Automated Hypothesis Generation to Identify Signals Relevant in the Development of Mammalian Cell and Tissue Bioprocesses, With Validation in a Retinal Culture System

Abstract

We have developed an accessible software tool (receptoR) to predict potentially active signaling pathways in one or more cell type(s) of interest from publicly available transcriptome data. As proof-of-concept, we applied it to mouse photoreceptors, yielding the previously untested hypothesis that activin signaling pathways are active in these cells. Expression of the type 2 activin receptor (Acvr2a) was experimentally confirmed by both RT-qPCR and immunochemistry, and activation of this signaling pathway with recombinant activin A significantly enhanced the survival of magnetically sorted photoreceptors in culture. Taken together, we demonstrate that our approach can be easily used to mine publicly available transcriptome data and generate hypotheses around receptor expression that can be used to identify novel signaling pathways in specific cell types of interest. We anticipate that receptoR (available at https://www.ucalgary.ca/ungrinlab/receptoR) will enable more efficient use of limited research resources.

Scaffold-Free Retinal Pigment Epithelium Microtissues Exhibit Increased Release of PEDF

“…Similar to the ARPE-19 microtissue, cells in the hESC-derived RPE-µTs secrete 3X more PEDF ( p < 0.01) and less than 1% of VEGF ( p < 0.01) than standard adherent culture ( Figure 5 f). Based on these secreted factor profiles, we predicted that CM from RPE-µT cultures would increase photoreceptor survival, using a dissociated mouse retina assay [ 61 ]. After 48 hours, photoreceptor-enriched in vitro cultures demonstrated superior survival with RPE-µT CM compared to CM collected from monolayer cultures (1.41 ± 0.11-fold increase, p < 0.05) and this effect was exaggerated by 72 h with a nearly two-fold increase in cell survival, using RPE-µT CM ( p < 0.01; Figure 5 g).…”

“…Photoreceptors were isolated, enriched and cultured as previously described [ 61 ]. Briefly, mouse retinas were dissected from the eyes of postnatal day 4 CD1 mice obtained through a secondary-use protocol approved by the Animal Care Committee at the University of Calgary according to IACUCC standards.…”