Decoding the role of intracellular calcium signaling and membrane potential in iPSC differentiation to endothelial cells

Introduction: Induced pluripotent stem cell (iPSC)-derived endothelial cells (dEC) are crucial for developing vascularized tissues and modeling vascular diseases, and therefore have potential uses in patient-specific regenerative therapies and drug screening tools. As such, numerous studies have reported on novel protocols to target endothelial differentiation, most of which use either growth factors, transcription factors, or a combination of both. During the differentiation process, cells undergo several physiological and transcriptomic changes, one of which is the change in the membrane potential resulting from, in particular, fluctuations in intracellular calcium ion concentration. In fact, previous studies have shown that membrane voltage and calcium signaling regulates the loss of pluripotency in iPSC . The role of calcium signaling is of particular interest in endothelial differentiation, as studies have shown the critical role of calcium signaling in regulating endothelial cells in vascular development, suggesting that calcium signaling can be used to fine-tune the differentiation protocol.
Therefore, in this study, we explore the role of membrane potential and calcium signaling in the iPSC differentiation into endothelial cells by mapping out the changes to these parameters as cells undergo differentiation (Figure 1). We map the different stages of pluripotency onto membrane potential and calcium concentration space and demonstrate an improvement in differentiation efficiency by manipulating these parameters. We also develop a computational pipeline to decode the unique calcium signaling patterns associated with endothelial cells stimulated with growth factors used in differentiation, which has the potential to fine-tune the differentiation of mesodermal lineage cells into endothelial cells.

Materials and

Methods: To generate the iPSC-derived endothelial cells, we relied on the existing differentiation protocol for dEC published by Harding and colleagues. We induce mesodermal identity through 2 days of treatment with 6μM of CHIR99021, a Wnt-pathway activator. Then we induce endothelial progenitor characteristics through 2 days of treatment with 50ng/mL of VEGF-A, FGF2, and BMP4 along with 10nM of ouabain, a small molecule that causes depolarization. To generate fully mature endothelial cells, cells are passaged and replated to density of 50,000 cells/cm2. Lastly, we treat the cells with 100ng/mL of VEGF-A for 4 days. Cells were either collected for flow cytometry analysis or live imaged for calcium signals.
Cells were analyzed for membrane potential and calcium concentration at days 1, 3, and 5, corresponding to pluripotent, mesodermal, and early endothelial stages, respectively. For each day, cells were stained for 30 minutes with 12 μM of RH421 dye, a fast-responding membrane potential dye. Then the cells were directly stained for 1 hour with 10 μM of Calbryte 520AM dye, a cell-permeable intracellular calcium dye (Figure 2A). The fluorescent images were segmented and quantified at a single-cell level to calculate the shifts in membrane potential and calcium concentration.

Results, Conclusions, and Discussions: We measured the calcium concentration and membrane potential of cells that are in the iPSC, mesodermal, and early endothelial stages of differentiation and found that the membrane potential increases as cells reach mesodermal stage, then drops slightly as cells enter endothelial stage (Figure 2B). Furthermore, we observed that calcium concentration increases only at the endothelial stage (Figure 2C). These results suggested that the membrane potential plays an important role in the early differentiation stages. We further validated this by treating the cells with 10nM of ouabain, a depolarizing small molecule, during days 3 and 4, when cells transition to endothelial cells and membrane potential decreases. We have found a significant increase in the yield of endothelial cells at day 5, indicating that manipulating membrane potential can improve differentiation yields (Figure 2D).
To determine the role of calcium signaling in endothelial induction in mesodermal cells, we first measured the changes in calcium signaling in native endothelial cells in response to treatment with VEGF-A, the primary growth factor used in endothelial induction. We designed an algorithm that segments the fluorescent images, extracts temporal calcium signaling data, and calculates the frequencies of the peaks of calcium signals (Figure 3A) and found a significant increase in calcium spiking frequency from approximately 0.007 Hz to 0.013 Hz with treatment with VEGF-A (Figure 3B). We further found that the iPSC-derived endothelial cells, when treated with VEGF-A following the differentiation, spikes at approximately 0.013 Hz as well, indicating that there is an intrinsic calcium spiking frequency triggered by VEGF-A (Figure 3C).
We demonstrated the usefulness of this intrinsic frequency by designing a microfluidic chip that can deplete ions from the cell growth channel using an external electric field in order to manipulate calcium signaling. We have shown that the microfluidic chip can influence the calcium signaling patterns, and also induce synchronization across the cell population within the chip (Figure 3D). Taken together, our results suggest that the membrane potential and calcium signaling play an interconnected and crucial role in regulating iPSC differentiation to endothelial cells, which can potentially be used to improve the existing differentiation protocols.

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