Optogenetic calcium ion regulation and its application in human pluripotent stem cells

Abstract

Calcium ions ($Ca^{2+}$) orchestrate numerous cellular mechanisms, with their concentration in a cell elaborately controlled in both spatial and temporal manners. A multifaceted and multidimensional regulatory toolset is thus particularly essential to explore this versatile and universal second messenger. Here, human pluripotent stem cell (hPSC) lines that consistently express optogenetic $Ca^{2+}$ modulator monSTIM1 were generated via CRISPR-Cas9-mediated genome editing. All established cell lines exhibited light-responsive increment of intracellular Ca2+ concentration ([$Ca^{2+}$]i), which was reversibly controllable in accordance with the presence or absence of the light. I hypothesized that by differentiating monSTIM1-kncokin hPSCs into multiple lineages, it can be widely utilized for studies on biological mechanisms relevant to $Ca^{2+}$. In $ \beta$-cells of pancreatic islet-like organoids derived from monSTIM1 transgenic human embryonic stem cells (hESCs), as in the PSC stage, [$Ca^{2+}$]i transients were induced temporarily in response to the episodic irradiations. Transgenic $ \beta$-cells were then capable of releasing secretory insulin vesicles upon light illumination, which was quantitatively parallel to the insulin secretion provoked by a normal secretagogue, glucose. Moreover, monSTIM1 transgenic induced pluripotent stem cells (iPSCs) originated from neonatal diabetes (ND) patients were also successfully differentiated into pancreatic organoids, and thereby the feasibility of the system for further clinical applications was verified. Concurrently, neural progenitor cells (NPCs) differentiated from monSTIM1 transgenic hESCs exhibited light-inducible $Ca^{2+}$ influx as well, and the resulting [$Ca^{2+}$]i transient reversibly activated either of the two archetypal transcription factors downstream of $Ca^{2+}$, NFAT or CREB. Based on these observations altogether, I demonstrate the practicality of this gain-of-function cellular model encompassing the versatility and universality of $Ca^{2+}$, which, therefore, is expected to be exploitable in multidisciplinary studies.