Context-Dependent Roles of Hes1 in the Adult Pancreas and Pancreatic Tumor Formation.

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.

Chorioallantoic membrane assay revealed the role of TIPARP (2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible poly (ADP-ribose) polymerase) in lung adenocarcinoma-induced angiogenesis.

BACKGROUND: The chorioallantoic membrane (CAM) assay is a well-established technique to evaluate tumor invasion and angiogenesis and may overcome the shortcoming of the patient-derived xenograft (PDX) mouse model. Currently, few reports have described lung cancer invasion and angiogenesis in the CAM assay. We therefore used the CAM assay in the evaluation of lung cancer. METHOD: Lung cancer cell line-derived organoids or lung cancer cell lines were transplanted into the CAM on embryonic development day (EDD) 10, and an analysis was performed on EDD 15. Microscopic and macroscopic images and movies of the grafts on the CAM were captured and analyzed. The relationships between the graft and chick vessels were evaluated using immunohistochemistry. RESULTS: We transplanted lung cancer cell lines and cell line-derived organoid into a CAM to investigate angiogenesis and invasion. They engrafted on the CAM at a rate of 50-83%. A549-OKS cells showed enhanced cell invasion and angiogenesis on the CAM in comparison to A549-GFP cells as was reported in vitro. Next, we found that A549-TIPARP cells promoted angiogenesis on the CAM. RNA-seq identified 203 genes that were upregulated more than twofold in comparison to A549-GFP cells. A pathway analysis revealed many upregulated pathways related to degradation and synthesis of the extracellular matrix in A549-TIPARP cells. CONCLUSIONS: The CAM assay can be used to evaluate and research invasion and angiogenesis in lung cancer. The elevated expression of TIPARP in lung cancer may induce angiogenesis by remodeling the extracellular matrix.

Blockage of retinoic acid signaling via RARγ suppressed the proliferation of pancreatic cancer cells by arresting the cell cycle progression of the G1-S phase.

BACKGROUND: Our study and several studies have reported that in some cancers, including pancreatic ductal adenocarcinoma (PDAC), the expression of squamous lineage markers, such as esophagus-tissue-specific genes, correlated with a poor prognosis. However, the mechanism by which the acquisition of squamous lineage phenotypes leads to a poor prognosis remains unclear. We previously reported that retinoic acid signaling via retinoic acid receptor γ (RARγ signaling) determines the differentiation lineage into the esophageal squamous epithelium. These findings hypothesized that the activation of RARγ signaling contributed to acquiring squamous lineage phenotypes and malignant behavior in PDAC. METHODS: This study utilized public databases and immunostaining of surgical specimens to examine RARγ expression in PDAC. We evaluated the function of RARγ signaling by inhibitors and siRNA knockdown using a PDAC cell line and patient-derived PDAC organoids. The mechanism of the tumor-suppressive effects by blocking RARγ signaling was examined by a cell cycle analysis, apoptosis assays, RNA sequencing and Western blotting. RESULTS: RARγ expression in pancreatic intraepithelial neoplasia (PanIN) and PDAC was higher than that in the normal pancreatic duct. Its expression correlated with a poor patient prognosis in PDAC. In PDAC cell lines, blockade of RARγ signaling suppressed cell proliferation by inducing cell cycle arrest in the G1 phase without causing apoptosis. We demonstrated that blocking RARγ signaling upregulated p21 and p27 and downregulated many cell cycle genes, including cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6. Furthermore, using patient-derived PDAC organoids, we confirmed the tumor-suppressive effect of RARγ inhibition and indicated the synergistic effects of RARγ inhibition with gemcitabine. CONCLUSIONS: This study clarified the function of RARγ signaling in PDAC progression and demonstrated the tumor-suppressive effect of selective blockade of RARγ signaling against PDAC. These results suggest that RARγ signaling might be a new therapeutic target for PDAC.

Microenvironment pH-Induced Selective Cell Death for Potential Cancer Therapy Using Nanofibrous Self-Assembly of a Peptide Amphiphile.

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.

Suppression of induced pluripotent stem cell generation by the p53-p21 pathway.

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.

Impact of National and International Stem Cell Banking Initiatives on progress in the field of cell therapy: IABS-JST Joint Workshop: Summary for Session 5.

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.

Report of the international conference on regulatory endeavors towards the sound development of human cell therapy products.

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.

Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells.

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.

[Application of iPSC technology to cancer treatment].

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.

Microplate-Based Cryopreservation of Adherent-Cultured Human Cell Lines Using Amino Acids and Proteins.

With the progression of regenerative medicine and cell therapy, the importance of cryopreservation techniques for cultured cells continues to rise. Traditional cryoprotectants, such as dimethyl sulfoxide and glycerol, are effective in cryopreserving suspended cells, but they do not demonstrate sufficient efficacy for two-dimensional (2D)-cultured cells. In the past decade, small molecules and polymers have been studied as cryoprotectants. Some L-amino acids have been reported to be natural and biocompatible cryoprotectants. However, the cryoprotective effects of D-amino acids have not been investigated for such organized cells. In the present study, the cryoprotective effects of D- and L-amino acids and previously reported cryoprotectants were assessed using HepG2 cells cultured on a microplate without suspending the cells. d-Proline had the highest cryoprotective effect on 2D-cultured cells. The composition of the cell-freezing solution and freezing conditions were then optimized. The d-proline-containing cell-freezing solution also effectively worked for other cell lines. To minimize the amount of animal-derived components, fetal bovine serum in the cell freezing solution was substituted with bovine serum albumin and StemFit (a commercial supplement for stem cell induction). Further investigations on the mechanism of cryopreservation suggested that d-proline protected enzymes essential for cell survival from freeze-induced damage. In conclusion, an effective and xeno-free cell-freezing solution was produced using d-proline combined with dimethyl sulfoxide and StemFit for 2D-cultured cells.

Molecular Aggregation Strategy for Inhibiting DNases.

This study highlights the novel potential of molecular aggregates as inhibitors of a disease-related protein. Enzyme inhibitors have been studied and developed as molecularly targeted drugs and have been applied for cancer, autoimmune diseases, and infections. In many cases, enzyme inhibitors that are used for therapeutic applications interact directly with enzymes in a molecule-to-molecule manner. We found that the aggregates of a small compound, Mn007, inhibited bovine pancreatic DNase I. Once Mn007 molecules formed aggregates, they exhibited inhibitory effects specific to DNases that require divalent metal ions. A DNase secreted by Streptococcus pyogenes causes streptococcal toxic shock syndrome (STSS). STSS is a severe infectious disease with a fatality rate exceeding 30% in patients, even in this century. S. pyogenes disrupts the human barrier system against microbial infections through the secreted DNase. Until now, the discovery/development of a DNase inhibitor has been challenging. Mn007 aggregates were found to inhibit the DNase secreted by S. pyogenes, which led to the successful suppression of S. pyogenes growth in human whole blood. To date, molecular aggregation has been outside the scope of drug discovery. The present study suggests that molecular aggregation is a vast area to be explored for drug discovery and development because aggregates of small-molecule compounds can inhibit disease-related enzymes.

Secondary Publication: Proposal for Points of Consideration for Pluripotent Stem Cell Culture.

Human pluripotent stem cells, such as human embryonic stem cells and human induced pluripotent stem cells, are used in basic research and various applied fields, including drug discovery and regenerative medicine. Stem cell technologies have developed rapidly in recent years, and the supply of culture materials has improved. This has facilitated the culture of human pluripotent stem cells and has enabled an increasing number of researchers and bioengineers to access this technology. At the same time, it is a challenge to share the basic concepts and techniques of this technology among researchers and technicians to ensure the reproducibility of research results. Human pluripotent stem cells differ from conventional somatic cells in many aspects, and many points need to be considered in their handling, even for those experienced in cell culture. Therefore, we have prepared this proposal, "Points of Consideration for Pluripotent Stem Cell Culture," to promote the effective use of human pluripotent stem cells. This proposal includes seven items to be considered and practices to be confirmed before using human pluripotent stem cells. These are laws/guidelines and consent/material transfer agreements, diversity of pluripotent stem cells, culture materials, thawing procedure, media exchange and cell passaging, freezing procedure, and culture management. We aim for the concept of these points of consideration to be shared by researchers and technicians involved in the cell culture of pluripotent stem cells. In this way, we hope the reliability of research using pluripotent stem cells can be improved, and cell culture technology will advance.

Re-generation of cytotoxic γδT cells with distinctive signatures from human γδT-derived iPSCs.

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.

Increased Interleukin-17-Producing γδT Cells in the Brain Exacerbate the Pathogenesis of Sepsis-Associated Encephalopathy and Sepsis-Induced Anxiety in Mice.

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.

CDX2-induced intestinal metaplasia in human gastric organoids derived from induced pluripotent stem cells.

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.

Epithelial-derived factors induce muscularis mucosa of human induced pluripotent stem cell-derived gastric organoids.

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-ß1 were secreted by the epithelium. HH and TGF-ß signal inhibition prevented subepithelial MM formation. A mechanical property of the substrate promoted SM differentiation around hGOs in the presence of TGF-ß. TGF-ß signaling was shown to influence the HH signaling and mechanical properties. In addition, clinical specimen findings suggested the involvement of TGF-ß signaling in MM formation in recovering gastric ulcers. HH and TGF-ß 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.

Increased expression of SPRR1A is associated with a poor prognosis in pancreatic ductal adenocarcinoma.

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.

In Situ Synthesis of an Anticancer Peptide Amphiphile Using Tyrosine Kinase Overexpressed in Cancer Cells.

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.

Identification and characterization of slow­cycling cells in Ewing sarcoma.

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 slow­cycling 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 label­retaining 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 non­SCCs were investigated. RNA sequencing also revealed several upregulated genes in SCCs as compared with non­SCCs; 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 label­retaining 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.