Report for newly appointed faculty startup
1. Name of project leader: Masamichi Imajo
2. Project title: Application of synthetic hydrogel to the control of cellular behavior andfunctions
3. Report
In multicellular organisms, cellular behavior and functions are strictly regulated byextracellular environment to achieve formation of complex organs and to maintain their
homeostasis. Thus, proper regulation of extracellular environment has long been
considered a key to successful manipulation of cellular functions in biomedicalengineering. Since polymer hydrogel mimics features of extracellular environment invarious tissues and can be used for cell culture and implantation, considerable efforts havebeen devoted to develop hydrogel with specific biological functions.
In ICReDD, I will aim to apply chemically synthesized materials to life science andmedical research to deepen our understanding of biological systems and to develop novel
therapeutics of incurable diseases. For these purposes, in FY2019, I aimed to develop
application of chemically synthesized hydrogel, in collaboration with the Prof. Gong’s
group, to biological and medical research. This start up fund covered the costs of various
cell lines, cell culture reagents, synthesis of hydrogel, molecular cell biology analyses, andPCs for data analysis, and so on. The achievements of this project are described below.
(1) Development of synthetic hydrogel that modulates cellular functions
To establish principles to design synthetic hydrogel with specific biological activities, I
first examined what physical parameters of hydrogel have critical effects on cellularbehavior. To this end, various kinds of hydrogels with distinct physical and chemical
properties were synthesized from different monomers and used as substrates for culturing
conventional cell lines, including non-cancerous and cancerous cells (Fig. 1). The results
showed that electric charge of hydrogel strongly affects cellular adhesion, survival,migration and proliferation in many cell lines. To strictly control electric charge ofFig. 1. An overview of thisproject. Various monomers withdifferent structures and charge
are polymerized to generatehydrogels harboring distinct
physical and chemicalproperties. The surface of thesehydrogels can be further
modified by biomolecules. The
hydrogels are used for cell
culture scaffolds, ECM mimetics
in transplantation, cell sorter,and so on, to develop innovative
methods to regulate cellularbehavior and functions.

hydrogel, we utilized the hydrogel composed of cationic ((3-acrylamidoproyl)trimethylammonium (APTMA)) and anionic (2-acrylamido-2-methylpropanesulfonate (AMPS))
monomers. Since APTMA-AMPS hydrogel has good biocompatibility and its electriccharge could be controlled by changing the ratio of two monomers, we found that
APTMA-AMPS hydrogel is an ideal substrate to control cellular functions throughchanging electric charge of the scaffold matrix. These findings would be a basis fordesigning hydrogel with specific biological activities in future studies.
(2) Elucidation of molecular mechanisms that mediate cellular responses to electriccharge of the scaffold matrix
The above findings that electric charge of hydrogel significantly affects cellular behaviors
have suggested that cells are equipped with some mechanisms to sense and respond toelectric charge of the surrounding materials. To reveal such mechanisms, I conductedlarge-scale integrated analysis of transcriptomic and proteomic changes caused byalteration of electric charge of the hydrogel scaffold. I identified more than 1,000transcripts and 3,000 proteins whose abundance is regulated by electric charge of thescaffold matrix. Notably, in several cancer cell lines, expression of genes comprising astem cell signature was increased by a certain range of electric charge, suggesting that
stemness of cancer cells is regulated by electric charge of the scaffold matrix. These results,
for the fist time, identified genes and proteins that respond to electric charge of thescaffold matrix, which should pave the way for future elucidation of cellular mechanisms
for sensing and responding to electric charge of the surrounding environment.
(3) Application of synthetic hydrogel that modulates functions of pluripotent stem cells
As described above, electric charge of hydrogel affects expression of genes comprising astem cell signature. Thus, the effects of electric charge of hydrogel on pluripotent stemcells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),
were investigated in collaboration with a postdoctoral researcher, Dr. Akira Hirota, in theProf. Tanaka group. We found that a certain range of electric charge promotes, whereas
opposite electric charge suppresses, differentiation of mouse ESCs. Importantly, on thehydrogel with certain electric charge that suppresses the differentiation, ESCs could
maintain their naive pluripotent state even in the presence of humoral factors inducing
the transition to the primed pluripotency. These results indicate that functions ofpluripotent stem cells can be controlled by modulating electric charge of the scaffold
matrix. In future studies, we will perform detailed analyses of the effects of electric charge
on the functions of human iPSCs and the underlying molecular mechanisms, thereby
aiming to develop novel methods that contribute to advancing the regenerative medicine.
(4) Establishment of a novel culture method for intestinal stem cells by using synthetichydrogel

Tissues exposed to environmental assault require constant renewal driven by tissueresident stem cells. In these tissues, defects in stem cell functions have been implicatedin many diseases, and transplantation of stem cells cultured and expanded in vitro has
been considered a promising strategy to promote tissue repair and to induce disease
remission. An example of such tissues is the intestinal epithelium, in which chronic
inflammation (inflammatory bowel disease (IBD)) has been shown to impair theepithelial barrier function and transplantation of intestinal stem cells (ISCs) could be anovel therapeutic approach to treat IBD (Fig. 2). However, the current methodology forculturing ISCs includes complicated procedures and is not suited to expand ISCs
sufficiently for in vivo transplantation, which hampers clinical implementation of the ISC
transplantation therapy of IBD. In this project, I aimed to develop a novel innovative
method that enables rapid and convenient culture of ISCs by using chemically synthesizedhydrogel. By modulating physical properties of hydrogel and formulation of culture media,
I found condition under which mouse ISCs can adhere to the hydrogel, maintain theirundifferentiated state, and proliferate vigorously. Thus, although further optimization of
the culture condition and validation of ISC functions are needed, I have successfullydeveloped a prototypic method for culturing ISCs. Since this novel culture method has
clear advantages over the conventional
culture method, I decided to apply for theAMED grant to further optimize the culture
condition, to adjust the method to the human
ISC culture, and to establish application ofcultured ISCs to the regeneration therapy for
IBD. Thus, the achievements of this grant
have provided a basis for the next important
project that will be supported by externalfunding in future.
4. Research achievement[Publication]
Masamichi Imajo. Analysis of Retinoic Acid
Receptor Signaling in Colorectal Cancer,
Methods Mol. Biol., Retinoid and Rexinoid
Signaling, 85-93, 2019

Fig. 2. A graphical abstract of aconcept of stem cell transplantation
therapy for IBD.

[Presentation]
Masamichi Imajo, Yu Muta, Michiyuki Matsuda. Alterations of ERK activity dynamics
underlying tumor-specific traits in the intestinal epithelium. The 38th Sapporo
International Cancer Symposium, 2019