Abstract
Mechanical forces play an important role in proper embryologic development, and similarly such forces can directly impact pluripotency and differentiation of mouse embryonic stem cells (mESC) in vitro. In addition, manipulation of the embryoid body (EB) microenvironment, such as by incorporation of microspheres or microparticles, can similarly influence fate determination. In this study, we developed a mechanical stimulation regimen using permanent neodymium magnets to magnetically attract cells within an EB. Arginine-Glycine-Aspartic Acid (RGD)-conjugated paramagnetic beads were incorporated into the interior of the EBs during aggregation, allowing us to exert force on individual cells using short-term magnetization. EBs were stimulated for one hour at different magnetic field strengths, subsequently exerting a range of force intensity on the cells at different stages of early EB development. Our results demonstrated that following exposure to a 0.2 Tesla magnetic field, ESCs respond to magnetically mediated strain by activating Protein Kinase A (PKA) and increasing phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) expression. The timing of stimulation can also be tailored to guide ESC differentiation: the combination of bone morphogenetic protein 4 (BMP4) supplementation with one hour of magnetic attraction on Day 3 enhances cardiomyogenesis by increasing contractile activity and the percentage of sarcomeric α-actin-expressing cells compared to control samples with BMP4 alone. Interestingly, we also observed that the beads alone had some impact on differentiation by increasingly slightly, albeit not significantly, the percentage of cardiomyocytes. Together these results suggest that magnetically mediated strain can be used to enhance the percentage of mouse ESC-derived cardiomyocytes over current differentiation protocols.
Highlights
Myocardial Infarction (MI) is one of the most prevalent diseases in America, accounting for approximately 50% (7.6 million) of the 15.4 million Americans suffering from coronary heart disease [1]
We confirmed the bead incorporation procedure by light microscopy and Prussian Blue staining on sectioned embryoid body (EB), in which the latter stains ferric ions a blue color
In comparison to the control EBs with no beads (Fig. 1 A, A9), the EBs with the magnetic beads were darker in light micrographs (Fig. 1B), and the beads appeared distributed throughout the entire EB (Fig. 1B9)
Summary
Myocardial Infarction (MI) is one of the most prevalent diseases in America, accounting for approximately 50% (7.6 million) of the 15.4 million Americans suffering from coronary heart disease [1]. As an alternative to invasive surgical treatment options, cell therapy holds promise in promoting recovery following heart failure. For this strategy to be effective in a clinical setting, an adequate cardiomyocyte cell source must be identified. Differentiation of cardiomyocytes from progenitor cells such as pluripotent stem cells (PSCs) has the most potential to derive a large enough population to be a clinically relevant source. While the highest purity of the differentiated cardiomyocyte population is generated from ESC monolayer culture (up to 90–95% cardiomyocytes from hESCs) [3], establishing a protocol to efficiently differentiate ESCs within an embryoid body (EB) could potentially increase the final cell yield, which is a bottleneck for the clinical use of ESCderived cardiomyocytes. The highest cardiomyocyte yield from murine EB culture is approximately 20% [4]
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