Innovation Showcase
Time: Friday, June 28th, 2019: 11:30AM-12:30PM
Location: Concourse F - Level 1

Laminin E8 Technology for Stem Cell Proliferation and Differentiation:
From Molecular Mechanism to Clinical Application

Dr. Ryuhei Hayashi, Professor, Graduate School of Medicine, Osaka University
Dr. Yukimasa Taniguchi, Assistant Professor, Institute for Protein Research, Osaka University

Dr. Ryuhei Hayashi’s technique for differentiating human pluripotent stem cells into ocular cells has recently been approved by Japan’s Ministry of Health, Labour and Welfare for cell therapy treatment of corneal epithelial stem cell deficiency. His group has developed the culture system for self-formed ectodermal autonomous multi-zone (SEAM), cell colonies that are arranged as four concentric areas that mimic the spatiotemporal development of the whole eye. This was accomplished with laminin E8, a recombinant laminin fragment, as the culture substrate. The group has since recently uncovered the specific isoforms of laminin E8 responsible for the induction of pluripotent stem cells to the corneal epithelium, the retina, and the epithelium of the lens. The outcome of their research underscores the importance of selecting appropriate laminin isoforms for cell culturing.

The binding affinity of substrates and integrins determines the nature of expanded human pluripotent stem cell colonies in terms of cell motility, cell-cell interactions and cell density. Currently, how binding between laminin and integrin is achieved is poorly understood. Dr. Yukimasa Taniguchi will present his group’s latest findings on the molecular mechanism of laminin E8-integrin interactions underlying stem cell culturing.

Registration Form for Innovation Showcase

Scraper-Free Detachment Method Using EDTA without Trypsin for Human Induced Pluripotent Stem Cells Cultured on Laminin-511E8

Dr. Fumi Ebisu, Project Leader, R&D, Matrixome Inc.

Poster #: W-3235
Session: Poster I – Odd
Date: June 26, 2019
Time: 18:30 (6:30 PM)

Recombinant E8 fragment of laminin-511 (LM511E8) is used in the culturing of human pluripotent stem cells (hPSCs) because it sustains long-term single cell passaging, preserves pluripotency and maintains cells in an undifferentiated state. LM511E8 is a truncated form of laminin-511, and its ability to offer these features comes from its binding activity with integrin α6β1, an isoform predominantly expressed on hPSCs. However, because of their strong interaction with each other, trypsinization followed by scraping is required to harvest the cultured cells. Nevertheless, cell scraping causes mechanical damage to the cells, reducing cell viability. In addition, cell scraping cannot be used when cells are cultured on multi-layer flasks or in automated cell culture systems. In this study we aim to develop a scraper-free cell detachment method for human induced pluripotent stem cells (hiPSCs) cultured on LM511E8. By referring to the protocols available to date, we examined whether incubation of hiPSCs with 5 mM EDTA/PBS(-) at 37˚C for 15 min enabled to detach the cells with high efficiency.  The inclusion of trypsin to EDTA/PBS(-) was also tested to see if there were any additional gain to cell detachment efficiency. We found that more than 95% of hiPSCs were detached without compromising cell viability when they were incubated with 5 mM EDTA/PBS(-) alone. Surprisingly, the addition of trypsin decreased the detachment efficiency.  This decrease was rescued when enough trypsin inhibitor was added to neutralize the enzyme, therefore the lowered detachment rate must be a result of its proteolytic activity, suggesting an ill-defined mechanism operating in hiPSCs to render them less susceptible for cell detachment by depletion of divalent cations.