Assessing the Behavior of Human Induced Pluripotent Stem Cells (hiPSCs) Through Micropatterning and Electrotaxis

Abstract

The development of human induced pluripotent stem cells (hiPSCs) in the early 2000s has facilitated stem cell research by circumventing ethical issues surrounding use of human embryonic stem cells (hESCs). While stem cell research and therapy have advanced significantly over the last two decades, the specific mechanisms of wound repair have remained unclear. When a wound is induced, an endogenous electric field is generated, directing specific cells to the site of the wound and beginning the healing process. The precise mechanisms of cell migration to the wound are unclear in vivo, primarily due to the presence of multiple layers of skin cells; however, research on cell monolayers indicates that the actin cytoskeleton and E-cadherin in adherens junctions are likely implicated. To investigate how hiPSCs would behave during wound repair in vivo, we performed electrotaxis of hiPSCs using the Cohen lab’s CORGI device. This revealed that hiPSCs do not migrate under a directed electric field, but rather that they exhibit what we hypothesized— due to brightening of cell-cell boundaries under phase contrast microscopy—was hydraulic fracturing of cell-cell boundaries in the direction of the electric field. In order to facilitate analysis of hiPSC colonies and determine the components involved, we employed both electrotaxis and micropatterning. We used two methods of micropatterning—contact micropatterning and light-induced molecular adsorption patterning (LIMAP) using a digital micromirror device (DMD)—and extensively optimized these methods to control the cell microenvironment and ensure proper cell adhesion and growth. Furthermore, we found that E-cadherin does not appear to overlap with the junction brightening phenomenon, but this might also be due to hiPSC junctions that are not vertical as originally assumed, or due to changing compositions of cell junctions due to, for instance, water influx.