Kyushu University [Masaki Kawamata (Assistant Professor) Medical Institute of Bioregulation, Department of Molecular and Cellular Biology]

Masaki Kawamata

Last modified date：2023.11.22

Assistant Professor / Division of Organogenesis and Regeneration / Department of Molecular and Cellular Biology / Medical Institute of Bioregulation

Papers

1.	Masaki Kawamata, Hiroshi I. Suzuki, Ryota Kimura, Atsushi Suzuki, Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs., Nature Biomedical Engineering, 10.1038/s41551-023-01011-7, 7, 762-691, Accepted: 17 February 2023, 2022.04, [URL], The precise regulation of the activity of Cas9 is crucial for safe and efficient editing. Here we show that the genome-editing activity of Cas9 can be constrained by the addition of cytosine stretches to the 5′-end of conventional single-guide RNAs (sgRNAs). Such a ‘safeguard sgRNA’ strategy, which is compatible with Cas12a and with systems for gene activation and interference via CRISPR (clustered regularly interspaced short palindromic repeats), leads to the length-dependent inhibition of the formation of functional Cas9 complexes. Short cytosine extensions reduced p53 activation and cytotoxicity in human pluripotent stem cells, and enhanced homology-directed repair while maintaining bi-allelic editing. Longer extensions further decreased on-target activity yet improved the specificity and precision of mono-allelic editing. By monitoring indels through a fluorescence-based allele-specific system and computational simulations, we identified optimal windows of Cas9 activity for a number of genome-editing applications, including bi-allelic and mono-allelic editing, and the generation and correction of disease-associated single-nucleotide substitutions via homology-directed repair. The safeguard-sgRNA strategy may improve the safety and applicability of genome editing..
2.	Masaki Kawamata, Hiroshi H Suzuki, Ryota Kimura, Atsushi Suzuki, Programmable downsizing of CRISPR-Cas9 activity for precise and safe genome editing, bioRxiv, https://doi.org/10.1101/2020.10.31.361733, 2020.11.
3.	Masaki Kawamata, Atsushi Suzuki, Cell fate modification toward the hepatic lineage by extrinsic factors, Journal of biochemistry, 10.1093/jb/mvx028, 162, 1, 11-16, 2017.07, The lineage of a somatic cell can be altered by targeting its signaling networks with small molecules and/or genetically altering the expression of key transcription factors. Depending on the combination of factors, fibroblasts can be fully reprogrammed into induced pluripotent stem (iPS) cells or directly converted into specific cell lineages, bypassing the pluripotent state. The generation of defined target cells will enormously benefit patients who require cell transplantation therapy. In the decade, since iPS cells were first generated, many cell types have been induced fromfibroblasts by direct conversion, including hepatocytes. Converted hepatocytelike cells have been shown to repopulate liver tissues after transplantation in mouse liver disease models, suggesting promise for future application in humans.Thus, to realize safe and efficient cell transplantation therapy, various methods for generating hepatocyte-like cells are being developed. In this review, we summarize the current methods for the generation of hepatocyte-like cells via cell fate modification using extrinsic factors..
4.	Maiko Terada, Masaki Kawamata, Ryota Kimura, Sayaka Sekiya, Go Nagamatsu, Katsuhiko Hayashi, Kenichi Horisawa, Atsushi Suzuki, Generation of Nanog reporter mice that distinguish pluripotent stem cells from unipotent primordial germ cells, Genesis, 10.1002/dvg.23334, 57, 11-12, 2019.11, Nanog is a core transcription factor specifically expressed not only in the pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced PSCs (iPSCs), but also in the unipotent primordial germ cells (PGCs). Although Nanog promoter/enhancer regions are well characterized by in vitro analyses, direct correlations between the regulatory elements for Nanog expression and in vivo expression patterns of Nanog have not been fully clarified. In this study, we generated Nanog-RFP transgenic (Tg) mice in which expression of red fluorescent protein (RFP) is driven by a 5.2 kb Nanog promoter/enhancer region. As expected, RFP was expressed in the inner cell mass of blastocysts, ESCs, and iPSCs. However, RFP fluorescence was not observed in PGCs, although Nanog was expressed in PGCs. Because RFP fluorescence was visible in the PGC-derived pluripotent EGCs in culture, it was suggested that the reporter gene expression was specifically activated in PSCs. In conclusion, we have generated a novel Nanog-RFP Tg mouse line that can selectively tag PSCs over unipotent PGCs..
5.	Takeshi Katsuda, Masaki Kawamata, Ayako Inoue, Tomoko Yamaguchi, Maki Abe, Takahiro Ochiya, Long-term maintenance of functional primary human hepatocytes using small molecules, FEBS Letters, 10.1002/1873-3468.13582, 594, 1, 114-125, 2020.01, The immediate deterioration of primary human hepatocytes (PHHs) during culture limits their utility in drug discovery studies. Here, we report that a cocktail of four small molecule signaling inhibitors, termed YPAC, is useful for maintaining various hepatic functions of PHHs, including albumin and urea productivity, glycogen storage, and cytochrome P450 (CYP) expression. Most importantly, we found that YPAC allows PHHs to retain enzymatic activities of CYP1A2, CYP2B6, and CYP3A4 even after 40 days of culture, and that inducibility of CYP3A4 activity in response to the prototypical inducers rifampicin and phenobarbital is also maintained. Our novel approach could facilitate drug discovery studies..
6.	Aya Yoshimura, Naoki Adachi, Hitomi Matsuno, Masaki Kawamata, Yusuke Yoshioka, Hisae Kikuchi, Haruki Odaka, Tadahiro Numakawa, Hiroshi Kunugi, Takahiro Ochiya, Yoshitaka Tamai, The Sox2 promoter-driven CD63-GFP transgenic rat model allows tracking of neural stem cell-derived extracellular vesicles, DMM Disease Models and Mechanisms, 10.1242/dmm.028779, 11, 1, 2018.01, Extracellular vesicles (EVs) can modulate microenvironments by transferring biomolecules, including RNAs and proteins derived from releasing cells, to target cells. To understand the molecular mechanisms maintaining the neural stem cell (NSC) niche through EVs, a new transgenic (Tg) rat strain that can release human CD63-GFP-expressing EVs from the NSCs was established. Human CD63-GFP expression was controlled under the rat Sox2 promoter (Sox2/ human CD63-GFP), and it was expressed in undifferentiated fetal brains. GFP signals were specifically observed in in vitro cultured NSCs obtained from embryonic brains of the Tg rats. We also demonstrated that embryonic NSC (eNSC)-derived EVs were labelled by human CD63-GFP. Furthermore, when we examined the transfer of EVs, eNSC-derived EVs were found to be incorporated into astrocytes and eNSCs, thus implying an EV-mediated communication between different cell types around NSCs. This new Sox2/human CD63-GFP Tg rat strain should provide resources to analyse the cell-to-cell communication via EVs in NSC microenvironments..
7.	Masaki Kawamata, Takeshi Katsuda, Yasuhiro Yamada, Takahiro Ochiya, In vitro reconstitution of breast cancer heterogeneity with multipotent cancer stem cells using small molecules., Biochem Biophys Res Commun, 2017.01, A small fraction of tumor cells are thought to possess the potential for both multiple-lineage differentiation and self-renewal, which underlies the cancer stem cell hypothesis. However, the differentiation mechanisms of these cells have not been elucidated due to a lack of appropriate culture methods. Here, we established a culture condition for maintaining multipotent tumor cells from rat breast tumors using 4 small molecules. Cultured tumor cells in this condition retained their intrinsic myoepithelial features, expressing p63 and CK14 and vimentin. In a xenograft model, the p63-expressing cells formed epithelial tumors containing glandular, squamous and sebaceous compartments. Upon withdrawal of the small molecules, p63 and CK14 expression was lost, with concurrent increase in expression of mesenchymal markers. These transited cells acquired drug resistance and invasiveness and showed massive sarcomatoid tumorigenicity. Epithelial features could not be recovered by re-exposure to the small molecules in the transited cells. Here, we have identified multipotent cancer cells within primary mammary tumors and demonstrated that their plasticity is maintained by the small molecules..
8.	Takeshi Katsuda, Masaki Kawamata, Keitaro Hagiwara, Ryou-u Takahashi, Yusuke Yamamoto, Fernando D. Camargo, Takahiro Ochiya, Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity., Cell Stem Cell, 2017.01, A challenge for advancing approaches to liver regeneration is loss of functional differentiation capacity when hepatocyte progenitors are maintained in culture. Recent lineage-tracing studies have shown that mature hepatocytes (MHs) convert to an immature state during chronic liver injury, and we investigated whether this conversion could be recapitulated in vitro and whether such converted cells could represent a source of expandable hepatocytes. We report that a cocktail of small molecules, Y-27632, A-83-01, and CHIR99021, can convert rat and mouse MHs in vitro into proliferative bipotent cells, which we term chemically induced liver progenitors (CLiPs). CLiPs can differentiate into both MHs and biliary epithelial cells that can form functional ductal structures. CLiPs in long-term culture did not lose their proliferative capacity or their hepatic differentiation ability, and rat CLiPs were shown to extensively repopulate chronically injured liver tissue. Thus, our study advances the goals of liver regenerative medicine..
9.	Aya Yoshimura, Masaki Kawamata, Yusuke Yoshioka, Takeshi Katsuda, Yoshitaka Nagai, Naoki Adachi, Tadahiro Numakawa, Hiroshi Kunugi, Takahiro Ochiya, Yoshitaka Tamai, Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids, Sci Rep, 6, 31172, 2016.08.
10.	James Hong, Hong He, Phuoc Bui, Ben Ryba-White, Mohammad A.K. Rumi, Michael J. Soares, Debasree Dutta, Soumen Paul, Masaki Kawamata, Takahio Ochiya, Qi-Long Ying, Pavan Rajanahalli, Mark L. Weiss, A Focused Microarray for Screening Rat Embryonic Stem Cell Lines, Stem Cells Dev, 22, 3, 431-443, 2013.02.
11.	Masaki Kawamata, Takahiro Ochiya, Two distinct knockout approaches highlight a critical role for p53 in rat development, Sci Rep, 2, 945, 2012.12.
12.	Masaki Kawamata, Takahiro Ochiya, Generation of genetically modified rats from embryonic stem cells, PNAS, 107, 32, 14223-14228, 2010.08.
13.	Fumitaka Takeshita, Lubna Patrawala, Mitsuhiko Osaki, Ryou-u Takahashi, Yusuke Yamamoto, Nobuyoshi Kosaka, Masaki Kawamata, Kevin Kelnar, Andreas G. Bader, David Brown, Takahiro Ochiya, Systemic delivery of synthetic microRNA-16 inhibits the growth of metastatic prostate tumors via downregulation of multiple cell-cycle genes, Mol Ther, 18, 1, 181-187, 2010.01.
14.	Shinobu Ueda, Masaki Kawamata, Takumi Teratani, Taku Shimizu, Yoshitaka Tamai, Hiromasa Ogawa, Katsuyuki Hayashi, Hiroyuki Tsuda, Takahiro Ochiya, Establishment of rat embryonic stem cells and making of chimera rats, PLoS ONE, 3, 7, e2800, 2008.07.
15.	Agnieszka Banas, Takumi Teratani, Yusuke Yamamoto, Makoto Tokuhara, Fumitaka Takeshita, Mitsuhiko Osaki, Masaki Kawamata, Takashi Kato, Hitoshi Okochi, Takahiro Ochiya, In vivo therapeutic potential of human Adipose Tissue Mesenchymal Stem Cells (AT-MSCs) after transplantation into mice with liver injury, Stem Cells, 26, 10, 2705-2712, 2008.10.
16.	Masaki Kawamata, Yutaka Tonomura, Tadashi Kimura, Yukihiko Sugimoto, Teruyuki Yanagisawa, Katsuhiko Nishimori, Oxytocin-induced phasic and tonic contractions are modulated by the contractile machinery rather than the quantity of oxytocin receptor, Am J Physiol Endocrinol Metab, 292, 4, 992-999, 2007.04.
17.	Masaki Kawamata, Hiroaki Inoue, Katsuhiko Nishimori, Male-specific function of Dmrt7 by sexually dimorphic translation in mouse testis, Sex Dev, 1, 5, 297-304, 2007.11.
18.	Masaki Kawamata, Masahide Yoshida, Yukihiko Sugimoto, Tadashi Kimura, Yutaka Tonomura, Yuki Takayanagi, Teruyuki Yanagisawa, Katsuhiko Nishimori, Infusion of oxytocin induces successful delivery in prostanoid FP-receptor-deficient mice, Mol Cell Endocrinol, 283, 1-2, 32-37, 2008.02.
19.	Masaki Kawamata, Minori Mitsui-Saito, Tadashi Kimura, Yuki Takayanagi, Teruyuki Yanagisawa, Katsuhiko Nishimori, Vasopressin-induced contraction of uterus is mediated solely by the oxytocin receptor in mice, but not in humans, Eur J Pharmacol, 472, 3, 229-234, 2003.07.
20.	Masaki Kawamata, Yutaka Tonomura, Tadashi Kimura, Teruyuki Yanagisawa, Katsuhiko Nishimori, The differential coupling of oxytocin receptors to uterine contractions in murine estrous cycle, Biochem Biophys Res Commun, 321, 3, 695-699, 2004.08.
21.	Yuki Takayanagi, Masahide Yoshida, Isadora F. Bielsky, Heather E. Ross, Masaki Kawamata, Tatsushi Onaka, Teruyuki Yanagisawa, Tadashi Kimura, Martin M. Matzuk, Larry J. Young, Katsuhiko Nishimori, Pervasive social deficits, but normal parturition, in oxytocin receptor-deficient mice, PNAS, 102, 44, 16096-16101, 2005.11.
22.	Masaki Kawamata, Katsuhiko Nishimori, Mice deficient in Dmrt7 show infertility with spermatogenic arrest at pachytene stage, FEBS Lett, 580, 27, 6442-6446, 2006.11.

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