Multiplex live single-cell transcriptional analysis demarcates cellular functional heterogeneity

  1. Ayhan Atmanli  Is a corresponding author
  2. Dongjian Hu
  3. Frederik Ernst Deiman
  4. Annebel Marjolein van de Vrugt
  5. Lauren Deems Black
  6. Ibrahim Domian
  1. Massachusetts General Hospital, United States
  2. Tufts University, United States

Abstract

A fundamental goal in the biological sciences is to determine how individual cells with varied gene expression profiles and diverse functional characteristics contribute to development, physiology, and disease. Here, we report a novel strategy to assess gene expression and cell physiology in single living cells. Our approach utilizes fluorescently-labeled mRNA-specific anti-sense RNA probes and dsRNA-binding protein to identify the expression of specific genes in real-time at single-cell resolution via FRET. We use this technology to identify distinct myocardial subpopulations expressing the structural proteins myosin heavy chain α and myosin light chain 2a in real-time during early differentiation of human pluripotent stem cells. We combine this live-cell gene expression analysis with detailed physiologic phenotyping to capture the functional evolution of these early myocardial subpopulations during lineage specification and diversification. This live-cell mRNA imaging approach will have wide ranging application wherever heterogeneity plays an important biological role.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Ayhan Atmanli

    Cardiovascular Research Center, Massachusetts General Hospital, Boston, United States
    For correspondence
    ayhan.atmanli@gmail.com
    Competing interests
    Ayhan Atmanli, Inventor of pending patent (PCT/US2016/029972).
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6951-8893
  2. Dongjian Hu

    Cardiovascular Research Center, Massachusetts General Hospital, Boston, United States
    Competing interests
    No competing interests declared.
  3. Frederik Ernst Deiman

    Cardiovascular Research Center, Massachusetts General Hospital, Boston, United States
    Competing interests
    No competing interests declared.
  4. Annebel Marjolein van de Vrugt

    Cardiovascular Research Center, Massachusetts General Hospital, Boston, United States
    Competing interests
    No competing interests declared.
  5. Lauren Deems Black

    Department of Biomedical Engineering, Tufts University, Medford, United States
    Competing interests
    No competing interests declared.
  6. Ibrahim Domian

    Cardiovascular Research Center, Massachusetts General Hospital, Boston, United States
    Competing interests
    Ibrahim Domian, Inventor of a pending patent (PCT/US2016/029972).

Funding

American Heart Association (Predoctoral Fellowship)

  • Ayhan Atmanli

National Heart, Lung, and Blood Institute (Progenitor Cell Biology Consortium (PCBC) Jump Start Award)

  • Ayhan Atmanli

National Heart, Lung, and Blood Institute (U01HL100408-01)

  • Ibrahim Domian

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Sean Wu, Stanford

Publication history

  1. Received: June 23, 2019
  2. Accepted: October 7, 2019
  3. Accepted Manuscript published: October 8, 2019 (version 1)
  4. Version of Record published: November 18, 2019 (version 2)

Copyright

© 2019, Atmanli et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,142
    Page views
  • 265
    Downloads
  • 5
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Ayhan Atmanli
  2. Dongjian Hu
  3. Frederik Ernst Deiman
  4. Annebel Marjolein van de Vrugt
  5. Lauren Deems Black
  6. Ibrahim Domian
(2019)
Multiplex live single-cell transcriptional analysis demarcates cellular functional heterogeneity
eLife 8:e49599.
https://doi.org/10.7554/eLife.49599

Further reading

    1. Cell Biology
    2. Developmental Biology
    Juan Lu et al.
    Research Article Updated

    Phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate (PIP2) are key phosphoinositides that determine the identity of the plasma membrane (PM) and regulate numerous key biological events there. To date, mechanisms regulating the homeostasis and dynamic turnover of PM PI4P and PIP2 in response to various physiological conditions and stresses remain to be fully elucidated. Here, we report that hypoxia in Drosophila induces acute and reversible depletion of PM PI4P and PIP2 that severely disrupts the electrostatic PM targeting of multiple polybasic polarity proteins. Genetically encoded ATP sensors confirmed that hypoxia induces acute and reversible reduction of cellular ATP levels which showed a strong real-time correlation with the levels of PM PI4P and PIP2 in cultured cells. By combining genetic manipulations with quantitative imaging assays we showed that PI4KIIIα, as well as Rbo/EFR3 and TTC7 that are essential for targeting PI4KIIIα to PM, are required for maintaining the homeostasis and dynamic turnover of PM PI4P and PIP2 under normoxia and hypoxia. Our results revealed that in cells challenged by energetic stresses triggered by hypoxia, ATP inhibition and possibly ischemia, dramatic turnover of PM PI4P and PIP2 could have profound impact on many cellular processes including electrostatic PM targeting of numerous polybasic proteins.

    1. Cell Biology
    2. Medicine
    Eric N Jimenez-Vazquez et al.
    Research Article

    Background:

    Patients with cardiomyopathy of Duchenne Muscular Dystrophy (DMD) are at risk of developing life-threatening arrhythmias, but the mechanisms are unknown. We aimed to determine the role of ion channels controlling cardiac excitability in the mechanisms of arrhythmias in DMD patients.

    Methods:

    To test whether dystrophin mutations lead to defective cardiac NaV1.5–Kir2.1 channelosomes and arrhythmias, we generated iPSC-CMs from two hemizygous DMD males, a heterozygous female, and two unrelated control males. We conducted studies including confocal microscopy, protein expression analysis, patch-clamping, non-viral piggy-bac gene expression, optical mapping and contractility assays.

    Results:

    Two patients had abnormal ECGs with frequent runs of ventricular tachycardia. iPSC-CMs from all DMD patients showed abnormal action potential profiles, slowed conduction velocities, and reduced sodium (INa) and inward rectifier potassium (IK1) currents. Membrane NaV1.5 and Kir2.1 protein levels were reduced in hemizygous DMD iPSC-CMs but not in heterozygous iPSC-CMs. Remarkably, transfecting just one component of the dystrophin protein complex (α1-syntrophin) in hemizygous iPSC-CMs from one patient restored channelosome function, INa and IK1 densities, and action potential profile in single cells. In addition, α1-syntrophin expression restored impulse conduction and contractility and prevented reentrant arrhythmias in hiPSC-CM monolayers.

    Conclusions:

    We provide the first demonstration that iPSC-CMs reprogrammed from skin fibroblasts of DMD patients with cardiomyopathy have a dysfunction of the NaV1.5–Kir2.1 channelosome, with consequent reduction of cardiac excitability and conduction. Altogether, iPSC-CMs from patients with DMD cardiomyopathy have a NaV1.5–Kir2.1 channelosome dysfunction, which can be rescued by the scaffolding protein α1-syntrophin to restore excitability and prevent arrhythmias.

    Funding:

    Supported by National Institutes of Health R01 HL122352 grant; ‘la Caixa’ Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH [2019039]; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant [2013032] to DM and OB.