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

Gerbin, Kaytlyn A.; Grancharova, Tanya; Donovan-Maiye, Rory; Hendershott, Melissa C.; Brown, Jackson M.; Dinh, Stephanie Q.; Gehring, Jamie L.; Hirano, Matthew; Johnson, Gregory R.; Nath, Aditya; Nelson, Angelique M.; Roco, Charles M.; Rosenberg, Alexander; Sluzewski, M.; Viana, Matheus P.; Yan, Calysta; Zaunbrecher, Rebecca J.; Metzler, Kimberly R. Cordes; Menon, Vilas; Palecek, Sean P.; Seelig, Georg; Gaudreault, Nathalie; Knijnenburg, Theo; Rafelski, Susanne M.; Theriot, Julie A.; Gunawardane, Ruwanthi N.

doi:10.1101/2020.05.26.081083

Cited by 15 publications

(22 citation statements)

References 134 publications

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“…In our previous study, we found that, similar to mouse PSC-CMs, human PSC-CMs appear to have a maturation state similar to fetal CMs and appear arrested at the onset of perinatal maturation (5). Here, we focused on four published datasets of CMs generated from induced PSCs - Friedman, Nguyen, and Lukowski et al (38) (FNL); Churko et al (39) (C); Gerbin, Grancharova, Donovan-Maye, and Hendershott et al (40) (GGDH); and Ruan and Liao et al (41) (RL). We initially aimed to perform trajectory reconstruction with Monocle 3 as done with the mouse PSC-CMs.…”

Section: Resultsmentioning

confidence: 99%

Trajectory reconstruction identifies dysregulation of perinatal maturation programs in pluripotent stem cell-derived cardiomyocytes

Kannan¹,

Miyamoto

Lin

et al. 2021

Preprint

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A primary limitation in the clinical application of pluripotent stem cell derived cardiomyocytes (PSC-CMs) is the failure of these cells to achieve full functional maturity. In vivo, cardiomyocytes undergo numerous adaptive changes during perinatal maturation. By contrast, PSC-CMs fail to fully undergo these developmental processes, instead remaining arrested at an embryonic stage of maturation. To date, however, the precise mechanisms by which directed differentiation differs from endogenous development, leading to consequent PSC-CM maturation arrest, are unknown. The advent of single cell RNA-sequencing (scRNA-seq) has offered great opportunities for studying CM maturation at single cell resolution. However, perinatal cardiac scRNA-seq has been limited owing to technical difficulties in the isolation of single CMs. Here, we used our previously developed large particle fluorescence-activated cell sorting approach to generate an scRNA-seq reference of mouse in vivo CM maturation with extensive sampling of perinatal time periods. We subsequently generated isogenic embryonic stem cells and created an in vitro scRNA-seq reference of PSC-CM directed differentiation. Through trajectory reconstruction methods, we identified a perinatal maturation program in endogenous CMs that is poorly recapitulated in vitro. By comparison of our trajectories with previously published human datasets, we identified a network of nine transcription factors (TFs) whose targets are consistently dysregulated in PSC-CMs across species. Notably, we demonstrated that these TFs are only partially activated in common ex vivo approaches to engineer PSC-CM maturation. Our study represents the first direct comparison of CM maturation in vivo and in vitro at the single cell level, and can be leveraged towards improving the clinical viability of PSC-CMs.Significance StatementThere is a significant clinical need to generate mature cardiomyocytes from pluripotent stem cells. However, to date, most differentiation protocols yield phenotypically immature cardiomyocytes. The mechanisms underlying this poor maturation state are unknown. Here, we used single cell RNA-sequencing to compare cardiomyocyte maturation pathways in endogenous and pluripotent stem cell-derived cardiomyocytes. We found that in vitro, cardiomyocytes fail to undergo critical perinatal gene expression changes necessary for complete maturation. We found that key transcription factors regulating these changes are poorly expressed in vitro. Our study provides a better understanding of cardiomyocyte maturation both in vivo and in vitro, and may lead to improved approaches for engineering mature cardiomyocytes from stem cells.

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Section: Resultsmentioning

confidence: 99%

Trajectory reconstruction identifies dysregulation of perinatal maturation programs in pluripotent stem cell-derived cardiomyocytes

Kannan¹,

Miyamoto

Lin

et al. 2021

Preprint

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“…Our studies add a new dimension to these analyses by incorporating the spatial organization of cell structures, that is, where and when these parts come together in space and time to drive that function. Our approach relies on the well-established tight linkage between 3D cell organization and cell function, aspiring to use this to identify cell types and states from images, and relate them to single cell gene expression profiles (Gerbin et al, 2020). These approaches were built with live cell imaging in mind and thus are poised to incorporate dynamics.…”

Section: Discussionmentioning

confidence: 99%

“…These approaches were built with live cell imaging in mind and thus are poised to incorporate dynamics. Recently, studies combining quantitative measures of sarcomere organization with gene expression in the same individual cardiomyocytes demonstrates the importance of incorporating the spatial cell organization metrics for a more complete classification of cell states (Gerbin et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

Robust integrated intracellular organization of the human iPS cell: where, how much, and how variable

Viana¹,

Chen²,

Knijnenburg³

et al. 2020

Preprint

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SummaryDespite the intimate link between cell organization and function, the principles underlying intracellular organization and the relation between organization, gene expression and phenotype are not well understood. We address this by creating a benchmark for mean cell organization and the natural range of cell-to-cell variation. This benchmark can be used for comparison to other normal or abnormal cell states. To do this, we developed a reproducible microscope imaging pipeline to generate a high-quality dataset of 3D, high-resolution images of over 200,000 live cells from 25 isogenic human induced pluripotent stem cell (hiPSC) lines from the Allen Cell Collection. Each line contains one fluorescently tagged protein, created via endogenous CRISPR/Cas9 gene editing, representing a key cellular structure or organelle. We used these images to develop a new multi-part and generalizable analysis approach of the locations, amounts, and variation of these 25 cellular structures. Taking an integrated approach, we found that both the extent to which a structure’s individual location varied (“stereotypy”) and the extent to which the structure localized relative to all the other cellular structures (“concordance”) were robust to a wide range of cell shape variation, from flatter to taller, smaller to larger, or less to more polarized cells. We also found that these cellular structures varied greatly in how their volumes scaled with cell and nuclear size. These analyses create a data-driven set of quantitative rules for the locations, amounts, and variation of 25 cellular structures within the hiPSC as a normal baseline for cell organization.

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“…It should be noted, however, that we do not see entropy score as the end-all for maturation quantification. In addition to potential discrepancies between transcript and protein level expression (53), the mature cellular phenotype encompasses numerous functional parameters that may be only partially captured at the transcriptomic level (54). We envision entropy score as complementing existing celltype-specific functional assays to advance a more complete assessment of single cell maturation status.…”

Section: Discussionmentioning

confidence: 99%

Transcriptomic entropy benchmarks stem cell-derived cardiomyocyte maturation against endogenous tissue at single cell level

Kannan

Farid²,

Lin³

et al. 2020

Preprint

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While pluripotent stem cell-derived cardiomyocytes (PSC-CMs) offer tremendous potential for a range of clinical applications, their use has been constrained by the failure to mature these cells to a fully adult-like phenotype. Extensive efforts are currently underway with the goal to mature PSC-CMs. However, comprehensive metrics to benchmark the maturation status and trajectory of PSC-CMs have not been established. Here, we developed a novel approach to quantify CM maturation through single cell transcriptomic entropy. We found that transcriptomic entropy is robust across datasets regardless of differences in isolation protocols, library preparation methods, and other potential batch effects. We analyzed over 40 single cell RNA-sequencing (scRNA-seq) datasets and over 45,000 CMs to establish a cross-study, cross-species reference of CM maturation based on transcriptomic entropy. We subsequently computed the maturation status of PSC-CMs by direct comparison to in vivo development. Our study presents a robust, interpretable, and easy-to-use metric for quantifying CM maturation. cardiomyocyte | transcriptomic | single cell

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

Cited by 15 publications

References 134 publications

Trajectory reconstruction identifies dysregulation of perinatal maturation programs in pluripotent stem cell-derived cardiomyocytes

Trajectory reconstruction identifies dysregulation of perinatal maturation programs in pluripotent stem cell-derived cardiomyocytes

Robust integrated intracellular organization of the human iPS cell: where, how much, and how variable

Transcriptomic entropy benchmarks stem cell-derived cardiomyocyte maturation against endogenous tissue at single cell level

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