| 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, 5, 672-691, 2023.04, 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. |
Takeshi Goya, Kenichi Horisawa, Miyako Udono, Yasuyuki Ohkawa, Yoshihiro Ogawa, Sayaka Sekiya, Atsushi Suzuki, Direct conversion of human endothelial cells into liver cancer-forming cells using nonintegrative episomal vectors, Hepatology Communications, 10.1002/hep4.1911, 2022.02, Liver cancer is an aggressive cancer associated with a poor prognosis. Development of therapeutic strategies for liver cancer requires fundamental research using suitable experimental models. Recent progress in direct reprogramming technology has enabled the generation of many types of cells that are difficult to obtain and provide a cellular resource in experimental models of human diseases. In this study, we aimed to establish a simple one-step method for inducing cells that can form malignant human liver tumors directly from healthy endothelial cells using nonintegrating episomal vectors. To screen for factors capable of inducing liver cancer-forming cells (LCCs), we selected nine genes and one short hairpin RNA that suppresses tumor protein p53 (TP53) expression and introduced them into human umbilical vein endothelial cells (HUVECs), using episomal vectors. To identify the essential factors, we examined the effect of changing the amounts and withdrawing individual factors. We then analyzed the proliferation, gene and protein expression, morphologic and chromosomal abnormality, transcriptome, and tumor formation ability of the induced cells. We found that a set of six factors, forkhead box A3 (FOXA3), hepatocyte nuclear factor homeobox 1A (HNF1A), HNF1B, lin-28 homolog B (LIN28B), MYCL proto-oncogene, bHLH transcription factor (L-MYC), and Kruppel-like factor 5 (KLF5), induced direct conversion of HUVECs into LCCs. The gene expression profile of these induced LCCs (iLCCs) was similar to that of human liver cancer cells, and these cells effectively formed tumors that resembled human combined hepatocellular-cholangiocarcinoma following transplantation into immunodeficient mice. Conclusion: We succeeded in the direct induction of iLCCs from HUVECs by using nonintegrating episomal vectors. iLCCs generated from patients with cancer and healthy volunteers will be useful for further advancements in cancer research and for developing methods for the diagnosis, treatment, and prognosis of liver cancer.. |
| 3. |
Hiroki Inada, Miyako Udono, Kanae Matsuda-Ito, Kenichi Horisawa, Yasuyuki Ohkawa, Shizuka Miura, Takeshi Goya, Junpei Yamamoto, Masao Nagasaki, Kazuko Ueno, Daisuke Saitou, Mikita Suyama, Yoshihiko Maehara, Wataru Kumamaru, Yoshihiro Ogawa, Sayaka Sekiya, Atsushi Suzuki, Direct reprogramming of human umbilical vein- and peripheral blood-derived endothelial cells into hepatic progenitor cells, Nature Communications, 10.1038/s41467-020-19041-z, 11, 1, 5292, 2020.10, Recent advances have enabled the direct induction of human tissue-specific stem and progenitor cells from differentiated somatic cells. However, it is not known whether human hepatic progenitor cells (hHepPCs) can be generated from other cell types by direct lineage reprogramming with defined transcription factors. Here, we show that a set of three transcription factors, FOXA3, HNF1A, and HNF6, can induce human umbilical vein endothelial cells to directly acquire the properties of hHepPCs. These induced hHepPCs (hiHepPCs) propagate in long-term monolayer culture and differentiate into functional hepatocytes and cholangiocytes by forming cell aggregates and cystic epithelial spheroids, respectively, under three-dimensional culture conditions. After transplantation, hiHepPC-derived hepatocytesand cholangiocytes reconstitute damaged liver tissues and support hepatic function. The defined transcription factors also induce hiHepPCs from endothelial cells circulating in adult human peripheral blood. These expandable and bipotential hiHepPCs may be useful in the study and treatment of human liver diseases.. |
| 4. |
Kenichi Horisawa, Miyako Udono, Kazuko Ueno, Yasuyuki Ohkawa, Masao Nagasaki, Sayaka Sekiya, Atsushi Suzuki, The dynamics of transcriptional activation by hepatic reprogramming factors, Molecular Cell, 10.1016/j.molcel.2020.07.012, 79, 4, 660-676, 2020.08, Specific combinations of two transcription factors (Hnf4a plus Foxa1, Foxa2, or Foxa3) can induce direct conversion of mouse fibroblasts into hepatocyte-like cells. However, the molecular mechanisms underlying hepatic reprogramming are largely unknown. Here, we show that the Foxa protein family members and Hnf4a sequentially and cooperatively bind to chromatin to activate liver-specific gene expression. Although all Foxa proteins bind to and open regions of closed chromatin as pioneer factors, Foxa3 has the unique potential of transferring from the distal to proximal regions of the transcription start site of target genes, binding RNA polymerase II, and co-traversing target genes. These distinctive characteristics of Foxa3 are essential for inducing the hepatic fate in fibroblasts. Similar functional coupling of transcription factors to RNA polymerase II may occur in other contexts whereby transcriptional activation can induce cell differentiation.. |
| 5. |
Shizuka Miura, Atsushi Suzuki, Induction of steatohepatitis and liver tumorigenesis by enforced Snail expression in hepatocytes, The American Journal of Pathology, 10.1016/j.ajpath.2020.02.005, 190, 6, 1271-1283, 2020.06, Snail is a transcription factor that regulates many cellular events involved in development, homeostasis, and disease. In hepatocellular carcinoma (HCC), Snail induces epithelial-to-mesenchymal transition that confers invasive properties on tumor cells during HCC progression and malignancy. Snail activation observed in HCC mouse models suggests its involvement in not only progression, but also onset of HCC. However, it remains unclear whether Snail directly contributes to HCC initiation or whether it supports HCC initiation promoted by other oncogenes. In this study, we generated mouse models for liver-specific and hepatocyte-specific overexpression of Snail to reveal the independent roles of Snail in liver homeostasis and disease. Our data demonstrated that enforced Snail expression resulted in liver and hepatocyte enlargement, inflammatory cell infiltration in the liver, lipid accumulation in hepatocytes, substantial increases in serum alanine transaminase and bile acids, yellow discoloration of tissues caused by bilirubin accumulation, and liver tumorigenesis. Snail overexpression suppressed mRNA expression of the tight junction components claudins and occludin and that of proteins associated with bile acid metabolism, leading to disruption of the biliary canaliculus formed among hepatocytes and excretion of abnormal amounts of unusual bile acids from hepatocytes. In conclusion, enforced Snail expression in hepatocytes is sufficient for induction of steatohepatitis and liver tumorigenesis through disruption of the biliary canaliculus and bile acid homeostasis in the liver.. |
| 6. |
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, e23334, 2019.11. |
| 7. |
Yasuo Takashima, Kenichi Horisawa, Miyako Udono, Yasuyuki Ohkawa, Atsushi Suzuki, Prolonged inhibition of hepatocellular carcinoma cell proliferation by combinatorial expression of defined transcription factors, Cancer Science, 10.1111/cas.13798, 109, 11, 3543-3553, 2018.11, Hepatocellular carcinoma (HCC) accounts for a large proportion of liver cancer cases and has an extremely poor prognosis. Therefore, novel innovative therapies for HCC are strongly desired. As gene therapy tools for HCC, 2 hepatic transcription factors (TF), HNF4A and HNF1A, have been used to suppress proliferation and to extinguish cancer-specific characteristics of target cells. However, our present data demonstrated that single transduction of HNF4A or HNF1A had only a limited effect on suppression of HCC cell proliferation. Thus, in this study, we examined whether combinations of TF could show more effective antitumor activity, and found that combinatorial transduction of 3 hepatic TF, HNF4A, HNF1A and FOXA3, suppressed HCC cell proliferation more stably than single transduction of these TF. The combinatorial transduction also suppressed cancer-specific phenotypes, such as anchorage-independent growth in culture and tumorigenicity after transplantation into mice. HCC cell lines transduced with the 3 TF did not recover their proliferative property after withdrawal of anticancer drugs, indicating that combinatorial expression of the 3 TF suppressed the growth of all cell subtypes within the HCC cell lines, including cancer stem-like cells. Transcriptome analyses revealed that the expression levels of a specific gene set involved in cell proliferation were only decreased in HCC cells overexpressing all 3 TF. Moreover, combined transduction of the 3 TF could facilitate hepatic differentiation of HCC cell lines. Our strategy for inducing stable inhibition and functional differentiation of tumor cells using a defined set of TF will become an effective therapeutic strategy for various types of cancers.. |
| 8. |
Junpei Yamamoto, Miyako Udono, Shizuka Miura, Sayaka Sekiya, Atsushi Suzuki, Cell aggregation culture induces functional differentiation of induced hepatocyte-like cells through activation of Hippo signaling, Cell Reports, 10.1016/j.celrep.2018.09.010, 25, 1, 183-198, 2018.10, Recent progress in direct lineage reprogramming has enabled the generation of induced hepatocyte-like (iHep) cells and revealed their potential as an alternative to hepatocytes for medical applications. However, the hepatic functions of iHep cells are insufficient compared with those of primary hepatocytes. Here, we show that cell-aggregate formation can rapidly induce growth arrest and hepatic maturation of iHep cells through activation of Hippo signaling. During formation of iHep cell aggregates, Yap inactivation is induced by actin reorganization and intercellular adhesion, leading to upregulation of Hnf1α expression in the absence of the Yap/Tead/Chd4 transcriptional repressor unit. Hnf1α then acts as a central transcription factor that regulates liver-enriched gene expression in iHep cell aggregates and induces functional differentiation of iHep cells. Moreover, iHep cell aggregates efficiently reconstitute injured liver tissues and support hepatic function after transplantation. Thus, iHep cell aggregates may provide insights into basic research and potential therapies for liver diseases. Yamamoto et al. show that cell-aggregate formation induces functional differentiation of hepatocyte-like cells, designated iHep cells, which are directly induced from mouse fibroblasts. Hepatic maturation of iHep cells is regulated by activation of Hippo signaling that leads to upregulation of Hnf1α expression for induction of liver-enriched gene expression.. |
| 9. |
Shizuka Miura, Atsushi Suzuki, Generation of Mouse and Human Organoid-Forming Intestinal Progenitor Cells by Direct Lineage Reprogramming, Cell Stem Cell, 10.1016/j.stem.2017.08.020, 21, 4, 456-471.e5, 2017.10, Intestinal organoids hold great promise as a valuable tool for studying and treating intestinal diseases. The currently available sources of human intestinal organoids, tissue fragments or pluripotent stem cells, involve invasive procedures or complex differentiation protocols, respectively. Here, we show that a set of four transcription factors, Hnf4α, Foxa3, Gata6, and Cdx2, can directly reprogram mouse fibroblasts to acquire the identity of fetal intestine-derived progenitor cells (FIPCs). These induced FIPCs (iFIPCs) form spherical organoids that develop into adult-type budding organoids containing cells with intestinal stem cell properties. The resulting stem cells produce all intestinal epithelial cell lineages and undergo self-renewing cell divisions. After transplantation, the induced spherical and budding organoids can reconstitute colonic and intestinal epithelia, respectively. The same combination of four defined transcription factors can also induce human iFIPCs. This alternative approach for producing intestinal organoids may well facilitate application for disease analysis and therapy development. Miura and Suzuki describe direct conversion of mouse fibroblasts to cells resembling fetal intestine-derived progenitor cells that can give rise to intestinal stem cell organoids and reconstitute injured colonic tissues after transplantation. They also show that a similar approach can work to make human induced intestinal progenitor cells.. |
| 10. |
Sayaka SEKIYA, Shizuka MIURA, Kanae MATSUDA-ITO, Atsushi SUZUKI, Myofibroblasts Derived from Hepatic Progenitor Cells Create the Tumor Microenvironment, Stem Cell Reports, 10.1016/j.stemcr.2016.11.002, 7, 6, 1130-1139, 2016.12, 本研究では、成体マウス肝前駆細胞が、肝細胞や胆管上皮細胞だけでなく、筋線維芽細胞にも分化できることを見出した。通常、培養下における筋線維芽細胞への分化頻度はとても低いが、p53欠損肝前駆細胞から形成される腫瘍では、ドナー細胞由来の筋線維芽細胞が数多く観察され、上皮系腫瘍組織を取り囲む間質組織として「腫瘍微小環境」の様態を呈していた。したがって、p53を欠損した肝前駆細胞は、自らが腫瘍を形成するだけでなく、腫瘍形成をサポートする微小環境をも自ら作り出していることが判明した。. |
| 11. |
Maiko TERADA, Kenichi HORISAWA, Shizuka MIURA, Yasuo TAKASHIMA, Yasuyuki OHKAWA, Sayaka SEKIYA, Kanae MATSUDA-ITO, Atsushi SUZUKI, Kupffer cells induce Notch-mediated hepatocyte conversion in a common mouse model of intrahepatic cholangiocarcinoma, Scientific Reports, 10.1038/srep34691, 6, 2016.10, 本研究では、肝内胆管がんにおける肝細胞の運命転換において、肝臓中に存在するマクロファージであるクッパー細胞が障害部位に集積し、肝細胞を刺激してNotchシグナルの活性化を促していることを明らかにした。. |
| 12. |
Yasuo Takashima, Maiko Terada, Miyako Udono, Shizuka Miura, Junpei Yamamoto, Atsushi Suzuki, Suppression of lethal-7b and miR-125a/b Maturation by Lin28b Enables Maintenance of Stem Cell Properties in Hepatoblasts, Hepatology, 10.1002/hep.28548, 64, 1, 245-260, 2016.07, In liver development, hepatoblasts that act as hepatic stem/progenitor cells proliferate and differentiate into both hepatocytes and cholangiocytes to form liver tissues. Although numerous factors contribute to this event, little is known about the roles of microRNAs in hepatoblast proliferation and differentiation. In this study, we focused on the lineage-28 (Lin28) family proteins, which are required for microRNA regulation in pluripotent stem cells and cancer cells, and investigated their roles as regulatory factors for the properties of hepatoblasts. Conclusion: Lin28b was specifically expressed in hepatoblasts, and its suppression induced growth arrest and cholangiocyte differentiation of hepatoblasts; mechanistically, Lin28b positively regulates the expression of Lin28b itself and cell cycle–related proteins in hepatoblasts by suppressing the maturation of target microRNAs, lethal-7b and miR-125a/b, enabling maintenance of the stem cell properties of hepatoblasts, such as their capabilities for proliferation and bi-lineage differentiation, during liver development. (Hepatology 2016;64:245–260).. |
| 13. |
Yasuo TAKASHIMA, Maiko TERADA, Masuyo KAWABATA, Atsushi SUZUKI, Dynamic three-dimensional morphogenesis of intrahepatic bile ducts in mouse liver development, Hepatology, 10.1002/hep.27436, 61, 3, 1003-1011, 2015.03, 肝臓の発生では、肝幹細胞（肝芽細胞）から分化した胆管上皮細胞が門脈周囲にductal plateと呼ばれる細胞層を形成し、発生プログラムにしたがって徐々に肝内胆管を形成していく。肝内胆管は肝臓内で管構造を形成するため、その形態形成過程を詳しく調べるには、肝発生初期から胆管上皮細胞を連続的かつ立体的に解析する必要がある。しかしながら、肝内胆管の発生は、これまで主に組織切片を用いた二次元平面で解析されており、三次元形態形成過程の解析や形態計測学に基づいた定量的な解析は行われていなかった。そこで我々は、マウスの胎仔から成体に至るまでの肝内胆管の立体構造をコンピューター上で再構築することでモデル化し、成長する管構造の長さや枝の数、結合予測値、門脈からの離散距離といった形態計測学的な事項について解析を行い、肝内胆管の発生過程を詳細かつ定量的に調べた。その結果、三次元再構築モデルを用いた時空間的な観察と形態計測学的解析による定量的な解析によって、立体的かつ動的な胆管形成モデルを構築することに成功した。. |
| 14. |
Shizuka MIURA, Atsushi SUZUKI, Rapid cell-fate conversion of mouse fibroblasts into hepatocyte-like cells, Inflammation and Regeneration, http://doi.org/10.2492/inflammregen.34.211, 34, 5, 211-216, 2014.12, これまでの研究で、我々はマウスの線維芽細胞にHnf4αとFoxa（Foxa1、Foxa2、Foxa3のいずれかひとつ）という肝細胞分化に関連した2種類の転写因子を導入することで、線維芽細胞を直接肝細胞の性質を有する細胞（iHep細胞）へと変化させることに成功した。本研究では、線維芽細胞からiHep細胞へのリプログラミングと細胞増殖の関係を明らかにすべく、リプログラミング過程における細胞増殖の様子を動画解析するとともに、遺伝子・タンパク質の経時的発現解析を行った。その結果、線維芽細胞にiHep細胞誘導因子を導入すると、わずか48時間でiHep細胞が出現し、増殖を開始することが明らかとなった。. |
| 15. |
Shizuka MIURA, Atsushi SUZUKI, Acquisition of lipid metabolic capability in hepatocyte-like cells directly induced from mouse fibroblasts, Frontiers in Cell and Developmental Biology, 10.3389/fcell.2014.00043, 2, 43, 1-6, 2014.08, これまでの研究で、我々はマウスの線維芽細胞にHnf4αとFoxa（Foxa1、Foxa2、Foxa3のいずれかひとつ）という肝細胞分化に関連した2種類の転写因子を導入することで、線維芽細胞を直接肝細胞の性質を有する細胞（iHep細胞）へと変化させることに成功した。そこで本研究では、iHep細胞の脂質代謝に関する機能を解析した。その結果、iHep細胞は肝細胞と同様に中性脂肪の合成や蓄積と分泌が可能であり、既知の脂肪酸合成阻害薬にも反応できることが示された。. |
| 16. |
Sayaka SEKIYA, Atsushi SUZUKI, Hepatocytes, rather than cholangiocytes, can be the major source of primitive ductules in the chronically injured mouse liver, American Journal of Pathology, 184, 5, 1468-1478, 2014.05, 本研究では、慢性的な肝障害によって門脈周囲に出現する偽胆管様構造（細胆管反応）の由来を明らかにすべく、誘導型Cre/loxPシステムを用いた細胞系譜追跡実験を行った。その結果、偽胆管を形成する細胞は、胆管上皮細胞の特徴をもつにも関わらず、Notchシグナルを介した肝細胞の運命転換によって、肝細胞から生じることが判明した。以上の結果は、慢性的な障害に対して再生を繰り返し行う肝臓では、正常な再生応答から逸脱した特殊な状況に陥ることによって肝細胞の分化状態が破綻し、肝細胞が胆管上皮細胞の特徴を有する偽胆管細胞に変化することを示している。我々は、このような現象を「疾患関連リプログラミング」と呼び、癌などの難治性疾患との関係に注目している。. |
| 17. |
Sayaka SEKIYA, Atsushi SUZUKI, Intrahepatic cholangiocarcinoma can arise from Notch-mediated conversion of hepatocytes, The Journal of Clinical Investigation, 122, 11, 3914-3918, 2012.11, 肝内胆管がんは、肝臓に発生する悪性腫瘍の中で2番目に多く、その発症率や死亡率は近年世界的に増加している。ウイルス性肝炎に起因する肝細胞がんとは異なり、その発症原因は不明で、放射線療法や化学療法による治療効果は低く、肝切除が唯一の治療法ともいえる。肝内胆管がんとは、肝臓内で胆管を形成する胆管上皮細胞から発生する悪性腫瘍と定義されているが、ウイルス性肝炎の患者にも肝内胆管がんの発生がしばしば見られることから、肝細胞の形質転換に由来する可能性も排除できない。そこで我々は、肝内胆管がんが従来の考え通りに胆管上皮細胞から生じるのか、それとも実際は肝細胞から生じる腫瘍なのかを検証すべく、それぞれの細胞を特異的に標識し、それらの子孫を正確に追跡できる遺伝子改変マウスを作製した。そして、作製したマウスに肝内胆管がんを発症させ、形成された肝内胆管がんが肝細胞と胆管上皮細胞のどちらを起源としているのかを調べた。その結果、これまでの常識を覆し、肝内胆管がんが、胆管上皮細胞ではなく肝細胞から生じる腫瘍であることを発見した。さらに、肝内胆管がんの形成過程において、肝細胞が胆管上皮細胞に似た細胞へと変化するためには、肝細胞におけるNotchシグナルの活性化が重要なことも明らかにした。. |
| 18. |
Sekiya S. and Suzuki A., Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors, Nature, 10.1038/nature10263, 475, 7356, 390-393, 2011.06, 肝細胞は多くの転写因子の働きによって胎生期に肝前駆細胞から分化するのが普通だが、稀に、障害を受けた膵臓の外分泌細胞や骨髄などに含まれる間葉系幹細胞から肝細胞が分化することがある。また、骨髄移植後に血液細胞が肝細胞と融合し、肝細胞として肝臓組織を構築することもある。これらの事象は、肝細胞以外の細胞を肝細胞に変化させる因子の存在を示唆しており、ある環境下ではそれらの因子が活性化して肝細胞以外の細胞を肝細胞に変化させていると考えられる。したがって、もし、このような肝細胞の運命決定因子を同定することができれば、それらを使って皮膚の線維芽細胞を肝細胞へと直接変化させることが可能になるかもしれない。そこで今回、我々は、肝細胞の運命決定を担う特定因子を同定し、マウスの線維芽細胞から肝細胞を直接作り出すことを試みた。その結果、線維芽細胞にHnf4αとFoxa（Foxa1、Foxa2、Foxa3のいずれかひとつ）という肝細胞分化に関連した2種類の転写因子を導入することで、線維芽細胞を肝細胞の性質をもった細胞（iHep細胞）へと直接変化させることに成功し、肝細胞の運命決定因子を同定した。作製したiHep細胞は肝細胞の形態的特徴や遺伝子・タンパク質発現を有し、肝細胞特有の機能をもったまま培養下での増殖や維持、凍結保存が可能であった。また、肝機能不全で死に至る高チロシン血症モデルマウスの肝臓へiHep細胞を移植すると、肝細胞として障害を受けた肝臓組織を機能的に再構築し、マウスの致死率を大幅に減少させることが可能であった。本法では、わずか2種類の転写因子を線維芽細胞に発現させるだけで、人工多能性幹細胞（iPS細胞）を経由することなく、線維芽細胞から直接肝細胞を作製可能なことから、移植医療や創薬研究、バイオ人工肝臓の開発などへの応用が期待される。. |
| 19. |
Sekiya S. and Suzuki A., Glycogen synthase kinase 3β-dependent Snail degradation directs hepatocyte proliferation in normal liver regeneration, Proc Natl Acad Sci USA, 108, 27, 11175-11180, 2011.06. |
