Dataset Information

Semi-Automated Approach for Retinal Tissue Differentiation.

ABSTRACT: Purpose:Three-dimensional strategy for the differentiation of pluripotent stem cells to the retina has been widely used to study retinal development, although the cell production and drug discovery applications are limited by the throughput. Here we attempted to scale up the protocol using a semiautomated approach. Methods:For the experiments we used the Rx-GFP mouse embryonic stem cell (mES) reporter cell line, specific for early retinal development and human embryonic stem cell line Brn3b-tdTomato, specific for retinal ganglion cells. To increase the throughput, we implemented automated media exchange using Thermo WellWash Versa with Thermo RapidStack robot. To analyze the rate of retinal differentiation in mouse stem-cell derived organoids we imaged the plates at day 10 of differentiation using Life Technologies EVOS Fl Auto. The automated image analysis of fluorescent images was performed with custom Python OpenCV script. Results:The implementation of a semiautomated approach significantly reduced the operator time needed: 34 minutes versus two hours for 960 organoids over the course of 25 days without any change in differentiation pattern and quantity of retinal differentiation. Automated image analysis showed that Forskolin treatment starting from day 1 leads to a significant increase in retinal field induction efficiency. Conclusions:Semiautomated approach can be applied to retinal tissue differentiation to increase the throughput of the protocol. We demonstrated that automated image analysis can be used to evaluate differentiation efficiency, as well as for troubleshooting and to study factors affecting retinal differentiation. Translational Relevance:Using robotic approach reduces the risk of human error and allows to perform all cycle of cell production in enclosed conditions, which is critical for GMP cell manufacture.

SUBMITTER: Kegeles E

PROVIDER: S-EPMC7521179 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Semi-Automated Approach for Retinal Tissue Differentiation.

Kegeles Evgenii E Perepelkina Tatiana T Baranov Petr P

Translational vision science & technology 20200923 10

<h4>Purpose</h4>Three-dimensional strategy for the differentiation of pluripotent stem cells to the retina has been widely used to study retinal development, although the cell production and drug discovery applications are limited by the throughput. Here we attempted to scale up the protocol using a semiautomated approach.<h4>Methods</h4>For the experiments we used the Rx-GFP mouse embryonic stem cell (mES) reporter cell line, specific for early retinal development and human embryonic stem cell ...[more]

PMID: 33024617

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Project description:Retinal ganglion cell (RGC) loss is one of the earliest and most important cellular changes in glaucoma. The DARC (Detection of Apoptosing Retinal Cells) technology enables in vivo real-time non-invasive imaging of single apoptosing retinal cells in animal models of glaucoma and Alzheimer's disease. To date, apoptosing RGCs imaged using DARC have been counted manually. This is time-consuming, labour-intensive, vulnerable to bias, and has considerable inter- and intra-operator variability.A semi-automated algorithm was developed which enabled automated identification of apoptosing RGCs labeled with fluorescent Annexin-5 on DARC images. Automated analysis included a pre-processing stage involving local-luminance and local-contrast "gain control", a "blob analysis" step to differentiate between cells, vessels and noise, and a method to exclude non-cell structures using specific combined 'size' and 'aspect' ratio criteria. Apoptosing retinal cells were counted by 3 masked operators, generating 'Gold-standard' mean manual cell counts, and were also counted using the newly developed automated algorithm. Comparison between automated cell counts and the mean manual cell counts on 66 DARC images showed significant correlation between the two methods (Pearson's correlation coefficient 0.978 (p < 0.001), R Squared = 0.956. The Intraclass correlation coefficient was 0.986 (95% CI 0.977-0.991, p < 0.001), and Cronbach's alpha measure of consistency = 0.986, confirming excellent correlation and consistency. No significant difference (p = 0.922, 95% CI: -5.53 to 6.10) was detected between the cell counts of the two methods.The novel automated algorithm enabled accurate quantification of apoptosing RGCs that is highly comparable to manual counting, and appears to minimise operator-bias, whilst being both fast and reproducible. This may prove to be a valuable method of quantifying apoptosing retinal cells, with particular relevance to translation in the clinic, where a Phase I clinical trial of DARC in glaucoma patients is due to start shortly.

Project description:Purpose: To assess the accuracy and efficacy of a semi-automated deep learning algorithm (DLA) assisted approach to detect vision-threatening diabetic retinopathy (DR). Methods: We developed a two-step semi-automated DLA-assisted approach to grade fundus photographs for vision-threatening referable DR. Study images were obtained from the Lingtou Cohort Study, and captured at participant enrollment in 2009-2010 ("baseline images") and annual follow-up between 2011 and 2017. To begin, a validated DLA automatically graded baseline images for referable DR and classified them as positive, negative, or ungradable. Following, each positive image, all other available images from patients who had a positive image, and a 5% random sample of all negative images were selected and regraded by trained human graders. A reference standard diagnosis was assigned once all graders achieved consistent grading outcomes or with a senior ophthalmologist's final diagnosis. The semi-automated DLA assisted approach combined initial DLA screening and subsequent human grading for images identified as high-risk. This approach was further validated within the follow-up image datasets and its time and economic costs evaluated against fully human grading. Results: For evaluation of baseline images, a total of 33,115 images were included and automatically graded by the DLA. 2,604 images (480 positive results, 624 available other images from participants with a positive result, and 1500 random negative samples) were selected and regraded by graders. The DLA achieved an area under the receiver operating characteristic curve (AUC), sensitivity, specificity, and accuracy of 0.953, 0.970, 0.879, and 88.6%, respectively. In further validation within the follow-up image datasets, a total of 88,363 images were graded using this semi-automated approach and human grading was performed on 8975 selected images. The DLA achieved an AUC, sensitivity, and specificity of 0.914, 0.852, 0.853, respectively. Compared against fully human grading, the semi-automated DLA-assisted approach achieved an estimated 75.6% time and 90.1% economic cost saving. Conclusions: The DLA described in this study was able to achieve high accuracy, sensitivity, and specificity in grading fundus images for referable DR. Validated against long-term follow-up datasets, a semi-automated DLA-assisted approach was able to accurately identify suspect cases, and minimize misdiagnosis whilst balancing safety, time, and economic cost.

Dataset Information

Semi-Automated Approach for Retinal Tissue Differentiation.

Publications

Semi-Automated Approach for Retinal Tissue Differentiation.

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