Cell states beyond transcriptomics: Integrating structural organization and gene expression in hiPSC-derived cardiomyocytes

Abstract

Highlights d Automated image-based classification of subcellular organization in single cells d Integrated analysis of gene expression and cell structure to classify cell state d Curated dataset containing over 30,000 hiPSC-derived cardiomyocytes d Open-source images, analysis code, and quantitative tools

A comprehensive analysis of gene expression changes in a high replicate and open-source dataset of differentiating hiPSC-derived cardiomyocytes

“…Notably, this analysis revealed that gene sets as small as two to twelve genes enabled prediction of time point with high accuracy, similar to the accuracy achieved by using over 1000 of the most highly variable genes. These data indicate that a small subset of carefully-chosen gene targets may be informative for downstream studies where gene set size is limited, such as in functional knock-out assays, in vivo experiments, or image-based RNA FISH studies 71 . In summary, we used scRNA-seq to profile gene expression in cardiomyocytes and non-cardiomyocytes during cardiomyocyte differentiation and extended culture in vitro.…”

“…Two differentiation protocols were used, referred to as “Protocol 1” and “Protocol 2” in the text. Protocol 1 is based on a previously reported small molecule differentiation method with modifications 75 , 76 as described in Gerbin et al 71 . HiPSC cultures were dissociated into single cells using Accutase and plated into matrigel-coated 6 well-plates at 125–350 k cells per well in mTeSR1 supplemented with 1% P/S and 10 µM Rock Inhibitor (Day -3).…”

“…Gene validation by RNA FISH was performed using the HCR v3.0 method, following the HCR v3.0 protocol for “Mammalian cells on a slide” with modifications to adapt for samples on glass bottom multiwell plates (Molecular Instruments, www.molecularinstruments.com/hcr-rnafish-protocols ), as previously described 71 . Fixed wells were washed 4 times with 500 µL 2X SSC (Invitrogen #15557-044), incubated for 30–60 min at 37 °C in probe hybridization buffer (Molecular Instruments), and hybridized overnight with 1.2 pmol of each probe set mixture containing 400 U/mL RNAse inhibitor (Enzymatics #Y9240L) at 37 °C (250 µL per well).…”

“…molec ulari nstru ments. com/ hcr-rnafi sh-proto cols), as previously described 71 Manual cell annotations in Napari. Multi-channel Z-stacks were loaded into Napari (https:// napari.…”

Advances in spatial transcriptomic data analysis

“…Owing to the rapid development of ST technologies, it is now possible to perform both transcriptomic profiling and morphology analyses for the same cells, thereby providing a great opportunity to systematically investigate the relationship between these two fundamentally different approaches. A few methods have recently been developed that focus on integration of both modalities (He et al 2020;Tan et al 2020;Gerbin et al 2021). Although not directly related to spatial transcriptomics, an interesting finding from living imaging analysis indicates that changes in morphology might even predict cell fate or state before this can be observed in the transcriptomic output (Buggenthin et al 2017).…”