The road to cardiomyocyte maturation is paved by progressive developmental cues from epicardial and epicardial-derived cells

Introduction: The use of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes to study health and disease has exploded in recent years. Unfortunately, the cardiomyocytes (CM) generated from such protocols are immature. Methods for maturing hiPSC-CM commonly involve the immediate imposition of post-natal cues such as the addition of fatty acids to the media, the ramping of electrical stimulation and the addition of large changes in afterload. Accumulating evidence in vitro indicates that the exposure of hiPSC-CM to post-natal environmental conditions alone is not sufficient to attain full cardiac maturation. Here we instead mimic the progressive embryonic ventricular environment via the co-culture of hiPSC-CM with epicardial cells and epicardial-derived cells

The epicardium is an epithelial layer that originates during embryonic heart development in the proepicardial organ and migrates to cover the primary heart tube by E10.5-E12 in mice. It serves two major functions in embryonic development 1) the secretion of mitogens that drive cardiomyocyte proliferation, and 2) the population of the heart with vascular smooth muscles cells and cardiac fibroblasts through a process known as the epicardial epithelial-to-mesenchymal transition (EMT). The epicardium is so important for proper cardiac development, that murine models with deficiencies in the epicardium have thin ventricular walls, impaired coronary artery angiogenesis, and embryonic or immediate perinatal lethality. These findings indicate that the epicardium is essential for proper ventricular morphogenesis. Thus, this study explores the impact of epicardial and epicardial-derived cells on hiPSC-CM maturation by mimicking embryonic heart development.

Materials and

Methods: Epicardial cells (EPC) and hiPSC-CM were both differentiated from hiPSC using small molecule WNT modulation in 2D. Two in vitro models were explored 1) the 2D laminate co-culture of EPC with hiPSC-CM and 2) 3D engineered heart tissues (EHTs) containing epicardial cells. The 2D cultures were formulated by laying hiPSC-CM down in a monolayer culture and then layering the epicardial cells on top 24 hours later. The EHTs were made by combining hiPSC-CM and EPCs in a fibrin gel in a PDMS mold containing two PDMS posts to provide a prescribed static load. The 2D and 3D cultures either included TGF-Beta inhibitors (SB431542) to prevent epicardial EMT (CM + EPC(+SB)) or excluded the inhibitor to allow for spontaneous epicardial EMT (CM + EPC->DC). These were compared with hiPSC-CM monoculture controls with (CM(+SB)) and without (CM) TGF-Beta inhibitors. In 3D an additional condition containing epicardial-derived fibroblasts (EPD-FB) and EPC was included and termed CM Tri-culture.

End-point analysis for the 2D co-cultures consisted of flow cytometry for cell identity and proliferation assessment and functional assays such as calcium transient assessment using Rhod-2AM calcium-sensitive dye as well as traction force microscopy for single CM force generation. Additionally, the 2D co-cultures were singularized, fixed, and sequenced using the 10X Genomics Next GEM Fixed Single Cell Sequencing Kit. EHT endpoints included immunohistochemistry for composition and tissue morphology, calcium handling, optical mapping of voltage, whole tissue force generation, and q-RT-PCR for maturation transcripts.

Results, Conclusions, and Discussions: When EPCs were combined with CM in 2D direct co-cultures the percentage of proliferating CM increased at the same time as stark electrophysiologic improvements in calcium handling (Figure 1A-B). The increase in proliferation was seen via flow cytometry for CM with incorporated 5-ethynyl 2´-deoxyuridine (EdU) indicative of new DNA synthesis or via immunofluorescence staining for the transcription factor Ki67. The simultaneous increase in proliferation and calcium handling was interesting as CM proliferation and maturation are normally considered dichotomous. Notably, no increase in single-cell CM force generation was observed (Figure 1B). These results indicate that though EPCs might play a role in CM electrophysiologic maturation they may not spur mechanical and myofilament maturation. Single-cell transcriptomics revealed a significant shift in the bulk CM population in the CM + EPC(+SB) co-cultures as characterized by more fetal-like myofilament isoforms but with enhanced pathways associated with cardiac conduction and ventricular development (Figure 1C-D). However, when the epicardial cells were allowed to undergo EMT, the CM + EPC->DC, the bulk CM population shifted to a more adult-like myofilament composition (Figure 1C-D).

EHTs that contained epicardial cells showed limited proliferation but a similar increase in CM electrophysiologic function while EPCs themselves did not spur robust EHT force generation (Figure 1E-G). The effect of epicardial-derived cells on CM calcium handling was more robust in long-term 3D cultures. These results indicate that certain aspects of CM maturation are enhanced, especially cardiac conduction, by epicardial cells while an embryonic phenotype is maintained (i.e., proliferation and myofilament isoforms). Next, consistent with developmental progression and the initiation of EMT into fibroblasts, EPD-FBs were added to the EHTs containing epicardial cells (CM Tri-culture). In this case, we observed significant myofilament maturation and increased force generation by more than an order of magnitude (Figure 1F). Taken together, our results support the importance of mimicking developmental progression to slowly taper proliferation while gradually driving the maturation of hiPSC-CM to achieve robust in vitro function at the cell and tissue scale. One could envision the realization of full cardiac maturation lying in the staged imposition of fetal followed by postnatal cues in vitro.

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