduction in amacrine cells and other inner nuclear layer cells. Integrated epigenetic analysis showed that DNA methylation contributes to the defects in f-iPSC retinogenesis and that rod specific CTCF insulator protein binding sites may promote retinogenes in r-iPSCs. Taken together, our data suggest that the source of stem cells are important for producing retinal neurons in 3D organ cultures.

Lessons from Retinoblastoma: Implications for Cancer, Development, Evolution, and Regenerative Medicine

“…To compare cellular pliancy across neuronal classes at different stages of development, a system for cellular reprogramming was developed based on the rationale that the major barrier to cellular reprogramming involves epigenetic and organizational changes in the epigenome, which significantly impact cellular pliancy [61]. Therefore, the model is based on the premise that cells with low pliancy would be more difficult to reprogram than cells with high pliancy.…”

“…Therefore, the model is based on the premise that cells with low pliancy would be more difficult to reprogram than cells with high pliancy. For quantitative cellular reprogramming of individual neuronal classes, a series of genetically engineered mouse strains were developed which harbored a doxycycline-inducible cassette containing 4 reprogramming Yamanaka factors (Oct3/4, Myc, Klf4, and Sox2) and cell type–specific GFP reporter transgenes [61]. Individual retinal neuron types were subsequently purified by fluorescence activated cell sorting, and reprogramming was induced by adding doxycycline.…”

“…Individual retinal neuron types were subsequently purified by fluorescence activated cell sorting, and reprogramming was induced by adding doxycycline. Reprogramming was carried out in mosaic pellets (a mixture of wild type retinal cells and reprogrammable neurons) to ensure that neurons could survive for the 10–14 days when undergoing reprogramming, after which individual induced pluripotent stem cells (iPSCs) were cloned and characterized [61]. By performing the initial reprogramming and subsequent plating in limiting dilution assays, the efficiency of reprogramming could be quantitated.…”

“…Interestingly, cells with low pliancy that were more difficult to reprogram were more likely to retain epigenetic memory as measured by their ability to produce retina [61]. As shown in Figure 5, we have developed a quantitative system called ‘Standard Transmission Electron Microscopy – Retinae’ (STEM-RET) for scoring retinal differentiation from stem cells (SC), which is critical for comparing across different stem cell lines [61].…”

Generating minicorneal organoids from human induced pluripotent stem cells

“…This method exploits the inherent self-organizing capacity of differentiating progenitor cell populations, together with the surrounding niche cells, to generate complex tissue structures in vitro. This has been demonstrated successfully with the generation of neuroretinal tissues using PSCs (Eiraku et al, 2011;Gonzalez-Cordero et al, 2013;Assawachananont et al, 2014;Reichman et al, 2014;Zhong et al, 2014;Hiler et al, 2015;Kaewkhaw et al, 2015;Völkner et al, 2016). A recent report has described a method of generating immature corneal organoids from human iPSCs and shown them to express a few corneal markers (Foster et al, 2017).…”