hPSC-derived Photoreceptor Synaptic Puncta and Synaptic Tracing Datasets and Code

Published: 5 January 2023| Version 1 | DOI: 10.17632/ncmn2msdhx.1
Allison Ludwig


Data used for analysis of synaptic puncta formation and synaptic tracing of human pluripotent stem cell (hPSC)-derived photoreceptors (PRs). This data is deposited as supplemental material for a manuscript by Allison L. Ludwig, Steven J. Mayerl, Yu Gao, Mark Banghart, Cole Bacig, Maria A. Fernandez Zepeda, Xinyu Zhao, and David M. Gamm, entitled “Re-formation of synaptic connectivity in dissociated human stem cell-derived retinal organoid cultures.” Please see associated manuscript for further details. Summary: These data are the result of manual and automated cell counts from confocal and epifluorescence microscopy experiments using dissociated hPSC-PRs differentiated from the WA09 wild type stem cell line to explore synaptic re-connection following dissociation of early retinal organoids. The included files consist of raw cell count data (.csv files) and R code (.Rmd files) used for analysis of synaptic puncta formation and synaptic tracing of hPSC-PRs in the days following dissociation from retinal organoids. The results of these experiments demonstrate detection of synaptic puncta within hPSC-PRs as early as 2 days after dissociation, with a stepwise increase in the proportion of hPSC-PRs with organized synaptic puncta on days 5 and 20 post-dissociation. Moreover, we observed consistent and measurable re-formation of synaptic connections after dissociation from retinal organoids, as demonstrated by monosynaptic retrograde rabies viral tracing, even after controlling for biomaterial transfer. These results demonstrate plasticity of hPSC-PRs and further underscores their potential to restore visual circuits.


Steps to reproduce

Original image data was collected by confocal and epifluorescence microscopy. Please see associated manuscript for detailed instructions regarding cell culture, dissociation, synaptic tracing, immunocytochemistry, and image analysis. Cell populations of interest were counted manually and/or quantified using a standardized automated high-content imaging workflow with the Operetta high content imaging system. Raw data from these counts are included in the attached .csv files, which were imported into R software for further analysis. R code used for statistical analyses is included in the attached .Rmd files; code for corresponding plots to visualize statistical analyses is also included. Descriptive statistics and corresponding bar graphs were performed by importing the attached .csv files into GraphPad Prism software.


University of Wisconsin Madison


Stem Cell, Synapse Formation, Synaptic Plasticity, Photoreceptor, Retina Synapse, Synaptogenesis, Organoid