Department of Biomaterials
Graduate School of Biomedical and Health Sciences, Hiroshima University
Research interests
BIOMATERIALS AND BIODEVICES FOR REGENERATIVE MEDICINE
Stem cell-based regenerative medicine could potentially aid in the reconstruction of tissues and organs damaged during various diseases and traumatic injuries. However, the widespread clinical application of this methodology requires technological advances in cell processing and transplantation. Our goal is to provide solutions to these challenges by employing innovative and advanced bioengineering approaches.
Bioreactors for stem cell culture | Engineered growth factors
To disseminate cell replacement therapy, additional technologies need to be developed to generate a large number of clinical-grade stem cells. Accordingly, bioreactors that enable a high-density stem cell culture in a closed system are necessary to address this unmet need.
Currently, we are developing bioactive solid supports as one of the most important bioreactor components. Our strategy is to immobilize growth factors, such as epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), onto the surface of solid supports. Growth factors are genetically modified with a small peptide with surface affinity to ensure the structural integrity of growth factors, as well as their stability on support surfaces [Langmuir 2005; Biomaterials 2008].
When seeded on the surface, stem cells expressing growth factor-specific receptors are selectively captured on the supports via growth factor−receptor interactions, activating intracellular signaling to promote stem cell proliferation and yielding pure stem cells in large quantities.
The feasibility of our strategy was demonstrated using rodents [Biomaterials 2007] and human neural stem cells (NSCs) [Biomaterials 2013]. In addition, this technology has been successfully applied to bone marrow stromal cells (BMSCs) [Sci Rep 2020] and NSCs derived from induced pluripotent stem cells (iPSCs) [Biotechnol Bioeng 2020].
Moreover, inspired by natural EGF signaling, immobilization of genetically engineered EGF, which can spontaneously dimerize, has been attempted [Bioconjugate Chem 2009]. Reportedly, the modification can significantly improve the efficiency of selective NSC expansion.
A parallel-plate bioreactor has been fabricated [Biomaterials 2011] using an EGF-immobilized glass plate and an oxygen-permeable polystyrene membrane. Our technology was found to be scalable for the mass production of NSCs in a closed system.
Alternatively, a growth factor fused to a small peptide presenting surface affinity for a commercially available polystyrene tissue culture plate (PSt-tag) can enable the preparation of a stem cell-specific culture setup by employing a simple procedure [Biomaterials 2011].
Biodevices for the quality control of stem cells | Antibody microarrays
For safe and efficient transplantation, the quality of graft cells must be thoroughly controlled. There are two aspects regarding cell quality: safety and reproducibility. We are particularly interested in the latter aspect, as cell populations produced through cell culture could vary in characteristics, such as the stem cell content. To characterize the cell populations, we propose an antibody microarray assay.
Antibody microarrays are fabricated by micropatterning an alkanethiol monolayer formed on a tiny glass chip, followed by the subsequent immobilization of multiple antibodies onto individual dots. A cell-binding assay on the microarray affords quantitative information on patterns of surface markers expressed on test cells [Biomaterials 2007].
The antibody microarray-based immunophenotyping method was successfully applied to the quality control of mesenchymal stem cells [ACS Appl Mater Interf 2015]. Other studies [Biomaterials 2005; Biomaterials 2005] have further demonstrated that antibody microarray-based methods can be used to identify unknown surface markers expressed on NSCs.
The throughput of the analysis relies on the time required for quantitative data acquisition, which is drastically improved by using the surface plasmon resonance (SPR) imaging technique [Anal Chem 2007] that allows the simultaneous and instant enumeration of bound cells for multiple antibody spots.
In addition, quantitative subset analysis [ACS Appl Bio Mater 2021] focusing on two or more surface markers can be performed using a microarray that has spots with multiple antibodies, as well as spots with single antibodies separately. The abundance ratio for every subset can be determined by numerical calculations based on the concept of the algebra of sets.
In addition to surface marker analysis, techniques for surface micropatterning and two-dimensional (2-D) antibody displays afford a wide variety of potential applications [ACS Appl Mater Interf 2009].
Optimization of cellular microenvironments | Materials informatics
In multicellular organisms, cell fate is closely regulated by soluble and insoluble factors in the extracellular space. Therefore, protein factors, such as growth factors and extracellular matrices, are attractive components of bioactive materials for application in tissue engineering. However, the diversity of these factors and the crosstalk between their signaling can complicate the precise selection of optimal components for biomaterial assembly.
We are conducting material informatics-based studies to gain deeper insights into cellular responses to multiple protein factors, which can aid in the molecular design of bioactive materials. Materials informatics is an emerging technology that efficiently develops new materials through data-driven machine learning.
In our approach, a large dataset was first acquired by high-throughput bioassays using a combinatorial microarray presenting diverse biomaterials [Biomaterials 2007; Bioconjugate Chem 2008; Biotechnol Bioeng 2015]. Our targets include stem cell responses to bioactive materials. The dataset thus obtained was applied to an unsupervised learning algorithm to predict cellular responses to different biomaterials.
In our previous study [Biomaterials 2011], various growth factor-tethering surfaces were examined for their effects on NSC proliferation and differentiation. The hierarchal clustering method was used to predict how NSCs change their phenotypes in response to growth factor cocktails.
Generally, the quality and size of datasets are critical for efficiently obtaining useful information using materials informatics approaches. In this sense, other microarray techniques for combinatorial overexpression [Nucleic Acids Res 2004; BBA General Subjects 2007] or knockdown [Anal Bioanal Chem 2008; Anal Biochem 2008] of multiple genes are also practical.
Fabrication of tissue engineering scaffolds | Engineered protein polymers
The fate of transplanted cells in host tissues significantly impacts therapeutic outcomes. However, it is difficult to precisely direct cell survival and integration after transplantation using current technologies. To control the behavior of transplanted cells, we are developing tissue engineering approaches that employ biomaterials incorporating engineered cell-adhesive polypeptides or growth factors for scaffolding cells.
Engineered protein-based building blocks for biomaterial assembly can be designed and created using recombinant DNA technology. This approach has allowed rational scaffold design. Given the various techniques and resources currently available, there is an infinite possibility for biomaterial design.
Based on our in vitro [Bioconjugate Chem 2012] and in vivo [Bioconjugate Chem 2013] studies, the collagen hydrogel incorporating engineered cell-adhesive polypeptides significantly improved the survival of NSCs transplanted into the brain by blocking the infiltration of inflammatory microglia and providing adhesive substrates to transplanted cells.
Similarly, a cell-adhesive domain fused with a cytokeratin-derived helical polypeptide was also found to be effective for improving the survival of NSCs in cytokeratin networks [Biomacromolecules 2008]. In this case, the helical polypeptide spontaneously participates in the cytokeratin-mediated self-assembly process.
Conversely, the proliferation of NSCs can be promoted in collagen hydrogels by incorporating engineered EGF [Biomaterials 2011] via specific interactions using a collagen-binding polypeptide domain fused to EGF [Bioconjugate Chem 2007]. Furthermore, the hyaluronic acid hydrogel incorporating engineered brain-derived neurotrophic factor (BDNF) can improve the survival of transplanted NSCs [Biomaterials 2009].
Our collaborators have previously demonstrated that a similar approach was effective for ectopically generating artificial lymph nodes with structural and functional similarity to their natural counterparts [Discovery Med 2011]. In this case, multiple chemokines carrying a collagen-binding peptide were loaded in a spatially controlled manner within a collagen sponge.