Human induced pluripotent stem cells generate light responsive retinal organoids with variable and nutrient dependent efficiency

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. © AlphaMed Press 2018

(a) Confocal fluorescence microscopy images of nonfixed spindles assembled either in the absence or additional presence of p50 or cc1 showing the localization of Eg5

The fluorescence of Eg5-paGFP (green) and of Cy5 microtubules (MT; red) was measured immediately after photoactivation of Eg5-paGFP in the entire spindle. (b) Fluorescence intensity profiles for photoactivated Eg5-paGFP and Cy5 microtubules along the spindle axis for the spindles shown in section a. (right) Mean ratios of the total fluorescence intensity of photoactivated Eg5-paGFP divided by the total fluorescence intensity of Cy5 microtubules (MT) for wild-type spindles, p50 spindles, and cc1 spindles are shown. The means were determined from five spindles per condition. The wild-type intensity ratio was set to 100. Error bars indicate standard deviation. (c) Confocal fluorescence microscopy images of fixed spindles assembled either in the absence (top) or presence (bottom) of cc1 showing the localization of dynein. Dynein heavy chain was detected by immunofluorescence (green), tubulin by using incorporated Alexa 568–tubulin (red), and DNA by Hoechst staining (blue). (d) Western blot analysis showing pull-down of p150 with Eg5 on anti-Eg5 beads and pull-down of Eg5 with p150 on anti-p150 beads in egg extract, either in the absence (−cc1) or presence (+cc1) of added p150 fragment cc1. Mock represents magnetic beads coated with an irrelevant antibody also incubated in egg extract. An anti-p150 antibody was used for detection in the top left three lanes and in the bottom right three lanes, whereas an anti-Eg5 antibody was used for detection in the top right three lanes and in the bottom left three lanes. All samples in a horizontal row were run on the same SDS gel. Molecular weight markers to the left of the blots are in kD.<p><b>Copyright information:</b></p><p>Taken from "Poleward transport of Eg5 by dynein–dynactin in egg extract spindles"</p><p></p><p>The Journal of Cell Biology 2008;182(4):715-726.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518710.</p><p></p

Time-lapse videos of a confocal section through spindles containing Cy5 microtubules and photoactivatable Eg5-paGFP were recorded simultaneously in the Cy5 and GFP channel of a confocal fluorescence microscope

Between the first and the second image of a time series, one or several rectangular stripes in the confocal section were bleached and simultaneously photoactivated. To convert the images of the two time series into fluorescence intensity profiles, the area of the spindle in the Cy5 microtubule image before the photobleach was determined by applying an intensity threshold to the image (I). This area served then as a mask for the entire time series of the Cy5 microtubule images and the Eg5-paGFP (illustrated here only for the second frame of the time series). The intensity values were projected onto the spindle axis. This created fluorescence intensity profiles (II and V). The turnover of microtubules and of Eg5 was extracted from time series of these intensity profiles (II and V; see Materials and methods). To determine the exact position of the maximum amount of bleached microtubules, the Cy5 intensity profiles were subtracted from the prebleach profile, resulting in an inverted intensity difference profile (III). The positions of the maximum of the intensity difference profiles of Cy5 microtubules and of the intensity profiles of photoactivated Eg5-paGFP were determined from a Gaussian fit (blue) to the profiles (IV and VI) and used to calculate the velocity of the movements of the peaks (see Materials and methods).<p><b>Copyright information:</b></p><p>Taken from "Poleward transport of Eg5 by dynein–dynactin in egg extract spindles"</p><p></p><p>The Journal of Cell Biology 2008;182(4):715-726.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518710.</p><p></p

(a) Spindles in egg extract depleted from endogenous Eg5 and supplemented with Eg5-paGFP and Cy5-tubulin before and after photoactivation of Eg5-paGFP and simultaneous photobleaching of Cy5 microtubules in two different spindle regions

Time series of confocal fluorescence microscopy images of Eg5-paGFP (green) and Cy5 microtubules (MT; red) at the indicated times, and fluorescence intensity profiles () for Cy5 microtubules and Eg5-paGFP of the same spindle at the indicated times. (b) Intensity difference profiles for bleached Cy5 microtubules and intensity profiles for photoactivated Eg5-paGFP at the indicated times. Only parts of entire profiles are shown for the midzone, halfzone, and pole region. Intensity difference profiles for Cy5 microtubules appear inverted as compared with the intensity profiles (). Note the splitting of the initial single peak of the red microtubule profile in the midzone into two peaks of the blue microtubule profile after 56 s. A corresponding split is not observed in the Eg5 profiles. The intensity profile for the halfzone is derived from the spindle shown in section a, whereas the intensity profiles for the midzone and pole are derived from spindles not shown because of different imaging requirements for the different regions in the spindle. (c) Displacements of the peaks of photoactivated Eg5-paGFP (green) and of photobleached Cy5 microtubules (red) along the spindle axis with time. Linear regression fits (lines) to the experimental values (dots) yielded the velocities as indicated. (d, left) Box plots of the speeds of Eg5 movement (22 measurements in 19 spindles) and of microtubule flux (MT; 16 measurements). (right) Scatter diagram of the speeds of microtubule (MT) flux as a function of the speeds of Eg5 in the halfzone. Each data point represents two simultaneous measurements in the same spindle region.<p><b>Copyright information:</b></p><p>Taken from "Poleward transport of Eg5 by dynein–dynactin in egg extract spindles"</p><p></p><p>The Journal of Cell Biology 2008;182(4):715-726.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518710.</p><p></p

(a) Fluorescence decay of photoactivated Eg5-paGFP (green) and fluorescence recovery of Cy5 microtubules (red) in the midzone, the halfzone, and the pole region within the first 2 min after photoactivation and photobleaching

A monoexponential fit (lines) to the experimental values (dots) yielded half-lives as indicated. (b) Box plots of half-lives and residuals of fluorescence decays of photoactivated Eg5-paGFP in the three different spindle regions. 4–12 measurements were made per region.<p><b>Copyright information:</b></p><p>Taken from "Poleward transport of Eg5 by dynein–dynactin in egg extract spindles"</p><p></p><p>The Journal of Cell Biology 2008;182(4):715-726.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518710.</p><p></p