Novel strategy to improve hepatocyte differentiation stability through synchronized behavior‐driven mechanical memory of iPSCs

Abstract

Cellular homeostasis is assumed to be regulated by the coordination of dynamic behaviors. Lack of efficient methods for synchronizing large quantities of cells makes studying cell culture strategies for bioprocess development challenging. Here, we demonstrate a novel application of botulinum hemagglutinin (HA), an E‐cadherin function‐blocking agent, to synchronize behavior‐driven mechanical memory in human induced pluripotent stem cell (hiPSC) cultures. Application of HA to hiPSCs resulted in a decrease in actin bundling and disruption of colony formation in a concentration‐and time‐dependent manner. Interestingly, cytoskeleton rearrangement in cells with prolonged exposure to HA resulted in mechanical memory synchronization with Yes‐associated protein, which increased pluripotent cell homogeneity. Synchronized hiPSCs have higher capability to differentiate into functional hepatocytes than unsynchronized hiPSCs, resulting in improved efficiency and robustness of hepatocyte differentiation. Thus, our strategy for cell behavior synchronization before differentiation induction provides an approach against the instability of differentiation of pluripotent cells.

A simple tool for the synchronous differentiation of human induced pluripotent stem cells into pancreatic progenitors

“…Flow cytometric analysis was conducted as described previously. [ 32 ] Briefly, cells were dissociated into single cell suspension using TrypLE Select. The cells were fixed and permeabilized according to the manufacturer's protocol using the Cytofix/Cytoperm kit (BD Biosciences) for 30 min at 4°C.…”

“…Flow cytometric analysis was conducted as described previously. [32] Briefly, cells were dissociated into single cell suspension using S1.…”

“…Intra-and inter-run variation analyses were performed as previously described. [32] The coefficient of variation (CV, %), also known as the ratio of the standard deviation (SD) to the mean, was used as a relative measure of variation as an indicator of culture reliability.…”

“…[29,30] To this end, we have developed a culture strategy for stem cells using botulinum hemagglutinin (HA). [31][32][33] HA, a nontoxic component of the botulinum neurotoxin complex, has been shown to bind E-cadherin and carbohydrates and disrupt cell-to-cell adhesion. [34][35][36] Although addition of HA in culture temporarily inhibits E-cadherin-mediated cell adhesion, HA is invariably reduced by spontaneous degradation and removed from cells during culture medium change.…”

“…A recent study has revealed that the combination of optimal concentration and exposure duration of HA leads to spatial rearrangement of the actin cytoskeleton through the disruption of E‐cadherin‐mediated cell adhesion in iPSC cultures. [ 32 ] Consequently, the mechanical memory synchronized with YAP acting improves the production of homogenous pluripotent cells without affecting the cell's ability to proliferate. These results demonstrate that synchronous mechanical memory using HA have potential to act as a culture tool for suppressing spatial heterogeneity in iPSC culture.…”