Research topics

During kidney development, nephron progenitor cells develop into the nephron, a functional unit of the kidney, via mesenchymal-epithelial transition (MET). We previously established an in vitro system in which METs can be artificially induced to human iPSCs-derived nephron progenitor cells by the stimulation of canonical-Wnt signalling. Interestingly, we further demonstrated that those MET events occur constantly and singularly within a uniform nephron progenitor population.
Here, in this research project, we aim to elucidate the mechanism that control such rare MET events using an in vitro MET occurrence system. For this purpose, we will construct a system for real-time monitoring of MET events, and then use a machine learning system to prospectively detect cells just before the onset of MET and analyze their characteristics.
This study will help to elucidate the mechanism of determining the number of nephrons in the human kidney, and the findings can be applied to the development of a new method for efficient artificial induction of kidney tissue in the future.

• 1. *Takasato, M., Er, P.X., Chiu, H.S., and Little, M.H. (2016). Generation of kidney organoids from human pluripotent stem cells. Nature Protocols 11, 1681–1692.
• 2. *Takasato, M., Er, P.X., Chiu, H.S., Maier, B., Baillie, G.J., Ferguson, C., Parton, R.G., Wolvetang, E.J., Roost, M.S., Chuva de Sousa Lopes, S.M., et al. (2015). Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature 526, 564–568.
• 3. Takasato, M., and *Little, M.H. (2015). The origin of the mammalian kidney: implications for recreating the kidney in vitro. Development 142, 1937–1947.

• Laboratory for Human Organogenesis
• Laboratory for Developmental Dynamics
• Laboratory for Biologically Inspired Computing

We have many technologies on microdevice development for rapid and high-throughput analysis of cells and has applied these technologies to life sciences. In BDR, we have fabricated and provided many microdevices for cell and tissue analysis to many laboratories or projects. Recently, this concept has been extended to molecules, namely nano scale and by combining with technologies for molecular measurement, simulation, or analysis in BDR, we aim to pioneer a new scientific field.
However, nanoscale channels are not only difficult to be fabricated, but also difficult to flow liquid in them. The scale is just between the molecules and bulk. So, it was suggested that water and other molecules behaviors are different from bulk. This field is difficult area not only as engineering but also science, but results with large impact is expected.
The final goal of this project is to construct a platform to manipulate single molecules in nanoscale. Before that, we aim to establish fundamental technologies including fabrication, fluid control, and measurement method in nanochannel with Okada TL.

• Laboratory for Cell Polarity Regulation

In November 2020, clinical research for the treatment of photoreceptor degeneration using neural retina sheets induced to differentiate from human iPS cells began. The neural retinal sheet used in this study was researched and developed at our institution, but at present, the retinal sheet for transplantation ismanually cut out fromtheneural retina toremove pigment epithelium and ciliary body.Butnot only does it require skillful techniques to cut out, but the size that can be cut out is as small as about 1 mm x 1mm, and many neural retinalsheets are required to cover a wide range of lesions, which requires enormous cost and labor. One solution is to develop a large neural retinal sheet. However, the technique for producing a largeretinal organoid that ensure qualityandsafetyhas not been establisheddue to the difficulties of controlling the differentiation from spatially and temporally heterogeneous cells.Therefore, in this project, we aim to develop a large-scale next-generation transplantation retinal sheet that suppresses differentiation into cells other than photoreceptor cells by reconstructing the retinal sheet only from retinal progenitor cells in a specific differentiation stage. Single cell (sc) RNA-seq is performed to understand the cell characteristics of retinal progenitor cells necessary for that purpose.

• Laboratory for Retinal Regeneration
• Laboratory for Prediction of Cell Systems Dynamics

The tissue stem cells are the most important cell population in adult tissueshomeostasis. In particular, the self-renewal and differentiation capacities of tissue stem cells are indispensable for tissue metabolism and regeneration from damage. The airway epithelium is exposed to invasivebacteria, viruses, and chemical stimulus thatmay damage the tissue. Airway epithelium has a relatively slow metabolism under normal conditions, but upon injury, cells proliferate rapidly to repair the damaged area. This type of tissue regeneration is called facultative regeneration in which stem cells activate their self-renewal capacity in response to injury. Multiple tissue stem cell populationshave been identified in airway epithelium that expected to respond to various type of damages to protect pulmonary system.
Recent innovations in stem cell culture techniques have made it possible to reconstitute mini-organs, called organoids, by culturingadult tissue stem cells for long periods of time in the laboratory. In lung organoid culture, lung tissue stem cells are isolated and grown into spheres by culturing them on a three-dimensional scaffold, which is suitable for studying lung stem cell dynamics. In this research project, we seek novel stem cells by combining scRNA-seq analysis with image analysis of lung tissue stem cells and organoids, which will be performed in collaboration with the Dr. Shiroguchi’s Laboratory. The candidates of the novel stem cell markers will be characterized using mouse lungs. In particular,in vivostem cell properties will be verified based on cell dynamics during metabolism and injury regeneration.

• Laboratory for Lung Development and Regeneration
• Laboratory for Prediction of Cell Systems Dynamics

Neural organoid is the three-dimensional neural tissue that is derived from pluripotent stem cells (PSCs). It is elusive how the neural epithelium develops during self-organizing formation of neural organoids. In this research subject, we will first induce neural organoids using knock-in human PSCs that can determine the generation of neural epithelium, and then perform 4D imaging to capture how the epithelial structure is constructed. Finally, we will elucidate the genetic background of the self-organizing formation of epithelial structure by single-cell RNA-seq analysis at the key point of the process. Combining the information of imaging and RNA-seq, it will be possible to explain how the complexed epithelial structure emerge.

• RIKEN BDR-Otsuka Pharmaceutical Collaboration Center (Sakaguchi lab)
• Laboratory for Cell Polarity Regulation

Aging is a biological process that occurs over an entire lifetime. Because aging is a rather complex process that takes a long time and is sensitive to genetic and environmental factors, a comprehensive understanding of the aging process remains unsolved. This research plan aims to comprehensively understand the aging process by monitoring changes in appearance, gene expression, metabolism, and muscle function over an entire lifetime using a genetically homogeneous model organism C.elegans. Furthermore, we examine the links between obtained data using the machine learning approach. This study would help us deepen our knowledge of the aging process and might predict the state of aging of an individual animal based on its appearance.

• Laboratory for Molecular Biology of Aging
• Laboratory for Developmental Dynamics
• Laboratory for Comprehensive Bioimaging

The three-dimensional organization of the genome within the cell nucleus is complex and plays an important role in various genome activities, including the regulation of transcription for gene expression. However, many aspects of the relationship between this 3D genome and gene regulation remain unelucidated. This project aims to deeply understand the relationship between the 3D genome structure and gene expression dynamics by integrative analysis using omics technologies. Furthermore, we aim to develop single-cell multi-omics technologies that simultaneously acquire 3D genome and gene expression. Through this study, if we can understand cellular states from a new perspective utilizing the information of the 3D genome structure, which differs from gene expression information, it is expected that this will lead to the development of technology for predicting cell differentiation lineages in the future, utilizing machine learning and other advanced techniques based on that information.

• Laboratory for Developmental Epigenetics
• Laboratory for Developmental Dynamics

When conducting genetic functional analysis of biopolymers or aiming to obtain molecules with desired properties and functions, evolutionary molecular engineering techniques are extremely useful because it can screen target molecules from a vast library of molecules by reproducing "mutation → selection → amplification" in vitro. However, typical evolutionary molecular engineering often selects molecules based on binding affinity. Therefore, we need to devise methods for selecting molecules focusing on functions beyond binding affinity. 　In this research, we aim to develop a simple method for activity-dependently selecting molecular variants expressed in artificial cells, intended as a general-purpose system. The system is composed of 3 steps as follows. First, mutant genes are individually encapsulated into semi-permeable membrane vesicles that allow only small molecules of about 2 to 3 kDa or smaller to pass through. Next, mutant genes are transcribed and translated into the target molecule in each vesicle using the cell-free protein synthesis system, the PURE system [Shimizu Y, Nat. Biotechnol., 2001]. Finally, fluorescent, chromogenic, and luminescent substrates infiltrate from outside the vesicle, and vesicles containing mutants are screened based on the light intensity when these substrates react with the molecule. This method is expected to enable us to obtain molecules with desired functions from a library of over approximately 105. If this research is successful, it will be possible to quantitatively measure the activity of biopolymers under various physiological conditions, such as the type and concentration of salts and metal ions, and pH. This will enable the analysis of the functions of target molecules in a general-purpose manner. It is also expected to contribute to the acquisition of enzymes that exhibit higher activity than natural molecules, and to the development of novel probes, biosensors, and drug candidate molecules.

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• Laboratory for Cell-Free Protein Synthesis
• Laboratory for Cell Polarity Regulation