Single-cell protein expression of hiPSC-derived cardiomyocytes using Single-Cell Westerns

https://doi.org/10.1016/j.yjmcc.2020.09.012Get rights and content

Highlights

  • Single-Cell Westerns allow protein quantification of cardiomyocytes at single-cell resolution.

  • Single-Cell Westerns can detect and resolve off-target antibody binding.

  • A subpopulation of hiPSC-CMs expresses MLC2A followed by co-expression of MLC2A and MLC2V.

Abstract

The ability to reprogram human somatic cells into human induced pluripotent stem cells (hiPSCs) has enabled researchers to generate cell types in vitro that have the potential to faithfully recapitulate patient-specific disease processes and phenotypes. hiPSC-derived cardiomyocytes (hiPSC-CMs) offer the promise of in vitro patient- and disease-specific models for drug testing and the discovery of novel therapeutic approaches for treating cardiovascular diseases. While methods to differentiate hiPSCs into cardiomyocytes have been demonstrated, the heterogeneity and immaturity of these differentiated populations have restricted their potential in reproducing human disease and the associated target cell phenotypes. These barriers may be overcome through comprehensive single-cell characterization to dissect the rich heterogeneity of hiPSC-CMs and to study the source of varying cell fates. In this study, we optimized and validated a new Single-Cell Western method to assess protein expression in hiPSC-CMs. To better understand distinct subpopulations generated from cardiomyocyte differentiations and to track populations at single-cell resolution over time, we measured and quantified the expression of cardiomyocyte subtype-specific proteins (MLC2V and MLC2A) using Single-Cell Westerns. By understanding their heterogeneity through single-cell protein expression and quantification, we may improve upon current cardiomyocyte differentiation protocols, generate hiPSC-CMs that are more representative of in vivo derived cardiomyocytes for disease modeling, and utilize hiPSC-CMs for regenerative medicine purposes. Single-Cell Westerns provide a robust platform for protein expression analysis at single-cell resolution.

Introduction

As the leading global cause of death, cardiovascular disease (CVD) is estimated to result in 17.9 million deaths each year [1] – placing a heavy weight on the need for therapeutics that not only delay disease progression but also improve heart function and prevent failure. Current pharmaceutical, interventional, and/or surgical approaches may improve clinical outcomes but are limited in their success due to their inability to promote heart tissue repair and regeneration [2].

The irreversible loss of a massive number of cardiomyocytes (CMs) brought about by injury and disease leads to an inevitable and fatal decline in heart function and disease progression [3,4]. However the advent of human induced pluripotent stem cell (hiPSC) technology and subsequent ability to differentiate and establish CMs in culture, has created the possibility for the achievement of human heart regenerative therapies [5,6]. As a continuous and biologically relevant source of differentiated CMs, hiPSC-CMs are a valuable tool in the cardiovascular research community for not only treating CVD, but modeling human heart development and disease, investigating underlying mechanisms, and screening novel drugs for efficacy and cardiotoxicity [[7], [8], [9]]. However, since hiPSC-CMs are composed of distinct cell subpopulations that are heterogenous, immature, express fetal gene expression profiles, and demonstrate reduced contractile force generation compared to adult cardiomyocytes, the accuracy and utility of hiPSC-CM disease models remain limited [7,8,[10], [11], [12], [13], [14], [15], [16], [17]]. Furthermore, as hiPSC-CMs mature and as protein expression dynamics fluctuate, bulk sample analysis becomes insufficient in resolution. Because of their recognized heterogeneity resulting in ventricular-like, atrial-like, and nodal-like subpopulations [17,18], hiPSC-CMs need to be rigorously characterized and screened for maturity, identity, and function. To that end, various single-cell modalities are already being performed to understand this heterogeneity [19].

While sequencing technologies have advanced in throughput and scale to analyze single-cell transcriptomic and genomic information, the demand for quantitative, single-cell proteomic techniques have been difficult to meet due to the very low amount of protein present in any one cell. Moreover, the proteome's complexity and wide concentration range (fM to high nM) present additional challenges [20]. To meaningfully adapt biochemical protein characterization assays on a single-cell level, the incorporation of high sensitivity tools is essential. The Single-Cell Western (scWestern) blot system, Milo from ProteinSimple, is a microfluidic-based lab-on-chip electrophoresis device [21]. This platform is the first and only of its kind and may overcome the hurdles single-cell proteomic approaches have faced to date.

scWesterns operate on the scWest chip, a patterned, pre-cast polyacrylamide gel consumable, containing 6400 microwells. The scWest chip captures ~1000 single cells by a gravity-driven settling from a loaded cell suspension, resulting in single cells being contained in individual microwells (Fig. 1). The occupancy of the microwells roughly follows a Poisson distribution with the vast majority of occupied wells containing a single cell. The Milo platform lyses the captured cells and performs a rapid (~45-60-s), size-based SDS-PAGE separation on each single-cell lysate captured in the chip. After separation, protein bands are immobilized into the polyacrylamide gel via UV light and UV-sensitive chemistry. By probing the scWest chip with standard Western antibodies, protein expression for up to 12 targets per cell can be measured [21]. Chip fluorescence images are obtained using an open format microarray scanner. Data analysis is automated by the accompanying Scout™ Software, which detects separated proteins in each single cell in the form of peaks that are then quantified using peak area.

Single-Cell Westerns enable single-cell resolution profiling of protein expression to dissect subpopulation cellular heterogeneity. Herein, we apply the optimized assay to study hiPSC-CM heterogeneity over time, illustrating changes in protein expression throughout the differentiation timeline. To characterize CM subtype markers, protein expression of the myosin regulatory light chain 2 atrial isoform (MLC2A or MYL7) and of its ventricular isoform (MLC2V or MYL2) were performed. MLC2A, although observed throughout the developing heart, decreases in expression and is progressively enriched in the atria [[22], [23], [24]], whereas MLC2V expression becomes enriched within the ventricles: albeit low levels of both MLC2A and MLC2V persist in the outflow tract [22,24,25]. As MLC2A appears to be unrestricted to atrial tissue during development, it is important to consider the likely immature state and developmental stage of hiPSC-CMs linked to the expression levels of MLC2A [26]. Thus, to understand how the single-cell protein expression profile of MLC2A and MLC2V changes during hiPSC-CM differentiation, MLC2A and MLC2V were quantified using Single-Cell Westerns during three time points of differentiation. In addition, hiPSC-CMs derived from either TBX5 or HEY2 deficient iPSC lines demonstrated lower single-cell MLC2V expression in the MYL2 & MYL7 co-expressing cells at the same stage (day 30) of differentiation.

Section snippets

Media

  • 1.

    hiPSC media: Essential 8 (E8) media.

  • 2.

    hiPSC-CM media (d0-d6): RPMI 1640, B27 without insulin.

  • 3.

    hiPSC-CM media (d7-): RPMI 1640, B27 with insulin.

Solutions

  • 1.

    Primary antibody solution: 80 μL containing mouse anti-MLC2V at 1:5 (Synaptic Systems 310111AT2, Goettingen, Germany), rabbit anti-MLC2A at 1:10 (Pierce PA530789, Life Technologies), and goat anti-GAPDH at 1:10 (Sigma-Aldrich SAB2500450, St. Louis, MO), diluted in wash buffer (WB) containing 1× BSA Blocking Buffer (ProteinSimple).

  • 2.

    Secondary antibody

Calibration and optimization of Single-Cell Westerns for hiPSC-CMs

Cells loaded onto an scWest chip will settle into the microwells by gravity, for which optimal settling times may vary by sample. For the optimization experiments performed herein, approximately 100,000 cardiomyocyte cells in 1 mL of 1× Suspension Buffer were settled on individual chips for 5 to 30 min. Increased settling times led to increased well occupancies as expected (Fig. 2). To confirm cell occupancy, one can inspect one block of 400 microwells on the chip under a standard, brightfield

Discussion

With applications in both basic and translational cardiac research, hiPSC-CMs are being used to understand cardiovascular disease and heart development. However, due to hiPSC-CMs being composed of molecularly distinct cell subpopulations and hiPSC-CM protein expression dynamics fluctuating as they mature, bulk sample analysis becomes insufficient in resolution. Single-cell proteomics of hiPSC-CMs can be limited by techniques that rely upon antibody binding for detection without the ability to

Glossary

hiPSC: human induced pluripotent stem cells that resemble embryonic stem cells, generated by the reprogramming of human somatic cells.

hiPSC-CM: human induced pluripotent stem cell-derived cardiomyocytes.

MLC2V: myosin light chain ventricular isoform encoded by MYL2, marker for ventricular cardiomyocytes.

MLC2A: myosin light chain atrial isoform encoded by MYL7, marker for atrial cardiomyocytes.

scWest: Single-Cell Westerns.

Sources of funding

Research reported in this publication was funded by the National Institutes of Health under the National Heart, Lung, and Blood Institute (NHLBI) R00 HL128906 (JMC), R01 HL126527 (JCW), and R01 HL145676 (JCW) and the National Institute of General Medical Sciences (NIGMS) R43GM112236.

Declaration of Competing Interest

The authors declare no competing interests for this work. E.J. was a previous employee of ProteinSimple. D.V., J.M., and K.G. are employees of ProteinSimple, but have no competing interests.

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