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Human gastric development has not been well studied. The generation of human pluripotent stem cell-derived gastric organoids (hGOs) comprising gastric marker-expressing epithelium without an apparent smooth muscle (SM) structure has been reported. We modified previously reported protocols to generate hGOs with muscularis mucosa (MM) from hiPSCs. Time course analyses revealed that epithelium development occurred prior to MM formation. Sonic hedgehog (SHH) and TGF-fl1 were secreted by the epithelium. HH and TGF-fl signal inhibition prevented subepithelial MM formation. A mechanical property of the substrate promoted SM differentiation around hGOs in the presence of TGF-fl. TGF-fl signaling was shown to influence the HH signaling and mechanical properties. In addition, clinical specimen findings suggested the involvement of TGF-fl signaling in MM formation in recovering gastric ulcers. HH and TGF-fl signaling from the epithelium to the stroma and the mechanical properties of the subepithelial environment may influence the emergence of MM in human stomach tissue.

Introduction of OCT3/4, KLF4 and SOX2 into SW480 using polycistronic vector.

Cancer stem cells (CSC) are capable of reconstructing cancer tissues, are involved in both recurrence and metastasis, and contribute to therapeutic resistance. Therefore, elucidating the molecular mechanism in CSCs is important to successfully treat unresectable cancers. Previously, we observed that colon cancer stem-like cells can be induced from human colon cancer cell lines by retrovirally introducing OCT3/4, SOX2, and KLF4, and we have designated such cells as induced cancer stem cells (iCSC). In the current study, we used iCSCs to evaluate the molecular mechanism of colon CSCs and developed new methods to control them. The spheres that were derived in vitro from the iCSCs, but not those from parental cells, mimicked human colon cancer tissues in terms of their immunohistologic patterns; therefore, sphere-forming ability was assessed as a measure of the tissue-reconstructing ability of iCSCs. Interestingly, the calcineurin inhibitor FK506 enhanced the sphere-forming ability of iCSCs, whereas GSK3 inhibition by RNAi, CHIR99021, and valproic acid (VPA) impeded the sphere-forming ability and expansion of iCSCs. FK506 and GSK3 inhibition showed the opposite effect regarding the NFATc3 localization of iCSCs. These data reveal the crucial role that NFAT localization, as regulated by calcineurin and GSK3, plays in the tissue-reconstructing ability of colon cancer stem cells and the potential of GSK3 inhibitors, such as VPA, in colon cancer stem cell–targeting therapy.Implications: This study identifies signaling pathways that contribute to the tissue-reconstructing capacity of colon CSCs and suggests that clinically used drugs could be repurposed to improve unresectable colon cancers. Mol Cancer Res; 15(10); 1455–66. ©2017 AACR.

Effects of calcineurin inhibitors and GSK3 inhibitors on 2nd V50 cells.

Effects of knock down of RCAN2 by siRNA on 2nd V50 cells.

Microarray Probe List

Knock down of GSK3α and GSK3β by siRNAs.

Co-culture of cancer cells, human umbilical vascular endothelial cells (HUVECs) and mesenchymal stem cells (MSCs).

Primer List

RNA isolation and quantitative reverse-transcriptase polymerase chain reaction

Self-assembly of synthetic molecules has been drawing broad attention as a novel emerging approach in drug discovery. Here, we report selective cell death induced by a novel peptide amphiphile that self-assembles to form entangled nanofibers (hydrogel) based on intracellular pH (pHi). We found that a palmitoylated hexapeptide (C16-VVAEEE) formed a hydrogel below pH 7. The formation of the nanofibrous self-assembly was responsive to a small pH change around pH 7. The cytotoxicity of C16-VVAEEE was correlated with pHi of cells. Microscope observation demonstrated the self-assembly of C16-VVAEEE inside HEK293 cells. In vivo experiments revealed that the transcutaneous administration of C16-VVAEEE showed remarkable anti-tumor activity. This study proposes that distinct microenvironment inside living cells can be used as a trigger for the intracellular self-assembly of a peptide amphiphile, which provide a new clue to drug discovery.

[Background & Aims] The Notch signaling pathway is an important pathway in the adult pancreas and in pancreatic ductal adenocarcinoma (PDAC), with hairy and enhancer of split-1 (HES1) as the core molecule in this pathway. However, the roles of HES1 in the adult pancreas and PDAC formation remain controversial. [Methods] We used genetically engineered dual-recombinase mouse models for inducing Hes1 deletion under various conditions. [Results] The loss of Hes1 expression in the adult pancreas did not induce phenotypic alterations. However, regeneration was impaired after caerulein-induced acute pancreatitis. In a pancreatic intraepithelial neoplasia (PanIN) mouse model, PanINs rarely formed when Hes1 deletion preceded PanIN formation, whereas more PanINs were formed when Hes1 deletion succeeded PanIN formation. In a PDAC mouse model, PDAC formation was also enhanced by Hes1 deletion after PanIN/PDAC development; therefore, Hes1 promotes PanIN initiation but inhibits PanIN/PDAC progression. RNA sequencing and chromatin immunoprecipitation-quantitative polymerase chain reaction revealed that Hes1 deletion enhanced epithelial-to-mesenchymal transition via Muc5ac up-regulation in PDAC progression. The results indicated that HES1 is not required for maintaining the adult pancreas under normal conditions, but is important for regeneration during recovery from pancreatitis; moreover, Hes1 plays different roles, depending on the tumor condition. [Conclusions] Our findings highlight the context-dependent roles of HES1 in the adult pancreas and pancreatic cancer.

Late-onset hypogonadism (LOH) syndrome, due to a partial lack of testosterone, decreases the quality of life of older men. Testosterone is mainly secreted by Leydig cells in the testes. Leydig cell transplantation is expected to be a promising alternative to conventional testosterone replacement therapy for LOH syndrome. We herein report a simple and robust protocol for directed differentiation of human induced pluripotent stem cells (hiPSCs) into Leydig-like cells by doxycycline-inducible overexpression of NR5A1 and treatment with a combination of 8-bromoadenosine-3′,5′-cyclic monophosphate (8-Br-cAMP) and forskolin. The differentiated cells expressed the steroidogenic enzyme genes STAR, CYP11A1, CYP17A1, and HSD3B2 and the specific markers of adult Leydig cells HSD17B3, INSL3, and LHCGR. Furthermore, we confirmed the secretion of functional testosterone from the cells into the culture supernatant by a testosterone-sensitive cell proliferation assay. These findings showed that the hiPSCs were able to be differentiated into Leydig-like cells, supporting the expectation that hiPSC-derived Leydig-like cells can be novel tools for treating LOH syndrome.

Pituitary develops from oral ectoderm in contact with adjacent ventral hypothalamus. Impairment in this process results in congenital pituitary hypoplasia (CPH); however, there have been no human disease models for CPH thus far, prohibiting the elucidation of the underlying mechanisms. In this study, we established a disease model of CPH using patient-derived induced pluripotent stem cells (iPSCs) and 3D organoid technique, in which oral ectoderm and hypothalamus develop simultaneously. Interestingly, patient iPSCs with a heterozygous mutation in the orthodenticle homeobox 2 (OTX2) gene showed increased apoptosis in the pituitary progenitor cells, and the differentiation into pituitary hormone-producing cells was severely impaired. As an underlying mechanism, OTX2 in hypothalamus, not in oral ectoderm, was essential for progenitor cell maintenance by regulating LHX3 expression in oral ectoderm via FGF10 expression in the hypothalamus. Convincingly, the phenotype was reversed by the correction of the mutation, and the haploinsufficiency of OTX2 in control iPSCs revealed a similar phenotype, demonstrating that this mutation was responsible. Thus, we established an iPSC-based congenital pituitary disease model, which recapitulated interaction between hypothalamus and oral ectoderm and demonstrated the essential role of hypothalamic OTX2.

Induced pluripotent stem cell (iPSC) technology offers a novel approach for conversion of human primary fibroblasts into melanocytes. During attempts to explore various protocols for differentiation of iPSCs into melanocytes, we found a distinct and self‐renewing cell lineage that could differentiate into melanocytes, named as melanocyte precursor cells (MPCs). The MPCs exhibited a morphology distinctive from that of melanocytes, in lacking either the melanosomal structure or the melanocyte‐specific marker genes MITF, TYR, and SOX10. In addition, gene expression studies in the MPCs showed high‐level expression of WNT5A, ROR2, which are non‐canonical WNT pathway markers, and its related receptor TGFβR2. In contrast, MPC differentiation into melanocytes was achieved by activating the canonical WNT pathway using the GSK3β inhibitor. Our data demonstrated the distinct characteristic of MPCs' ability to differentiate into melanocytes, and the underlying mechanism of interfacing between canonical WNT signaling pathway and non‐canonical WNT signaling pathway.

Objectives Small proline-rich protein 1A (SPRR1A) is recognized as a squamous differentiation marker but is also upregulated in some non-squamous cancers. However, its expression in pancreatic ductal adenocarcinoma (PDAC) has not been investigated. This study elucidated the expression of SPRR1A in PDAC and its effect on the prognosis and malignant behavior of PDAC. Methods We examined the SPRR1A expression by immunohistochemistry in 86 surgical PDAC cases and revealed the relationship between its expression and the prognosis of the PDAC patients. Furthermore, we overexpressed SPRR1A in pancreatic cancer cell lines (PK-1 and Panc-1) and assessed the phenotype and gene expression changes in vitro. Results Among the 84 cases, excluding 2 with squamous differentiation, 31 (36.9%) had a high SPRR1A expression. The overall survival (median 22.1 months vs. 33.6 months, p = 0.0357) and recurrence-free survival (median 10.7 months vs. 15.5 months, p = 0.0298) were significantly lower in the high-SPRR1A-expression group than in the low-SPRR1A-expression group. A multivariate analysis indicated that a high SPRR1A expression (HR 1.706, 95% CI 1.018 to 2.862, p = 0.0427) and residual tumor status (HR 2.687, 95% CI 1.487 to 4.855, p = 0.00106) were independent prognostic factors. The analysis of TCGA transcriptome data demonstrated that the high-SPRR1A-expression group had a significantly worse prognosis than the low-SPRR1A-expression group, which supported our data. SPRR1A overexpression in PK-1 and Panc-1 did not result in remarkable changes to in vitro phenotypes, such as the cell proliferation, chemo-resistance, EMT, migration or global gene expression. Conclusion Increased expression of SPRR1A is associated with a poor prognosis in PDAC and may serve as a novel prognostic marker. However, our in vitro study suggests that the SPRR1A expression may be a consequence, not a cause, of the aggressive behavior of PDAC.

Intestinal metaplasia is related to gastric carcinogenesis. Previous studies have suggested the important role of CDX2 in intestinal metaplasia, and several reports have shown that the overexpression of CDX2 in mouse gastric mucosa caused intestinal metaplasia. However, no study has examined the induction of intestinal metaplasia using human gastric mucosa. In the present study, to produce an intestinal metaplasia model in human gastric mucosa in vitro, we differentiated human-induced pluripotent stem cells (hiPSC) to gastric organoids, followed by the overexpression of CDX2 using a tet-on system. The overexpression of CDX2 induced, although not completely, intestinal phenotypes and the enhanced expression of many, but not all, intestinal genes and previously reported intestinal metaplasia-related genes in the gastric organoids. This model can help clarify the mechanisms underlying intestinal metaplasia and carcinogenesis in human gastric mucosa and develop therapies to restitute precursor conditions of gastric cancer to normal mucosa.

SUMMARY Fukuyama congenital muscular dystrophy (FCMD) is a severe, intractable genetic disease that affects the skeletal muscle, eyes, and brain and is attributed to a defect in alpha dystroglycan (αDG) O-mannosyl glycosylation. We previously established disease models of FCMD; however, they did not fully recapitulate the phenotypes observed in human patients. In this study, we generated induced pluripotent stem cells (iPSCs) from a human FCMD patient and differentiated these cells into three-dimensional brain organoids and skeletal muscle. The brain organoids successfully mimicked patient phenotypes not reliably reproduced by existing models, including decreased αDG glycosylation and abnormal radial glial cell (RG) fiber migration. The basic polycyclic compound Mannan-007 (Mn007) restored αDG glycosylation in all models tested and partially rescued the abnormal RG migration observed in cortical organoids. Therefore, our study underscores the importance of αDG O -mannosyl glycans for normal RG architecture and proper neuronal migration in corticogenesis.

Background: A protocol of differentiation form induced pluripotent stem cells (iPSCs) to hepatocytes has been previously reported, and such iPSCs-derived hepatocytes possessed morphological and biochemical characteristics similar to authentic hepatocytes. It is however unknown whether iPSCs-derived hepatocytes normally respond to metabolic regulatory hormones including insulin. Mutations in the gene for p85α subunit of PI3K (PIK3R1), an essential element of insulin action, give rise to severe insulin resistance in human. Such cases are very rare, and only ∼30 families with PIK3R1 mutations have been reported as of today. Methods and Results: We established a protocol of differentiation from iPSCs to hepatocytes that respond to insulin. In hepatocytes differentiated from iPSCs derived from healthy subject with this protocol, phosphorylation of AKT and the expression of the lipogenic genes FASN as well as SREBF1c were induced by insulin. Furthermore, the expression of the gluconeogenic gene G6PC was upregulated by a combination of cAMP and dexamethasone, which was markedly impaired by the addition of insulin. We then generated iPSCs from a severe insulin resistant diabetic patient with the PIK3R1 c.1945C>T mutation, a hot-spot mutation in this gene. Insulin-induced gene expression along with phosphorylation of AKT was impaired in hepatocytes differentiated from the patient-derived iPSCs. We also repaired the mutation of PIK3R1 in the patient-derived iPSCs by the CRISPR/Cas9 genome editing. Insulin responsiveness was recovered in hepatocytes differentiated from the resultant iPSCs in which the mutation was repaired. Conclusion: We established a differentiation protocol of hepatocytes in which insulin’s metabolic actions can be evaluated. The usefulness of this protocol was validated with the use of iPSCs derived from a severe insulin-resistant patient with PIK3R1 mutation. Disclosure T. Hamaguchi: None. Y. Hirota: Other Relationship; Self; Sanofi. T. Takeuchi: None. M. Koyanagi-Aoi: None. T. Aoi: None. W. Ogawa: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharma GmbH & Co.KG, Eli Lilly Japan K.K., Kowa Company, Ltd., Nippon Boehringer Ingelheim Co. Ltd., Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd. Other Relationship; Self; Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited.

BackgroundCancer stem cells (CSCs) are considered to be responsible for tumor initiation, formation, and poor prognosis of cancer patients. However, the rarity of CSCs in clinical samples makes it difficult to elucidate characteristics of CSCs, especially in osteosarcoma (OS). The aim of this study is to verify whether it is possible to generate CSC-like cells by transducing defined factors into an OS cell line.MethodsWe retrovirally transduced the Octamer-binding transcription factor 3/4 (OCT3/4), Kruppel-like factor 4 (KLF4), and SRY-box transcription factor 2 (SOX2) genes into the MG-63 human OS cell line (MG-OKS). Parental and GFP-transduced MG-63 cells were used as negative control. We assessed the properties of the generated cells in vitro and in vivo. Multiple comparisons among groups were made using a one-way analysis of variance (ANOVA) followed by post hoc testing with Tukey’s procedure.ResultsMG-OKS cells in vitro exhibited the significantly increased mRNA expression levels of CSC markers (CD24,CD26, andCD133), decreased cell growth, increased chemoresistance and cell migration, and enhanced sphere formation. Notably, MG-OKS cells cultured under osteogenic differentiation conditions showed strongly positive staining for both Alizarin Red S and alkaline phosphatase, indicating osteogenesis of the cells. Gene ontology analysis of microarray data revealed significant upregulation of epidermal-related genes. Tumors derived from MG-OKS cells in vivo were significantly larger than those from other cells in μCT analysis, and immunohistochemical staining showed that Ki-67, osteocalcin, and HIF-1α-positive cells were more frequently detected in the MG-OKS-derived tumors.ConclusionsIn this study, we successfully generated OS CSC-like cells with significantly enhanced CSC properties following transduction of defined factors.

Summary Fukuyama congenital muscular dystrophy (FCMD) is a severe, intractable genetic disease that affects the skeletal muscle, eyes, and brain and is attributed to a defect in alpha dystroglycan (αDG) O-mannosyl glycosylation. We previously established disease models of FCMD; however, they did not fully recapitulate the phenotypes observed in human patients. In this study, we generated induced pluripotent stem cells (iPSCs) from a human FCMD patient and differentiated these cells into three-dimensional brain organoids and skeletal muscle. The brain organoids successfully mimicked patient phenotypes not reliably reproduced by existing models, including decreased αDG glycosylation and abnormal radial glial (RG) fiber migration. The basic polycyclic compound Mannan-007 (Mn007) restored αDG glycosylation in the brain and muscle models tested and partially rescued the abnormal RG fiber migration observed in cortical organoids. Therefore, our study underscores the importance of αDG O-mannosyl glycans for normal RG fiber architecture and proper neuronal migration in corticogenesis., Graphical abstract, Highlights • FCMD muscle and brain defects result from reduced α-dystroglycan (α-DG) glycosylation • iPSC-derived brain organoids exhibit structural defects like those seen in FCMD patients • FCMD organoids exhibit decreased α-DG glycosylation and abnormal radial glial migrations • Mannan-007 partially restored α-DG glycosylation and radial glial migration defects, Pathophysiology; Neuroscience; Tissue Engineering

γδT cells constitute a small proportion of lymphocytes in peripheral blood. Unlike αβT cells, the anti-tumor activities are exerted through several different pathways in a MHC-unrestricted manner. Thus, immunotherapy using γδT cells is considered to be effective for various types of cancer. Occasionally, however, ex vivo expanded cells are not as effective as expected due to cell exhaustion. To overcome the issue of T-cell exhaustion, researchers have generated induced pluripotent stem cells (iPSCs) that harbor the same T-cell receptor (TCR) genes as their original T-cells, which provide nearly limitless sources for antigen-specific cytotoxic T lymphocytes (CTLs). However, these technologies have focused on αβT cells and require a population of antigen-specific CTLs, which are purified by cell sorting with HLA-peptide multimer, as the origin of iPS cells. In the present study, we aimed to develop an efficient and convenient system for generating iPSCs that harbor rearrangements of the TCRG and TCRD gene regions (γδT-iPSCs) without cell-sorting. We stimulated human whole peripheral blood mononuclear cell (PBMC) culture using Interleukin-2 and Zoledronate to activate γδT cells. Gene transfer into those cells with the Sendai virus vector resulted in γδT cell-dominant expression of exogenous genes. The introduction of reprogramming factors into the stimulated PBMC culture allowed us to establish iPSC lines. Around 70% of the established lines carried rearrangements at the TCRG and TCRD gene locus. The γδT-iPSCs could differentiate into hematopoietic progenitors. Our technology will pave the way for new avenues toward novel immunotherapy that can be applied for various types of cancer.

INTRODUCTION AND OBJECTIVES:Patients with bladder diseases often require augmentation or reconstruction of urinary bladder. Although gastrointestinal tissue or autologous bladder cells have been employed in treatments for the regeneration of bladder functions, these approaches often cause various adverse events. Recently, bladder urothelium derived from human induced pluripotent stem cells (hiPSCs) has received focus. However, previous studies have only shown the emergence of cells expressing some urothelial markers among derivatives of hiPSCs. This study aimed to develop a protocol for the directed differentiation of hiPSCs into mature stratified bladder urothelium.METHODS:We evaluated the effect of several small molecules, FGF10 and culture devices on the urothelial differentiation of hiPSCs by assessing markers from initial to terminal differentiation stage as well as formation of stratified structure. The marker gene expression was analyzed by Reverse transcription polymerase chain reaction (RT-PCR) a...

In 2006, induced pluripotent stem (iPS) cells were generated by Yamanaka and Takahashi for the first time from a mouse fibroblast culture by introducing four factors. In the 10 years since then, this breakthrough discovery has been making waves in the fields of biology and medical science. For example, various technologies for generating iPS cells have been developed, and we have cultivated a better understanding of the mechanisms involved in reprogramming. In addition, many researchers have explored the applications of iPS cells, such as drug discovery, the study of disease mechanisms and regenerative medicine, and the development of advanced technologies for the differentiation and qualification of the cells. Furthermore, the concept of iPS cell generation has inspired a number of studies that do not use iPS cells. We herein review and discuss the past, present and future of iPS cells and their related issues.

At present, most cases of unresectable cancer cannot be cured. Genetic mutations, EMT, and cancer stem cells are three major issues linked to poor prognosis in such cases, all connected by inter- and intra-tumor heterogeneity. Issues on inter-/intra-tumor heterogeneity of genetic mutation could be resolved with recent and future technologies of deep sequencers, whereas, regarding such issues as the "same genome, different epigenome/phenotype", we expect to solve many of these problems in the future through further research in stem cell biology. We herein review and discuss the three major issues in the biology of cancers, especially from the standpoint of stem cell biology.

As a series of endeavors to establish suitable measures for the sound development of regenerative medicine using human stem cell-based products, we studied scientific principles, concepts and basic technical elements to ensure the quality and safety of therapeutic products derived from allogeneic human induced pluripotent stem cells (iPS cells) or iPS cell-like cells, taking into consideration scientific and technological advances, ethics, regulatory rationale, and international trends in human stem cell-derived products. This led to the development of the Japanese official Notification No. 0907-5, “Guideline on Ensuring the Quality and Safety of Pharmaceuticals and Medical Devices Derived from the Processing of Allogeneic Human Induced Pluripotent Stem(-Like) Cells,” issued by Pharmaceuticals and Food Safety Bureau, Ministry of Health, Labour and Welfare of Japan, on September 7, 2012. The present paper addresses various aspects of products derived from allogeneic human iPS cells (or iPS cell-like cells), in addition to similar points to consider that are described previously for allogeneic human stem cell-based products. Major additional points include 1) possible existence of allogeneic human iPS cell-like cells that are different from iPS cells in specific biological features; 2) the use of allogeneic human iPS(-like) cells as appropriate starting materials for regenerative medicine, where necessary and significant; 3) establishment of an allogeneic human iPS(-like) cell line and its characterization; 4) establishment of well-characterized stable cell banks and relevant intermediate cell products, if necessary; 5) concerns about the presence of undifferentiated cells in final products; such cells may cause ectopic tissue formation and/or tumorigenesis; and 6) concerns about undesirable immunological reactions that may be caused by the final products. The ultimate goal of this guidance is to provide suitable medical opportunities as soon as possible to the patients with severe diseases that are difficult to treat with conventional modalities.

As a series of endeavors to establish suitable measures for the sound development of regenerative medicine using human stem cell-based products, we studied scientific principles, concepts, and basic technical elements to ensure the quality and safety of therapeutic products derived from the processing of human embryonic stem cells (hESCs), taking into consideration scientific and technological advances, ethics, regulatory rationale, and international trends in human stem cell-derived products. This led to the development of the Japanese official Notification No. 0907-6, “Guideline on Ensuring the Quality and Safety of Pharmaceuticals and Medical Devices Derived from the Processing of Human Embryonic Stem Cells,” issued by Pharmaceuticals and Food Safety Bureau, Ministry of Health, Labour and Welfare of Japan, on September 7, 2012. The present paper addresses various aspects of products derived from hESCs, in addition to similar points to consider that are described previously for allogeneic human stem cell-based products. Major additional points include 1) establishment of hESCs; 2) establishment of stable and well-characterized cell banks of hESCs and relevant intermediate cell products; 3) concerns about the presence of undifferentiated cells in final products, which may result in ectopic tissue formation and/or tumorigenesis; and 4) concerns about undesirable immunological reactions caused by the final products. The ultimate goal of this series of guidelines on regenerative medicine is to provide suitable medical opportunities as soon as possible to the patients with severe diseases that are difficult to treat with conventional modalities. If these guidelines are interpreted and employed in a flexible and meaningful way in this context, they should serve as a useful means to achieve their goals.

The regulation of human cell therapy products is a key factor in their development and use to treat human diseases. In that regard, there is a recognized need for a global effort to develop a set of common principles that may serve to facilitate a convergence of regulatory approaches to ensure the smooth and efficient evaluation of products. This conference, with experts from regulatory agencies, industry, and academia, contributed to the process of developing such a document. Elements that could form a minimum consensus package of requirements for evaluating human cell therapy products were the overall focus of the conference. The important regulatory considerations that are unique to human cell therapy products were highlighted. Sessions addressed specific points that are different from those of traditional biological/biotechnological protein products. Panel discussions complemented the presentations. The conference concluded that most of the current regulatory framework is appropriate for cell therapy, but there are some areas where the application of the requirements for traditional biologicals is inappropriate. In addition, it was agreed that there is a need for international consensus on core regulatory elements, and that one of the major international organizations should take the lead in formulating such a consensus document.

Cancer stem cells (CSC) are capable of reconstructing cancer tissues, are involved in both recurrence and metastasis, and contribute to therapeutic resistance. Therefore, elucidating the molecular mechanism in CSCs is important to successfully treat unresectable cancers. Previously, we observed that colon cancer stem-like cells can be induced from human colon cancer cell lines by retrovirally introducing OCT3/4, SOX2, and KLF4, and we have designated such cells as induced cancer stem cells (iCSC). In the current study, we used iCSCs to evaluate the molecular mechanism of colon CSCs and developed new methods to control them. The spheres that were derived in vitro from the iCSCs, but not those from parental cells, mimicked human colon cancer tissues in terms of their immunohistologic patterns; therefore, sphere-forming ability was assessed as a measure of the tissue-reconstructing ability of iCSCs. Interestingly, the calcineurin inhibitor FK506 enhanced the sphere-forming ability of iCSCs, whereas GSK3 inhibition by RNAi, CHIR99021, and valproic acid (VPA) impeded the sphere-forming ability and expansion of iCSCs. FK506 and GSK3 inhibition showed the opposite effect regarding the NFATc3 localization of iCSCs. These data reveal the crucial role that NFAT localization, as regulated by calcineurin and GSK3, plays in the tissue-reconstructing ability of colon cancer stem cells and the potential of GSK3 inhibitors, such as VPA, in colon cancer stem cell–targeting therapy. Implications: This study identifies signaling pathways that contribute to the tissue-reconstructing capacity of colon CSCs and suggests that clinically used drugs could be repurposed to improve unresectable colon cancers. Mol Cancer Res; 15(10); 1455–66. ©2017 AACR.

In order to assure the quality and safety of future advanced cell therapies it is vital to ensure that source materials including the donor cells have been assessed and demonstrated as suitable for use in the development and manufacture of such new medicines. Here we provide a brief overview of the key issues in the delivery of quality controlled and safety tested seed stocks of human pluripotent stem cell lines to support stem cell research and the development of advanced cell therapies. We also reflect on the importance of national and internationally coordinated cell banking systems in this process in order to promote more efficient development of cell therapies.

Induced pluripotent stem (iPS) cells are generated from somatic cells by introducing small sets of transcription factors. iPS cells demonstrate pluripotency and the ability to self-renew. In addition, iPS cells can be generated from donor individuals with particular characteristics. Based on these features, iPS cells are expected to be applicable in drug discovery, the study of disease mechanisms and cell therapy. From a technical point of view, "diversity" is the key word. At present, iPS cells can be derived using various techniques, resulting in diversity in the quality of iPS cells generated. Therefore, optimization of the derivation technology is one of the most important issues. Another "diversity" is in the propensities amongst iPS cell lines derived using similar techniques. Thus, strategies for selecting good quality lines remain to be established. Considering such technical hurdles, establishment of an iPS cell bank consisting of high quality and versatile iPS lines is a promising idea because of the merits of cost and quality control. Now, we are exploring relevant parameters for the quality control of banked cells. The challenges facing clinical application of iPS cells are new but not unprecedented. To realize clinical applications of iPS cells, we need to make these challenges clear and overcome them through partnership not only with industry, governments and universities, but also patients and society at large.

In the present study, we successfully generated lung cancer stem cell (CSC)-like cells by introducing a small set of transcription factors into a lung cancer cell line. In addition to properties that are conventionally referred to as CSC properties, the lung induced CSCs exhibited the ability to form lung cancer-like tissues in vitro with vascular cells and mesenchymal stem cells, which showed structures and immunohistological patterns that were similar to human lung cancer tissues. We named them “lung cancer organoids”. We found that interleukin-6 (IL-6), which was expressed in the lung induced CSCs, facilitates the formation of lung cancer organoids via the conversion of mesenchymal stem cells into alpha-smooth muscle actin (αSMA)-positive cells. Interestingly, the combination of anti-IL-6 antibody and cisplatin could destroy the lung cancer organoids, while cisplatin alone could not. Furthermore, IL-6 mRNA-positive cancer cells were found in clinical lung cancer samples. These results suggest that IL-6 could be a novel therapeutic target in lung cancer.

Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, the in vitro directed differentiation of human pluripotent stem cells into mature hepatocytes remains challenging. Little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. In the current study, we developed an improved hepatic differentiation protocol and compared 28 hiPSC lines originated from various somatic cells and derived using retroviruses, Sendai viruses, or episomal plasmids. This comparison indicated that the origins, but not the derivation methods, may be a major determinant of variation in hepatic differentiation. The hiPSC clones derived from peripheral blood cells consistently showed good differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts showed poor differentiation. However, when we compared hiPSCs from peripheral blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. These data underscore the importance of donor differences when comparing the differentiation propensities of hiPSC clones.

Induced pluripotent stem (iPS) cells can be generated from somatic cells by the introduction of Oct3/4 (also known as Pou5f1), Sox2, Klf4 and c-Myc, in mouse and in human. The efficiency of this process, however, is low. Pluripotency can be induced without c-Myc, but with even lower efficiency. A p53 (also known as TP53 in humans and Trp53 in mice) short-interfering RNA (siRNA) was recently shown to promote human iPS cell generation, but the specificity and mechanisms remain to be determined. Here we report that up to 10% of transduced mouse embryonic fibroblasts lacking p53 became iPS cells, even without the Myc retrovirus. The p53 deletion also promoted the induction of integration-free mouse iPS cells with plasmid transfection. Furthermore, in the p53-null background, iPS cells were generated from terminally differentiated T lymphocytes. The suppression of p53 also increased the efficiency of human iPS cell generation. DNA microarray analyses identified 34 p53-regulated genes that are common in mouse and human fibroblasts. Functional analyses of these genes demonstrate that the p53-p21 pathway serves as a barrier not only in tumorigenicity, but also in iPS cell generation.

Human embryonic stem (ES) cells are expected to be a resource for regenerative medicine because of their pluripotency and ability to self-renew. In 2007, human-induced pluripotent stem (iPS) cells were generated from somatic cells by introducing small sets of transcription factors, raising further hopes of clinical applications of pluripotent stem cells. Today, several promising projects using these cells have been launched.

Direct reprogramming of somatic cells provides an opportunity to generate patient- or disease-specific pluripotent stem cells. Such induced pluripotent stem (iPS) cells were generated from mouse fibroblasts by retroviral transduction of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. Mouse iPS cells are indistinguishable from embryonic stem (ES) cells in many respects and produce germline-competent chimeras. Reactivation of the c-Myc retrovirus, however, increases tumorigenicity in the chimeras and progeny mice, hindering clinical applications. Here we describe a modified protocol for the generation of iPS cells that does not require the Myc retrovirus. With this protocol, we obtained significantly fewer non-iPS background cells, and the iPS cells generated were consistently of high quality. Mice derived from Myc(-) iPS cells did not develop tumors during the study period. The protocol also enabled efficient isolation of iPS cells without drug selection. Furthermore, we generated human iPS cells from adult dermal fibroblasts without MYC.

To make sure that novel human cell-based products contribute to human health care, it is essential that, based on sound science at present, suitable measures be taken by the manufacturers and regulatory authorities on applying these products to the treatment of patients by taking into account specificity of starting cell lines, the manufacturing process, products, administration procedures, diseases in question, and patient population. As part of such an endeavor, we studied scientific principles, concepts, and basic technical elements to ensure the quality and safety of therapeutic products derived from autologous human somatic stem cells, taking into consideration scientific and technological advances, ethics, regulatory rationale, and international trends in human stem cell-derived products. This led to the development of the Japanese official Notification No. 0907-2, “Guideline on Ensuring the Quality and Safety of Pharmaceuticals and Medical Devices Derived from the Processing of Autologous Human Somatic Stem Cells,” issued by Pharmaceuticals and Food Safety Bureau, Ministry of Health, Labour and Welfare of Japan on September 7, 2012. The present paper describes the background information and the development of our study and the resulting guidance. For products derived from autologous somatic stem cells, major points to consider include 1) multipotency and self-replication ability of autologous human somatic stem cells and differences in cell characteristics of the final products from those of the starting cells; 2) a donor's infectious status; 3) the risk of proliferation/reactivation of viruses during the manufacturing processes; 4) robust process control to minimize unevenness of “custom-made” products; 5) a limited amount of samples for quality evaluation of products; and 6) robust application and function of the final products in a cell environment different from where the original cells were localized and were performing their natural endogenous functions. The ultimate goal of this guidance is to provide suitable medical opportunities as soon as possible to the patients.

Induced pluripotent stem cells(iPSCs)can be generated via reprogramming of somatic cells into pluripotent stem cells by introducing defined factors with appropriate culture conditions. iPSCs have 4 key properties. iPSCs have pluripotency and self-renewal ability, which are properties in common with embryonic stem cells. Additionally, iPSCs can be generated from various donor individuals with particular characteristics and also from various types of cells in a single donor. Regarding the medical applications of the technology, the best use of iPSCs should be based on a better understanding of these properties in accordance to different purposes. At present, the technology has been applied in various research fields. In cancer research, the technology has been expected to be useful, especially in immunotherapy, disease modeling, drug development. Thus, iPSC technology is a promising tool even in the field. Continuous challenges raise the hope for the development of novel cancer treatments using iPSC technologies in the future.

Background: Lung cancer stem cells are considered to be responsible for lung cancer progression and metastasis. However, little is known about how they actually promote aggressive behaviors of lung cancer. Methods: We transduced OCT3/4, SOX2, and KLF4 (hereafter, OSK) into a KRAS-mutated (G12S) human lung adenocarcinoma cell line (A549) using retrovirus vectors. Dome-shaped colonies appeared in the OSK transduced A549 cells from 10 to 15 days after transduction. The colonies were picked up and sub-cultured. We named them OSK-A549-Colony cells. We evaluated cancer stem cell properties in the OSK-A549-Colony cells in terms of their chemo resistance, and sphere formation ability. We also assessed organoids constructing ability by co-culture with mesenchymal stem cells (MSCs) and human umbilical vein epithelial cells (HUVECs) on low attachment plates. To clarify the molecular mechanisms that determined the properties of the OSK-A549-Colony cells, we performed microarray analysis. We further analyzed the molecular mechanisms of organoids construction by OSK-A549-Colony cells and searched for a method to destroy them. Results: The induced OSK-A549-colony cells were significantly more resistant to cisplatin than the parental A549 cells. Sphere forming ability was enhanced in the OSK-A549-Colony cells. Co-culture with MSCs and HUVECs showed that only the OSK-A549-Colony cells were able to construct consolidated organoids in vitro that mimicked bona-fide human lung cancer tissues. We named them “lung cancer organoids”. We found that interleukin-6 (IL-6), which was the most differentially expressed gene in the OSK-A549-Colony cells by microarray analysis, facilitated the formation of lung cancer organoids via the conversion of MSCs into alpha-smooth muscle actin (αSMA)-positive cells. Surprisingly, the combination of anti-IL-6 antibody and cisplatin could destroy the lung cancer organoids, while cisplatin alone could not. Conclusion: By transducing defined factors, A549 cells acquired lung cancer stem cell like properties. Analysis of chemosensitivity using the lung cancer organoids showed that the combination of IL-6 blockade and cisplatin could be a novel therapy targeting lung cancer stem cells. Citation Format: Hiroyuki Ogawa, Michiyo Koyanagi-Aoi, Yoshimasa Maniwa, Takashi Aoi. Interleukin-6 blockade, a novel cancer stem cell targeted therapy, attenuates lung cancer tissue construction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3902. doi:10.1158/1538-7445.AM2017-3902

Our work and the study of Bilican et al. highlight the need for complementary assays to detect subtle phenotypic differences between control and mutant induced pluripotent stem cell lines.

A method for stimulating the differentiation of human pluripotent stem cells into kidney lineages remains to be developed. Most cells in kidney are derived from an embryonic germ layer known as intermediate mesoderm. Here we show the establishment of an efficient system of homologous recombination in human pluripotent stem cells by means of bacterial artificial chromosome-based vectors and single-nucleotide polymorphism array-based detection. This system allowed us to generate human-induced pluripotent stem cell lines containing green fluorescence protein knocked into OSR1, a specific intermediate mesoderm marker. We have also established a robust induction protocol for intermediate mesoderm, which produces up to 90% OSR1(+) cells. These human intermediate mesoderm cells can differentiate into multiple cell types of intermediate mesoderm-derived organs in vitro and in vivo, thereby supplying a useful system to elucidate the mechanisms of intermediate mesoderm development and potentially providing a cell source for regenerative therapies of the kidney.

Developing new treatments that reduce prostate cancer progression is important. Therapeutic efficacy of conventional gene therapy for metastatic prostate cancer is still low. Lower induction rate of naked genes into target cell is due to reduced expression of adenovirus receptor on cancer cell and also due to high seroprevalence of anti-Ad antibodies in adults. Therefore, efficient Ad carrier systems that circumvent these problems should be developed. Gamma-delta T cells have demonstrated high affinity to cancer cells. CD46, which leads to broad tropism in Ad35 vectors, is expressed in hematopoietic cells, including γδ T cells. In this study, we demonstrate the potential of γδ T cells as “vehicles” for transporting Ad5/F35 vectors and genes into cancer cells.