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For a long time, ex vivo-expanded peripheral-blood-derived γδT cell (PBγδT)-based immunotherapy has been attractive, and clinical trials have been undertaken. However, the difficulty in expanding cytotoxic γδT cells to an adequate number has been a major limitation to the efficacy of treatment in most cases. We successfully re-generated γδT cells from γδT cell-derived human induced pluripotent stem cells (iPSCs). The iPSC-derived γδT cells (iγδTs) killed several cancer types in a major histocompatibility complex (MHC)-unrestricted manner. Single-cell RNA sequencing (scRNA-seq) revealed that the iγδTs were identical to a minor subset of PBγδTs. Compared with a major subset of PBγδTs, the iγδTs showed a distinctive gene expression pattern: lower CD2, CD5, and antigen-presenting genes; higher CD7, KIT, and natural killer (NK) cell markers. The iγδTs expressed granzyme B and perforin but not interferon gamma (IFNγ). Our data provide a new source for γδT cell-based immunotherapy without quantitative limitation.

Cell-selective killing using molecular self-assemblies is an emerging concept for cancer therapy. Reported molecular self-assemblies are triggered by hydrolysis of well-designed molecules inside or outside cancer cells. This hydrolysis can occur in cancer and normal cells because of the abundance of water in living systems. Here, we report the in situ synthesis of a self-assembling molecule using a tyrosine kinase overexpressed in cancer cells. We designed a tyrosine-containing peptide amphiphile (C16-E4Y) that is transformed into a phosphorylated peptide amphiphile (C16-E4pY) by the overexpressed tyrosine kinase. Phosphorylation of C16-E4Y promoted self-assembly to form nanofibers in cancer cells. C16-E4Y exhibited selective cytotoxicity toward cancer cells overexpressing the tyrosine kinase. Self-assembled C16-E4pY induced endoplasmic reticulum stress that caused apoptotic cell death. Animal experiments revealed that C16-E4Y has antitumor activity. These results show that an enzyme overexpressed in cancer cells is available for intracellular synthesis of an antitumor self-assembling drug that is cell-selective.

Overactivated microglia play a key role in sepsis-associated encephalopathy (SAE), although the involvement of T cells is unclear. γδT cells in the brain and meninges regulate normal fear responses via interleukin (IL)-17 in healthy mice. In our sepsis model, the mice showed exacerbated anxious behavior at 10 days post-induction (dpi). At 8 dpi, IL-17 mRNA was significantly upregulated in the brains of septic mice compared with those of control mice. Simultaneously, the number of γδT cells increased in the brains of septic mice in a severity-dependent manner. Additionally, IL-17-producing γδT cells, expressing both the C-X-C motif receptor (CXCR) 6 and the C-C motif receptor (CCR) 6, increased in mice brains, dependent on the severity of sepsis. The frequency of γδT cells in the meninges fluctuated similarly to that in the brain, peaking at 8 dpi of sepsis. Behavioral tests were performed on septic mice after the continuous administration of anti-γδTCR (α-γδTCR) or anti-IL-17A (α-IL-17A) antibodies to deplete the γδT cells and IL-17A, respectively. Compared with IgG-treated septic mice, α-γδTCR- and α-IL-17A-treated septic mice showed suppressed microglial activation and improvements in anxious behavior. These results suggested that CCR6+CXCR6+ IL-17-producing γδT cells in the brain and meninges promote the exacerbation of SAE and sepsis-induced psychological disorders in mice.

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.

Microarray Probe List

RNA isolation and quantitative reverse-transcriptase polymerase chain reaction

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.

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 The esophagus is known to be derived from the foregut. However, the mechanisms regulating this process remain unclear. In particular, the details of the human esophagus itself have been poorly researched. In this decade, studies using human induced pluripotent stem cells (hiPSCs) have proven powerful tools for clarifying the developmental biology of various human organs. Several studies using hiPSCs have demonstrated that retinoic acid (RA) signaling promotes the differentiation of foregut into tissues such as lung and pancreas. However, the effect of RA signaling on the differentiation of foregut into esophagus remains unclear. Methods We established a novel stepwise protocol with transwell culture and an air–liquid interface system for esophageal epithelial cell (EEC) differentiation from hiPSCs. We then evaluated the effect of all-trans retinoic acid (ATRA), which is a retinoic acid receptor (RAR)α, RARβ and RARγ agonist, on the differentiation from the hiPSC-derived foregut. Finally, to identify which RAR subtype was involved in the differentiation, we used synthetic agonists and antagonists of RARα and RARγ, which are known to be expressed in esophagus. Results We successfully generated stratified layers of cells expressing EEC marker genes that were positive for lugol staining. The enhancing effect of ATRA on EEC differentiation was clearly demonstrated with quantitative reverse transcription polymerase chain reaction, immunohistology, lugol-staining and RNA sequencing analyses. RARγ agonist and antagonist enhanced and suppressed EEC differentiation, respectively. RARα agonist had no effect on the differentiation. Conclusion We revealed that RARγ activation promotes the differentiation of hiPSCs-derived foregut into EECs.

[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.

Ewing sarcoma (ES) is an aggressive primary malignant bone tumor that predominantly affects children and young adults. Multimodal treatment approaches have markedly improved the survival of patients with localized ES. However, local recurrence and distant metastasis following curative therapies remain a main concern for patients with ES. Recent studies have suggested that slow‑cycling cells (SCCs) are associated with tumor progression, local recurrence and distant metastasis in various types of cancers. According to the results of these studies, it was hypothesized that SCCs may play a critical role in tumor progression, chemoresistance and local/distal recurrence in patients with ES. The present study applied a label‑retaining system using carboxyfluorescein diacetate succinimidyl ester (CFSE) to identify and isolate SCCs in ES cell lines. In addition, the properties of SCCs, including sphere formation ability, cell cycle distribution and chemoresistance, in comparison with non‑SCCs were investigated. RNA sequencing also revealed several upregulated genes in SCCs as compared with non‑SCCs; the identified genes not only inhibited cell cycle progression, but also promoted the malignant properties of SCCs. On the whole, the present study successfully identified SCCs in ES cells through a label‑retaining system using CFSE. Moreover, to the best of our knowledge, the present study is the first to describe the characteristic properties of SCCs in ES. The findings of this study, if confirmed, may prove to be useful in elucidating the underlying molecular mechanisms and identifying effective therapeutic targets for ES.

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.

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.

The esophagus is known to be derived from the foregut. However, the mechanisms regulating this process remain unclear. In particular, the details of the human esophagus itself have been poorly researched. In this decade, studies using human induced pluripotent stem cells (hiPSCs) have proven powerful tools for clarifying the developmental biology of various human organs. Several studies using hiPSCs have demonstrated that retinoic acid (RA) signaling promotes the differentiation of foregut into tissues such as lung and pancreas. However, the effect of RA signaling on the differentiation of foregut into esophagus remains unclear.We established a novel stepwise protocol with transwell culture and an air-liquid interface system for esophageal epithelial cell (EEC) differentiation from hiPSCs. We then evaluated the effect of all-trans retinoic acid (ATRA), which is a retinoic acid receptor (RAR)α, RARβ and RARγ agonist, on the differentiation from the hiPSC-derived foregut. Finally, to identify which RAR subtype was involved in the differentiation, we used synthetic agonists and antagonists of RARα and RARγ, which are known to be expressed in esophagus.We successfully generated stratified layers of cells expressing EEC marker genes that were positive for lugol staining. The enhancing effect of ATRA on EEC differentiation was clearly demonstrated with quantitative reverse transcription polymerase chain reaction, immunohistology, lugol-staining and RNA sequencing analyses. RARγ agonist and antagonist enhanced and suppressed EEC differentiation, respectively. RARα agonist had no effect on the differentiation.We revealed that RARγ activation promotes the differentiation of hiPSCs-derived foregut into EECs.

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.

The present study reports that a short oligopeptide D-P1, consisting of only five D-amino acids, self-assembled into entangled nanofibers to form a hydrogel that functioned as a scaffold for cell cultures. D-P1 (Ac-D-Phe-D-Phe-D-Phe-Gly-D-Lys) gelated aqueous buffer solution and water at a minimum gelation concentration of 0.5 wt%. The circular dichroism (CD) measurements demonstrated the formation of a β-sheet structure in the self-assembly of D-P1. We investigated the gelation properties and CD spectra of both the D- and L-forms of the oligopeptide, and found only a minimal difference between them. The D-P1 hydrogel was resistant to a protease, whereas the L-P1 hydrogel was rapidly degraded. Both oligopeptides exhibited nontoxic properties to human cancer cells and embryoid bodies (EBs) derived from human-induced pluripotent stem cells. Additionally, we succeeded in forming spheroids of HeLa cells on the D-P1 hydrogel, which indicates the potential of this hydrogel for 3-dimensional cell culture.

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 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 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.

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.

Oligomeric forms of amyloid-β peptide (Aβ) are thought to play a pivotal role in the pathogenesis of Alzheimer's disease (AD), but the mechanism involved is still unclear. Here, we generated induced pluripotent stem cells (iPSCs) from familial and sporadic AD patients and differentiated them into neural cells. Aβ oligomers accumulated in iPSC-derived neurons and astrocytes in cells from patients with a familial amyloid precursor protein (APP)-E693Δ mutation and sporadic AD, leading to endoplasmic reticulum (ER) and oxidative stress. The accumulated Aβ oligomers were not proteolytically resistant, and docosahexaenoic acid (DHA) treatment alleviated the stress responses in the AD neural cells. Differential manifestation of ER stress and DHA responsiveness may help explain variable clinical results obtained with the use of DHA treatment and suggests that DHA may in fact be effective for a subset of patients. It also illustrates how patient-specific iPSCs can be useful for analyzing AD pathogenesis and evaluating drugs., Cell Stem Cell, 12(4), pp.487-496; 2013

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.