LEFTY-PITX2 signaling pathway is critical for generation of mature and ventricular cardiac organoids in human pluripotent stem cell-derived cardiac mesoderm cells
Introduction
Cardiovascular disease (CVD) is a serious disease with high prevalence and mortality rates [1]. CVD represents a group of adult-onset diseases including ischemic heart disease, stroke, heart failure, and myocardial infarction [[1], [2], [3]]. Myocardial infarction occurs from the loss of ventricular cardiomyocytes (CMs), resulting in decreased cardiac function, and treatment requires many mature ventricular CMs [4]. Some studies have shown that anticancer therapies give rise to cardiotoxicities such as hypertension, heart failure, and left ventricular dysfunction [5,6]. However, human CMs have little or no regenerative capacity and limited supplies of CMs are available [7]. Therefore, a human cell-based CVD model system is urgently needed [8,9]. Recently, human pluripotent stem cell-derived CMs (hPSC-CMs) have been extensively used in studies of heart development, drug screening, disease modeling, and heart repair [8,[10], [11], [12], [13], [14], [15], [16], [17], [18]]. However, hPSC-CMs exhibit immature characteristics, similar to cells in embryonic or early fetal stages [19]. Therefore, developing an approach for the generation of mature and ventricular CMs that resemble adult CMs in structure and function is required.
Most studies on CMs have focused on the maturation of ventricular CMs [20]. CM maturation involves various structural, gene expression, metabolic, and functional changes in CMs as the heart transits from the fetal to the adult state [18,20]. Structural maturation of CMs is associated with increased sarcomere length, increased Z-line width, improved sarcomere alignment and transverse-tubule (t-tubule) formation [21,22]. Metabolic maturation of CMs enables a high and sustained rate of ATP production for sarcomere contraction and relaxation. CMs undergoing maturation show an increase in mitochondria number and size and switch to fatty acid utilization and oxidative phosphorylation using lipid substrates [3,[22], [23], [24]]. Electrophysiological maturation of CMs involves an increase in action potential duration (APD) and amplitude [18]. The Ca2+ handling feature in electrophysiological maturation also relies on RyR-mediated Ca2+ release with t-tubules and coupling between RyR and L-type Ca2+ channels [25].
Three-dimensional (3D) cell culture systems provide a more accurate microenvironment resembling conditions where cells naturally reside in comparison with the conventional 2D culture systems [[26], [27], [28]]. Cardiac organoids (COs) are stem cell-derived in vitro 3D tissues that have self-organizing capacity [29,30]. Self-organizing COs from hPSCs that intrinsically specify, pattern, and morph into chamber-like structures containing a cavity [29,30]. The cardiac progenitors self-organized into an anterior domain reminiscent of a cardiac crescent [31].
Two approaches have been mainly used to generate COs. One is to aggregate terminally differentiated CMs, endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts (FBs) in a 2D culture system [7,32,33]. The advantages of this CO generation method can utilize CMs at high purity (>80–90%) via the modulation of Wnt signaling [4] and also the ability to regulate the ratio of cardiac component cells. However, this method has disadvantages that a highly hydrated polymer network using a hydrogel or ECM is required for induction of self-patterning [[33], [34], [35]]. The second approach is to form COs by triggering cells towards cardiac cell lineages after spheroid formation using undifferentiated hPSCs [[36], [37], [38]]. Spheroid formation-derived COs have the advantages of showing a high self-organizing capacity, whereas differentiation efficiency into cardiac cell lineages is relatively reduced [20,39].
Several environmental cues regulate CMs, including key biophysical factors that affect CM maturation such as geometric constraints, extracellular matrix (ECM) viscoelasticity, mechanical strain, and electrical stimulation [20]. In addition, several biochemical factors that regulate CM maturation such as thyroid hormone, glucocorticoids, insulin-like growth factors, and fatty acids have been reported [20]. Transcriptional profiling studies [40,41] regarding mammalian heart development, repair, and regeneration have provided transcriptional regulation and gene regulatory networks for understanding CM maturation. However, the genes and regulatory mechanisms for generation of structurally and functionally matured COs are still largely unknown.
We hypothesized that the developmental plasticity of COs is critical for the generation of mature COs, like during in vivo heart generation. Cardiac mesoderm cells (CMCs) have a relatively high self-organizing capacity compared with CMs, and they also can differentiate into cardiac component cells including CMs, ECs, SMCs, and FBs. In contrast, terminally differentiated CMs have a high proportion of CMs, whereas they show low self-organizing capacity. In this study, we generated CMC-derived COs (CMC-COs) and CM-derived COs (CM-COs) and investigated the effects of the two COs in developmental plasticity on the differentiation into mature and ventricular CMs. Furthermore, we elucidated the genes and mechanisms involved in the generation of mature and ventricular COs by performing transcriptome analysis and gene knockdown experiment.
Section snippets
Maintenance and cardiac differentiation of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)
The H9 hESC line was obtained from the WiCell Research Institute (Madison, WI, USA) and the TMOi001-A episomal hiPSC line derived from CD34+ cord blood was purchased from Thermo Fisher Scientific (A18945, Waltham, MA, USA). Undifferentiated hPSCs were maintained on Matrigel (BD Biosciences, San Jose, CA, USA)-coated plates in E8 medium (Thermo Fisher Scientific) and grown to 70–80% confluence. Cells were passaged at a ratio of 1:4. hPSCs were dissociated into a single cell suspension with
Ventricular-like CMs were predominantly generated in hESC-derived CMC-COs compared with hESC-derived CM-COs
To generate COs with different developmental plasticity, we first induced differentiation of H9 hESCs into CMCs or CMs using an optimal CM differentiation protocol with Wnt modulation in modified chemically defined medium (Supplementary Fig. 1A). To determine the optimal differentiation day to specify CMCs or CMs from H9 hESCs, we examined gene expressions of mesendoderm markers (MIXL1 and T), mesoderm markers (VEGFR2 and MESP1), and CM markers (cTnT and MLC2a) in H9 hESC at different time
Discussion
In this study, we generated mature ventricular COs using CMCs having self-organizing capacity in both hESCs and hiPSCs. We found advanced structural, metabolic, functional, and molecular maturation of CMs in CMC-COs compared with CM-COs. CMC-COs exhibited (1) more organized sarcomere structures; (2) more organized mitochondria; (3) a well-arranged t-tubule structure; and (4) more organized and evenly distributed ICDs. CMC-COs showed increased gene expression of (1) ventricular CM markers; (2)
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) [2019M3A9H1103792].
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These authors contributed equally to this work.