Springer Science and Business Media LLC Acta Medica Okayama 0920-9069 75 3 2023 GSK-3α/β and MEK inhibitors assist the microenvironment of tumor initiation 243 253 EN Ghmkin Hassan Department of Cancer Stem Cell Engineering, Faculty of Interdisciplinary Science and Engineering in Health Systems, Institute of Academic and Research, Okayama University Said M. Afify Department of Cancer Stem Cell Engineering, Faculty of Interdisciplinary Science and Engineering in Health Systems, Institute of Academic and Research, Okayama University Maram H. Zahra Department of Cancer Stem Cell Engineering, Faculty of Interdisciplinary Science and Engineering in Health Systems, Institute of Academic and Research, Okayama University Hend M. Nawara Department of Cancer Stem Cell Engineering, Faculty of Interdisciplinary Science and Engineering in Health Systems, Institute of Academic and Research, Okayama University Kazuki Kumon Department of Cancer Stem Cell Engineering, Faculty of Interdisciplinary Science and Engineering in Health Systems, Institute of Academic and Research, Okayama University Yoshiaki Iwasaki Health Service Center, Okayama University David S. Salomon Center for Cancer Research, National Cancer Institute Akimasa Seno Department of Cancer Stem Cell Engineering, Faculty of Interdisciplinary Science and Engineering in Health Systems, Institute of Academic and Research, Okayama University Masaharu Seno Department of Cancer Stem Cell Engineering, Faculty of Interdisciplinary Science and Engineering in Health Systems, Institute of Academic and Research, Okayama University Induced pluripotent stem cells (iPSCs) are useful tools for modeling diseases and developing personalized medicine. We have been developing cancer stem cells (CSCs) from iPSCs with conditioned medium (CM) of cancer-derived cells as the mimicry of the microenvironment of tumor initiation. However, the conversion of human iPSCs has not always been efficient with only CM. In this study, human iPSCs reprogrammed from monocytes of healthy volunteers were cultured in a media containing 50% of the CM from human pancreatic cancer derived BxPC3 cells supplemented with a MEK inhibitor (AZD6244) and a GSK-3α/β inhibitor (CHIR99021). The survived cells were assessed for the characteristics of CSCs in vitro and in vivo. As a result, they exhibited CSC phenotypes of self-renewal, differentiation, and malignant tumorigenicity. Primary culture of the malignant tumors of the converted cells exhibited the elevated expression of CSC related genes CD44, CD24 and EPCAM maintaining the expression of stemness genes. In conclusion, the inhibition of GSK-3α/β and MEK and the microenvironment of tumor initiation mimicked by the CM can convert human normal stem cells into CSCs. This study could provide insights into establishing potentially novel personalized cancer models which could help investigate the tumor initiation and screening of personalized therapies on CSCs. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2045-2322 12 1 2022 Cancer stem cells induced by chronic stimulation with prostaglandin E2 exhibited constitutively activated PI3K axis 15628 EN Hideki Minematsu Laboratory of Nao‑Biotechnology, Division of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Said M. Afify Division of Biochemistry, Chemistry Department, Faculty of Science, Menoufa University Yuki Sugihara R&D Center, Katayama Chemicals Ind., Co. Ltd, Ina, Minoh Ghmkin Hassan Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Maram H. Zahra Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akimasa Seno Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaki Adachi R&D Center, Katayama Chemicals Ind., Co. Ltd, Ina, Minoh Masaharu Seno Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Previously, our group has demonstrated establishment of Cancer Stem Cell (CSC) models from stem cells in the presence of conditioned medium of cancer cell lines. In this study, we tried to identify the factors responsible for the induction of CSCs. Since we found the lipid composition could be traced to arachidonic acid cascade in the CSC model, we assessed prostaglandin E2 (PGE2) as a candidate for the ability to induce CSCs from induced pluripotent stem cells (iPSCs). Mouse iPSCs acquired the characteristics of CSCs in the presence of 10 ng/mL of PGE2 after 4 weeks. Since constitutive Akt activation and pik3cg overexpression were found in the resultant CSCs, of which growth was found independent of PGE2, chronic stimulation of the receptors EP-2/4 by PGE2 was supposed to induce CSCs from iPSCs through epigenetic effect. The bioinformatics analysis of the next generation sequence data of the obtained CSCs proposed not only receptor tyrosine kinase activation by growth factors but also extracellular matrix and focal adhesion enhanced PI3K pathway. Collectively, chronic stimulation of stem cells with PGE2 was implied responsible for cancer initiation enhancing PI3K/Akt axis. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2045-2322 12 1 2022 The significance of ErbB2/3 in the conversion of induced pluripotent stem cells into cancer stem cells 2711 EN Ghmkin Hassan Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Maram H. Zahra Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akimasa Seno Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaharu Seno Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Cancer stem cells (CSCs) are suggested to be responsible for drug resistance and aggressive phenotypes of tumors. Mechanisms of CSC induction are still under investigation. Our lab has established a novel method to generate CSCs from iPSCs under a cancerous microenvironment mimicked by the conditioned medium (CM) of cancer-derived cells. Here, we analyzed the transcriptome of CSCs, which were converted from iPSCs with CM from pancreatic ductal adenocarcinoma cells. The differentially expressed genes were identified and used to explore pathway enrichment. From the comparison of the CSCs with iPSCs, genes with elevated expression were related to the ErbB2/3 signaling pathway. Inhibition of either ErbB2 with lapatinib as a tyrosine kinase inhibitor or ErbB3 with TX1-85-1 or siRNAs arrested cell proliferation, inhibited the in vitro tumorigenicity, and lead to loss of stemness in the converting cells. The self-renewal and tube formation abilities of cells were also abolished while CD24 and Oct3/4 levels were reduced, and the MAPK pathway was overactivated. This study shows a potential involvement of the ErbB2/ErbB3 pathway in CSC generation and could lead to new insight into the mechanism of tumorigenesis and the way of cancer prevention. No potential conflict of interest relevant to this article was reported.

BMC Acta Medica Okayama 1756-9966 41 1 2022 Different pancreatic cancer microenvironments convert iPSCs into cancer stem cells exhibiting distinct plasticity with altered gene expression of metabolic pathways 29 EN Ghmkin Hassan Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Toshiaki Ohara Department of Pathology and Experimental Medicine, Medical School, Okayama University Said M. Afify Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kazuki Kumon Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Maram H. Zahra Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Xiaoying Fu Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mohamad Al Kadi Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University Akimasa Seno Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University David S. Salomon Mouse genetics program, Center for Cancer Research, National Cancer Institute Masaharu Seno Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Background<br> Cancer stem cells (CSCs) are generated under irregular microenvironment in vivo, of which mimic is quite difficult due to the lack of enough information of the factors responsible for cancer initiation. Here, we demonstrated that mouse induced pluripotent cells (miPSCs) reprogrammed from normal embryonic fibroblasts were susceptible to the microenvironment affected by cancer cells to convert into CSCs in vivo. <br> Methods Three different pancreatic cancer line cells, BxPC3, PANC1, and PK8 cells were mixed with miPSCs and subcutaneously injected into immunodeficient mice. Tumors were evaluated by histological analysis and cells derived from iPSCs were isolated and selected from tumors. The isolated cells were characterized for cancer stem cell characters in vitro and in vivo as well as their responses to anticancer drugs. The impact of co-injection of iPSCs with cancer cells on transcriptome and signaling pathways of iPSCs was investigated. <br> Results The injection of miPSCs mixed with human pancreatic cancer cells into immunodeficient mice maintained the stemness of miPSCs and changed their phenotype. The miPSCs acquired CSC characteristics of tumorigenicity and self-renewal. The drug responses and the metastatic ability of CSCs converted from miPSCs varied depending on the microenvironment of cancer cells. Interestingly, transcriptome profiles of these cells indicated that the pathways related with aggressiveness and energy production were upregulated from the levels of miPSCs.<br> Conclusions<br> Our result suggests that cancer-inducing microenvironment in vivo could rewire the cell signaling and metabolic pathways to convert normal stem cells into CSCs. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2045-2322 11 1 2021 Differentiation of cancer stem cells into erythroblasts in the presence of CoCl2 23977 EN Kazuki Kumon Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Said M. Afify Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ghmkin Hassan Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Shunsuke Ueno Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Sadia Monzur Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Hend M. Nawara Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Hagar A. Abu Quora Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mona Sheta Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yanning Xu Department of Pathology, Tianjin Central Hospital of Gynecology Obstetrics, Nankai University Affiliated Maternity Hospital, Tianjin Key Laboratory of Human Development and Reproductive Regulation Xiaoying Fu Department of Pathology, Tianjin University of Traditional Chinese Medicine Maram H. Zahra Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akimasa Seno Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaharu Seno Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Cancer stem cells (CSCs) are subpopulations in the malignant tumors that show self-renewal and multilineage differentiation into tumor microenvironment components that drive tumor growth and heterogeneity. In previous studies, our group succeeded in producing a CSC model by treating mouse induced pluripotent stem cells. In the current study, we investigated the potential of CSC differentiation into blood cells under chemical hypoxic conditions using CoCl2. CSCs and miPS-LLCcm cells were cultured for 1 to 7 days in the presence of CoCl2, and the expression of VEGFR1/2, Runx1, c-kit, CD31, CD34, and TER-119 was assessed by RT-qPCR, Western blotting and flow cytometry together with Wright-Giemsa staining and immunocytochemistry. CoCl2 induced significant accumulation of HIF-1 alpha changing the morphology of miPS-LLCcm cells while the morphological change was apparently not related to differentiation. The expression of VEGFR2 and CD31 was suppressed while Runx1 expression was upregulated. The population with hematopoietic markers CD34(+) and c-kit(+) was immunologically detected in the presence of CoCl2. Additionally, high expression of CD34 and, a marker for erythroblasts, TER-119, was observed. Therefore, CSCs were suggested to differentiate into erythroblasts and erythrocytes under hypoxia. This differentiation potential of CSCs could provide new insight into the tumor microenvironment elucidating tumor heterogenicity. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2409-9279 3 3 2020 Metastasis Model of Cancer Stem Cell-Derived Tumors 60 EN Hager Mansour Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Ghmkin Hassan Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Said M. Afify Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ting Yan Department of Pathology, Shanxi Key Laboratory of Carcinogenesis and Translational Research on Esophageal Cancer, Shanxi Medical University Akimasa Seno Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Masaharu Seno Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Metastasis includes the dissemination of cancer cells from a malignant tumor and seed in distant sites inside the body forming secondary tumors. Metastatic cells from the primary tumor can move even before the cancer is detected. Therefore, metastases are responsible for more than 90% of cancer-related deaths. Over recent decades there has been adequate evidence suggesting the existence of CSCs with self-renewing and drug-resistant potency within heterogeneous tumors. Cancer stem cells (CSCs) act as a tumor initiating cells and have roles in tumor retrieve and metastasis. Our group recently developed a unique CSC model from mouse induced pluripotent stem cells cultured in the presence of cancer cell-conditioned medium that mimics tumors microenvironment. Using this model, we demonstrated a new method for studying metastasis by intraperitoneal transplantation of tumors and investigate the metastasis ability of cells from these segments. First of all, CSCs were injected subcutaneously in nude mice. The developed malignant tumors were minimized then transplanted into the peritoneal cavity. Following this, the developed tumor in addition to lung, pancreas and liver were then excised and analyzed. Our method showed the metastatic potential of CSCs with the ability of disseminated and moving to blood circulation and seeding in distant organs such as lung and pancreas. This method could provide a good model to study the mechanisms of metastasis according to CSC theory. No potential conflict of interest relevant to this article was reported.

e-Century Publishing Corp. Acta Medica Okayama 2156-6976 11 7 2021 Microenvironment of mammary fat pads affected the characteristics of the tumors derived from the induced cancer stem cells 3475 3495 EN Hagar A. Abu Quora Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Maram H. Zahra Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Samah El-Ghlban Division of Biochemistry, Faculty of Science, Menoufia University Neha Nair Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Said M. Afify Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ghmkin Hassan Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Hend M. Nawara Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Mona Sheta Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Sadia Monzur Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Xiaoying Fu Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Amira Osman Department of Histology, Faculty of Medicine, Kafr Elsheikh University Akimasa Seno Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaharu Seno Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Breast cancer is the first common cause of cancer-related death in women worldwide. Since the malignancy and aggressiveness of breast cancer have been correlated with the presence of breast cancer stem cells, the establishment of a disease model with cancer stem cells is required for the development of a novel therapeutic strategy. Here, we aimed to evaluate the availability of cancer stem cell models developed from mouse induced pluripotent stem cells with the conditioned medium of different subtypes of breast cancer cell lines, the hormonal-responsive T47D cell line and the triple-negative breast cancer BT549 cell line, to generate in vivo tumor models. When transplanted into the mammary fat pads of BALB/c nude mice, these two model cells formed malignant tumors exhibiting pronounced histopathological characteristics similar to breast cancers. Serial transplantation of the primary cultured cells into mammary fat pads evoked the same features of breast cancer, while this result was perturbed following subcutaneous transplantation. The tumors formed in the mammary fat pads exhibited immune reactivities to prolactin receptor, progesterone receptor, green florescent protein, Ki67, CD44, estrogen receptor alpha/beta and cytokeratin 8, while all of the tumors and their derived primary cells exhibited immunoreactivity to estrogen receptor alpha/beta and cytokeratin 8. Cancer stem cells can be developed from pluripotent stem cells via the secretory factors of cancer-derived cells with the capacity to inherit tissue specificity. However, cancer stem cells should be plastic enough to be affected by the microenvironment of specific tissues. In summary, we successfully established a breast cancer tumor model using mouse induced pluripotent stem cells developed from normal fibroblasts without genetic manipulation. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2227-9059 9 5 2021 Paclitaxel-Based Chemotherapy Targeting Cancer Stem Cells from Mono- to Combination Therapy 500 EN Hend M. Nawara Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Said M. Afify Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ghmkin Hassan Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Maram H. Zahra Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akimasa Seno Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaharu Seno Department of Biotechnology and Drug Discovery, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Paclitaxel (PTX) is a chemotherapeutical agent commonly used to treat several kinds of cancer. PTX is known as a microtubule-targeting agent with a primary molecular mechanism that disrupts the dynamics of microtubules and induces mitotic arrest and cell death. Simultaneously, other mechanisms have been evaluated in many studies. Since the anticancer activity of PTX was discovered, it has been used to treat many cancer patients and has become one of the most extensively used anticancer drugs. Regrettably, the resistance of cancer to PTX is considered an extensive obstacle in clinical applications and is one of the major causes of death correlated with treatment failure. Therefore, the combination of PTX with other drugs could lead to efficient therapeutic strategies. Here, we summarize the mechanisms of PTX, and the current studies focusing on PTX and review promising combinations. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 1044579X 2021 Cooperation between NRF2-mediated transcription and MDIG-dependent epigenetic modifications in arsenic-induced carcinogenesis and cancer stem cells EN Zhuoyue Bi Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University Qian Zhang Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University Yao Fu Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University Akimasa Seno Faculty of Engineering, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Priya Wadgaonkar Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University Yiran Qiu Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University Bandar Almutairy Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University Liping Xu Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University Wenxuan Zhang Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University Chitra Thakur Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University Fei Chen Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University Environmental exposure to arsenic, a well-established carcinogen linked to a number of human cancers, is a public health concern in many areas of the world. Despite extensive studies on the molecular mechanisms of arsenic-induced carcinogenesis, how initial cellular responses, such as activation of stress kinases and the generation of reactive oxygen species, converge to affect the transcriptional and/or epigenetic reprogramming required for the malignant transformation of normal cells or normal stem cells remains to be elucidated. In this review, we discuss some recent discoveries showing how the transcription factor NRF2 and an epigenetic regulator, MDIG, contribute to the arsenic-induced generation of cancer stem-like cells (CSCs) as determined by applying CRISPR-Cas9 gene editing and chromosome immunoprecipitation followed by DNA sequencing (ChIP-seq). No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2227-9717 9 1 2020 Cancer Stem Cell Microenvironment Models with Biomaterial Scaffolds In Vitro 45 EN Ghmkin Hassan Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Said M. Afify Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Shiro Kitano Technical Research Institute, Toppan Printing Co., Ltd. Akimasa Seno Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Hiroko Ishii GSP Enterprise, Inc. Yucheng Shang Department of Applied Chemistry, Graduate School of Engineering, Osaka University Michiya Matsusaki Department of Applied Chemistry, Graduate School of Engineering, Osaka University Masaharu Seno Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Defined by its potential for self-renewal, differentiation and tumorigenicity, cancer stem cells (CSCs) are considered responsible for drug resistance and relapse. To understand the behavior of CSC, the effects of the microenvironment in each tissue are a matter of great concerns for scientists in cancer biology. However, there are many complicated obstacles in the mimicking the microenvironment of CSCs even with current advanced technology. In this context, novel biomaterials have widely been assessed as in vitro platforms for their ability to mimic cancer microenvironment. These efforts should be successful to identify and characterize various CSCs specific in each type of cancer. Therefore, extracellular matrix scaffolds made of biomaterial will modulate the interactions and facilitate the investigation of CSC associated with biological phenomena simplifying the complexity of the microenvironment. In this review, we summarize latest advances in biomaterial scaffolds, which are exploited to mimic CSC microenvironment, and their chemical and biological requirements with discussion. The discussion includes the possible effects on both cells in tumors and microenvironment to propose what the critical factors are in controlling the CSC microenvironment focusing the future investigation. Our insights on their availability in drug screening will also follow the discussion. No potential conflict of interest relevant to this article was reported.

Springer Nature Acta Medica Okayama 0007-0920 122 9 2020 A novel model of liver cancer stem cells developed from induced pluripotent stem cells 1378 1390 EN Said M. Afify Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Anna Sanchez Calle Division of Molecular and Cellular Medicine, National Cancer Center Research Institute Ghmkin Hassan Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Kazuki Kumon Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Hend M. Nawara Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Maram H. Zahra Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Hager M. Mansour Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Apriliana Cahya Khayrani Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Md Jahangir Alam Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Juan Du Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Akimasa Seno Okayama University Research Laboratory of Stem Cell Engineering in Detroit, IBio, Wayne State University Yoshiaki Iwasaki Department of Gastroenterology and Hepatology, Graduate School of Medicine, Okayama University Masaharu Seno Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Background</br> Liver cancer is the second most common cause of cancer-related death. Every type of tumours including liver cancer contains cancer stem cells (CSCs). To date, the molecular mechanism regulating the development of liver CSCs remains unknown.</br> Methods</br> In this study, we tried to generate a new model of liver CSCs by converting mouse induced pluripotent stem cells (miPSCs) with hepatocellular carcinoma (HCC) cell line Huh7 cells conditioned medium (CM). miPSCs treated with CM were injected into the liver of BALB/c nude mice. The developed tumours were then excised and analysed.</br> Results</br> The primary cultured cells from the malignant tumour possessed self-renewal capacity, differentiation potential and tumorigenicity in vivo, which were found rich in liver cancer-associated markers as well as CSC markers.</br> Conclusions</br> We established a model of liver CSCs converting from miPS and showed different stages of stemness during conversion process. Our CSC model will be important to assess the molecular mechanisms necessary to develop liver CSCs and could help in defeating liver cancer. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-6694 12 6 2020 Paclitaxel and Sorafenib: The Effective Combination of Suppressing the Self-Renewal of Cancer Stem Cells 1360 EN Hend M. Nawara Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Said M. Afify Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ghmkin Hassan Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Maram H. Zahra Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Marwa N. Atallah Vertebrates Embryology and Comparative Anatomy, Zoology Department, Faculty of Science, Menoufia University Hager Mansour Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Hagar A. Abu Quora Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Md Jahangir Alam Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Amira Osman Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Hiroki Kakuta Division of Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiroki Hamada Department of Life Science, Faculty of Science, Okayama University of Science Akimasa Seno Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaharu Seno Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Combination therapy, which is a treatment modality combining two or more therapeutic agents, is considered a cornerstone of cancer therapy. The combination of anticancer drugs, of which functions are different from the other, enhances the efficiency compared to the monotherapy because it targets cancer cells in a synergistic or an additive manner. In this study, the combination of paclitaxel and sorafenib in low concentration was evaluated to target cancer stem cells, miPS-BT549cmP and miPS-Huh7cmP cells, developed from mouse induced pluripotent stem cells. The synergistic effect of paclitaxel and sorafenib on cancer stem cells was assessed by the inhibition of proliferation, self-renewal, colony formation, and differentiation. While the IC(50)values of paclitaxel and sorafenib were approximately ranging between 250 and 300 nM and between 6.5 and 8 mu M, respectively, IC(50)of paclitaxel reduced to 20 and 25 nM, which was not toxic in a single dose, in the presence of 1 mu M sorafenib, which was not toxic to the cells. Then, the synergistic effect was further assessed for the potential of self-renewal of cancer stem cells by sphere formation ability. As a result, 1 mu M of sorafenib significantly enhanced the effect of paclitaxel to suppress the number of spheres. Simultaneously, paclitaxel ranging in 1 to 4 nM significantly suppressed not only the colony formation but also the tube formation of the cancer stem cells in the presence of 1 mu M sorafenib. These results suggest the combination therapy of paclitaxel and sorafenib in low doses should be an attractive approach to target cancer stem cells with fewer side effects. No potential conflict of interest relevant to this article was reported.

NATURE PUBLISHING GROUP Acta Medica Okayama 2045-2322 10 1 2020 Signaling Inhibitors Accelerate the Conversion of mouse iPS Cells into Cancer Stem Cells in the Tumor Microenvironment 9955 EN Juan Du Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Yanning Xu Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Saki Sasada Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Aung Ko Ko Oo Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Ghmkin Hassan Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Hafizah Mahmud Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Apriliana Cahya Khayrani Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Md Jahangir Alam Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Kazuki Kumon Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Ryo Uesaki Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Said M. Afify Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Hager M. Mansour Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Neha Nair Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Maram H. Zahra Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Akimasa Seno Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Nobuhiro Okada Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ling Chen Department of Pathology, Tianjin Central Hospital of Gynecology Obstetrics Ting Yan Department of Pathology, Shanxi Key Laboratory of Carcinogenesis and Translational Research on Esophageal Cancer, Shanxi Medical University Masaharu Seno Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Cancer stem cells (CSCs) are a class of cancer cells characterized by self-renewal, differentiation and tumorigenic potential. We previously established a model of CSCs by culturing mouse induced pluripotent stem cells (miPSCs) for four weeks in the presence of a conditioned medium (CM) of cancer cell lines, which functioned as the tumor microenvironment. Based on this methodology of developing CSCs from miPSCs, we assessed the risk of 110 non-mutagenic chemical compounds, most of which are known as inhibitors of cytoplasmic signaling pathways, as potential carcinogens. We treated miPSCs with each compound for one week in the presence of a CM of Lewis lung carcinoma (LLC) cells. However, one-week period was too short for the CM to convert miPSCs into CSCs. Consequently, PDO325901 (MEK inhibitor), CHIR99021 (GSK-3 beta inhibitor) and Dasatinib (Abl, Src and c-Kit inhibitor) were found to confer miPSCs with the CSC phenotype in one week. The tumor cells that survived exhibited stemness markers, spheroid formation and tumorigenesis in Balb/c nude mice. Hence, we concluded that the three signal inhibitors accelerated the conversion of miPSCs into CSCs. Similarly to our previous study, we found that the PI3K-Akt signaling pathway was upregulated in the CSCs. Herein, we focused on the expression of relative genes after the treatment with these three inhibitors. Our results demonstrated an increased expression of pik3ca, pik3cb, pik3r5 and pik3r1 genes indicating class IA PI3K as the responsible signaling pathway. Hence, AKT phosphorylation was found to be up-regulated in the obtained CSCs. Inhibition of Erk1/2, tyrosine kinase, and/or GSK-3 beta was implied to be involved in the enhancement of the PI3K-AKT signaling pathway in the undifferentiated cells, resulting in the sustained stemness, and subsequent conversion of miPSCs into CSCs in the tumor microenvironment. No potential conflict of interest relevant to this article was reported.

Amber Publication Acta Medica Okayama 2347-2367 8 1 2020 Upregulated CCL20 and CCR6 in Cancer Stem Cells Converted from Mouse iPS Cells 200 207 EN Juan Du Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Akimasa Seno Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama Universityalth Systems Saki Sasada Graduate School of Natural Science and Technology, Okayama University Yanning Xu Department of Pathology, Tianjin Central Hospital of Gynecology Obstetrics Aung Ko Ko Oo Graduate School of Natural Science and Technology, Okayama University Ghmkin Hassan Graduate School of Natural Science and Technology, Okayama University Shunsuke Ueno Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Said M. Afify Graduate School of Natural Science and Technology, Okayama University Maram H Zahra Graduate School of Natural Science and Technology, Okayama University Nobuhiro Okada Graduate School of Natural Science and Technology, Okayama University Ling Chen Department of Pathology, Tianjin Central Hospital of Gynecology Obstetrics Xiaoying Fu School of Integrative Medicine, Tianjin University of Traditional Chinese Medicine Heizo Tokutaka SOM Japan Ting Yan The Hong Kong University of Science and Technology Medical Center, Shenzhen Peking University Masaharu Seno Graduate School of Natural Science and Technology, Okayama University Background: Cancer stem cells (CSCs) as a class of malignant cancer cells play an important role in tumor progression. Previous studies by our group have demonstrated the establishment of the model of CSCs converting mouse iPS cells (miPSCs) into CSCs by treating the miPSCs with a conditioned medium (CM) of Lewis Lung Carcinoma (LLC) cells with or without the nonmutagenic chemical compounds. CSCs converted from miPSCs developed highly malignant adenocarcinoma when subcutaneously transplanted into the nude mice.</br> Methods: The miPSCs were treated with each compound for 1 week in the presence of a CM of LLC cells. We evaluated the gene expression in the resultant CSCs comparing that in miPSCs by microarray analysis. And the expression of chemokine (C-C motif) ligand 20 (CCL20) and C-C chemokine receptor type 6 (CCR6) in converted cells were evaluated by rt-qPCR. The CCR6 expression in converted cells and primary cells were determined by flow cytometry.</br> Results: As the result, the expression of CCL20 was found upregulated in the presence of CM supplemented with PD0325901. Then we assessed the expression of CCR6, which was considered to be stimulated by CCL20. Then the expression of CCR6 was also found up-regulated. Interestingly, IL17A expression was also observed only in the CSCs from the primary tumor implying the effect of tumor microenvironment. Moreover, significantly high level of CCR6 was showed in flow cytometric analysis.</br> Conclusion: These results suggest that a model of CSCs with CCL20-CCR6 autocrine loop was obtained as the result of the conversion of iPSCs. This CSC should be a good model to study targeting CCR6 as a G protein-coupled receptor (GPCR). No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-6694 12 4 2020 Revisiting Cancer Stem Cells as the Origin of Cancer-Associated Cells in the Tumor Microenvironment: A Hypothetical View from the Potential of iPSCs 879 EN Amira Osman Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Said M. Afify Graduate School of Natural Science and Technology, Okayama University Ghmkin Hassan Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Xiaoying Fu Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akimasa Seno Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaharu Seno Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University The tumor microenvironment (TME) has an essential role in tumor initiation and development. Tumor cells are considered to actively create their microenvironment during tumorigenesis and tumor development. The TME contains multiple types of stromal cells, cancer-associated fibroblasts (CAFs), Tumor endothelial cells (TECs), tumor-associated adipocytes (TAAs), tumor-associated macrophages (TAMs) and others. These cells work together and with the extracellular matrix (ECM) and many other factors to coordinately contribute to tumor growth and maintenance. Although the types and functions of TME cells are well understood, the origin of these cells is still obscure. Many scientists have tried to demonstrate the origin of these cells. Some researchers postulated that TME cells originated from surrounding normal tissues, and others demonstrated that the origin is cancer cells. Recent evidence demonstrates that cancer stem cells (CSCs) have differentiation abilities to generate the original lineage cells for promoting tumor growth and metastasis. The differentiation of CSCs into tumor stromal cells provides a new dimension that explains tumor heterogeneity. Using induced pluripotent stem cells (iPSCs), our group postulates that CSCs could be one of the key sources of CAFs, TECs, TAAs, and TAMs as well as the descendants, which support the self-renewal potential of the cells and exhibit heterogeneity. In this review, we summarize TME components, their interactions within the TME and their insight into cancer therapy. Especially, we focus on the TME cells and their possible origin and also discuss the multi-lineage differentiation potentials of CSCs exploiting iPSCs to create a society of cells in cancer tissues including TME. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2072-6694 12 1 2019 Hematopoietic Cells Derived from Cancer Stem Cells Generated from Mouse Induced Pluripotent Stem Cells 82 EN Ghmkin Hassan Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Said M. Afify Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Neha Nair Graduate School of Natural Science and Technology, Okayama University Kazuki Kumon Graduate School of Natural Science and Technology, Okayama University Amira Osman Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Juan Du Graduate School of Natural Science and Technology, Okayama University Hager Mansour Hagar A. Abu Quora Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Hend M. Nawara Graduate School of Natural Science and Technology, Okayama University Ayano Satoh Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Maram H. Zahra Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Nobuhiro Okada Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Akimasa Seno Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masaharu Seno Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Cancer stem cells (CSCs) represent the subpopulation of cancer cells with the ability to differentiate into other cell phenotypes and initiated tumorigenesis. Previously, we reported generating CSCs from mouse induced pluripotent stem cells (miPSCs). Here, we investigated the ability of the CSCs to differentiate into hematopoietic cells. First, the primary cells were isolated from malignant tumors that were formed by the CSCs. Non-adherent cells (NACs) that arose from adherent cells were collected and their viability, as well as the morphology and expression of hematopoietic cell markers, were analyzed. Moreover, NACs were injected into the tail vein of busulfan conditioned Balb/c nude mice. Finally, CSCs were induced to differentiate to macrophages while using IL3 and SCF. The round nucleated NACs were found to be viable, positive for hematopoietic lineage markers and CD34, and expressed hematopoietic markers, just like homing to the bone marrow. When NACs were injected into mice, Wright-Giemsa staining showed that the number of white blood cells got higher than those in the control mice after four weeks. CSCs also showed the ability to differentiate toward macrophages. CSCs were demonstrated to have the potential to provide progenies with hematopoietic markers, morphology, and homing ability to the bone marrow, which could give new insight into the tumor microenvironment according to the plasticity of CSCs. No potential conflict of interest relevant to this article was reported.

Spandidos Publications Acta Medica Okayama 1792-1074 18 3 2019 Cancer stem cell induction from mouse embryonic stem cells 2756 2762 EN Akimasa Seno Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Chikae Murakami Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University Bishoy El‑Aarag Laboratory of Nano-Biotechnology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Yoshiaki Iwasaki Health Service Center, Okayama University Toshiaki Ohara Department of Pathology and Experimental MedicineOkayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Masaharu Seno Laboratory of Nano-Biotechnology, Okayama University Graduate School of Interdisciplinary Science and Engineering in Health Systems Although cancers are often removed by surgery and treated by chemotherapy and/or radiation therapies, they often reoccur following treatment due to the presence of resistant residual cells such as cancer stem cells (CSCs). CSCs are characterized by their self-renewal, pluripotency, and tumorigenicity properties, and are promising therapeutic targets for the complete therapy of cancers; however, the number of CSCs in cancer tissue is typically too small to investigate fully. We have previously reported that CSCs could be established from induced pluripotent stem cells (iPSCs) using a conditioned medium during cancer cell culture. In the present study, mouse embryonic stem cells (mESCs) were observed to be converted to CSCs (mES-CSCs). This demonstrated that CSC induction does not exclusively occur following gene editing in somatic cells, and that conditioned medium from cancer cells may contain factors that can induce CSCs. Therefore, not only iPSCs but also mESCs, were demonstrated to be able to produce CSCs as one of the potentials of pluripotency of stem cells, suggesting that the conversion to CSCs is not specific to iPSCs. The resultant mES-CSCs would be also useful to generate tissue specific cancers and these naturally occurring cancers can contribute to drug screenings, but also undergo further investigation in order to reveal cancer mechanisms. No potential conflict of interest relevant to this article was reported.