||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..
||Inada H., Udono M., Matsuda-Ito K., Horisawa K., Ohkawa Y., Miura S., Goya T., Yamamoto J., Nagasaki M., Ueno K., Saitou D., Suyama M., Maehara Y., Kumamaru W., Ogawa Y., Sekiya S., Suzuki A., Direct reprogramming of human umbilical vein- and peripheral blood-derived endothelial cells into hepatic progenitor cells., Nat comm, 10.1038/s41467-020-19041-z, 11, 5292, 2020.12, Recent advances have enabled the direct induction of human tissue-specific stem and pro- genitor 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 tran- scription 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 hepatocytes and 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..
||Horisawa K., Udono M., Ueno K., Ohkawa Y., Nagasaki M., Sekiya S., Suzuki A., The Dynamics of Transcriptional Activation by Hepatic Reprogramming Factors., Mol Cell, 10.1016/j.molcel.2020.07.012, 79, 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..
||Terada, Maiko; Kawamata, Masaki; Kimura, Ryota; Sekiya, Sayaka; Nagamatsu, Go; Hayashi, Katsuhiko; Horisawa, Kenichi; Suzuki, Atsushi, 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.
||Takashima Y, Horisawa K, Udono M, Ohkawa Y, Suzuki A., Prolonged inhibition of hepatocellular carcinoma cell proliferation by combinatorial expression of defined transcription factors., Cancer Sci., 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 demon- strated 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 com- binations of TF could show more effective antitumor activity, and found that combi- natorial 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..
||Maiko Terada, Horisawa K, Shizuka Miura, 高島 康郎, Ohkawa, Y, Sayaka Sekiya, 松田 花菜江, Atsushi Suzuki, Kupffer cells induce Notch-mediated hepatocyte conversion in a common mouse model of intrahepatic cholangiocarcinoma., Sci Rep., doi: 10.1038/srep34691., 6, 34691-34691, 2016.10, Intrahepatic cholangiocarcinoma (ICC) is a malignant epithelial neoplasm composed of cells resembling cholangiocytes that line the intrahepatic bile ducts in portal areas of the hepatic lobule. Although ICC has been defined as a tumor arising from cholangiocyte transformation, recent evidence from genetic lineage-tracing experiments has indicated that hepatocytes can be a cellular origin of ICC by directly changing their fate to that of biliary lineage cells. Notch signaling has been identified as an essential factor for hepatocyte conversion into biliary lineage cells at the onset of ICC. However, the mechanisms underlying Notch signal activation in hepatocytes remain unclear. Here, using a mouse model of ICC, we found that hepatic macrophages called Kupffer cells transiently congregate around the central veins in the liver and express the Notch ligand Jagged-1 coincident with Notch activation in pericentral hepatocytes. Depletion of Kupffer cells prevents the Notch-mediated cell-fate conversion of hepatocytes to biliary lineage cells, inducing hepatocyte apoptosis and increasing mortality in mice. These findings will be useful for uncovering the pathogenic mechanism of ICC and developing prevenient and therapeutic strategies for this refractory disease..
||Niikura K, Horisawa K, Doi N, Endosomal escape efficiency of fusogenic B18 and B55 peptides fused with anti-EGFR single chain Fv as estimated by nuclear translocation., J Biochem., 10.1093/jb/mvv083., 159, 1, 123-132, 2016.01.