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.

Tumor necrosis factor-α downregulates the REIC/Dkk-3 tumor suppressor gene in normal human skin keratinocytes.

Our previous studies revealed that REIC/Dkk-3 was expressed various tissues, including skin keratinocytes. The aim of the present study was to identify the factors that regulate the expression of the dickkopf Wnt signaling pathway inhibitor 3 (REIC/Dkk­3) tumor suppressor gene in normal human skin keratinocytes (NHKs). Several growth factors and cytokines that have previously been reported to be involved in the growth and differentiation of keratinocytes were screened as potential regulators. Western blot analysis was performed using protein from NHKs cultured with/without various factors including the epidermal growth factor, tumor necrosis factor­α, transforming growth factor­ß, interleukin (IL)­1F9, IL­6, IL­8 and Ca2+. The results indicated that only TNF­α downregulated REIC/Dkk­3 expression in NHKs. Subsequently, TNF­α was confirmed to reduce the expression levels of REIC/Dkk­3 in mouse skin tissue and hair culture models. TNF­α­mediated downregulation of REIC/Dkk­3 expression in NHKs was abrogated by the addition of a TNF­α­specific antibody. In conclusion, the results indicate that TNF­α downregulates REIC/Dkk­3 expression in normal skin keratinocytes.

Spontaneous hair follicle germ (HFG) formation in vitro, enabling the large-scale production of HFGs for regenerative medicine.

Hair follicle morphogenesis is triggered by reciprocal interactions between hair follicle germ (HFG) epithelial and mesenchymal layers. Here, we developed a method for large-scale preparation of HFGs in vitro via self-organization of cells. We mixed mouse epidermal and mouse/human mesenchymal cells in suspension and seeded them in microwells of a custom-designed array plate. Over a 3-day culture period, cells initially formed a randomly distributed single cell aggregate and then spatially separated from each other, exhibiting typical HFG morphological features. These self-sorted hair follicle germs (ssHFGs) were shown to be capable of efficient hair-follicle and shaft generation upon intracutaneous transplantation into the backs of nude mice. This finding facilitated the large-scale preparation of approximately 5000 ssHFGs in a microwell-array chip made of oxygen-permeable silicone. We demonstrated that the integrity of the oxygen supply through the bottom of the silicone chip was crucial to enabling both ssHFG formation and subsequent hair shaft generation. Finally, spatially aligned ssHFGs on the chip were encapsulated into a hydrogel and simultaneously transplanted into the back skin of nude mice to preserve their intervening spaces, resulting in spatially aligned hair follicle generation. This simple ssHFG preparation approach is a promising strategy for improving current hair-regenerative medicine techniques.

Expression of tumor suppressor REIC/Dkk-3 by a newly improved adenovirus vector with insertion of a hTERT promoter at the 3'-side of the transgene.

Reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3) overexpression, induced using an adenovirus (Ad)-REIC, has been revealed to have a dramatic therapeutic effect on multiple types of cancer. To achieve an improved therapeutic effect from Ad-REIC on cancer, our group previously developed an enhanced gene expression system, the C-TSC cassette [cytomegalovirus (CMV)-RU5' located upstream (C); another promoter unit composed of triple tandem promoters, human telomerase reverse transcriptase (hTERT), simian virus 40 and CMV, located downstream of the cDNA (TSC); plus a polyadenylation (polyA) signal]. When applied to the conventional Ad-REIC, this novel system induced the development of an enhanced product, Ad-C-TSC-REIC, which exhibited a noticeable anticancer effect. However, there were difficulties in terms of Ad-C-TSC-REIC productivity in HEK293 cells, which are a widely used donor cell line for viral production. Productivity of Ad-C-TSC-REIC was significantly reduced compared with the conventional Ad-REIC, as the Ad-C-TSC-REIC had a significantly higher ability to induce apoptotic cell death of not only various types of cancer cell, but also HEK293 cells. The present study aimed to overcome this problem by modifying the C-TSC structure, resulting in an improved candidate: A C-T cassette (C: CMV-RU5' located upstream; T: another promoter unit composed of a single hTERT promoter, located downstream of the cDNA plus a polyA signal), which demonstrated gene expression comparable to that of the C-TSC system. The improved adenovirus REIC/Dkk-3 product with the C-T cassette, named Ad-C-T-REIC, exhibited a higher expression level of REIC/Dkk3, similar to that of Ad-C-TSC-REIC. Notably, the vector mitigated the cell death of donor HEK293 cells, resulting in a higher rate of production of its adenovirus. These results indicated that Ad-C-T-REIC has the potential to be a useful tool for application in cancer gene therapy.

NRF2 Regulates PINK1 Expression under Oxidative Stress Conditions.

Mutations of the PTEN-induced putative kinase 1 (PINK1) gene are a cause of autosomal recessive forms of Parkinson's disease. Recent studies have revealed that PINK1 is an essential factor for controlling mitochondrial quality, and that it protects cells from oxidative stresses. Although there has been considerable progress in the elucidation of various aspects of PINK1 protein regulation such as activation, stability and degradation, the transcriptional regulation of PINK1 mRNA under stress conditions remains unclear. In this study, we found that nuclear factor (erythroid-derived 2)-like 2 (NRF2), an antioxidant transcription factor, regulates PINK1 expression under oxidative stress conditions. Damaged mitochondria arising from stress conditions induced NRF2-dependent transcription of the PINK1 gene through production of reactive oxygen species (ROS). Either an ROS scavenger or forced expression of KEAP1, a potent inhibitory partner to NRF2, restricted PINK1 expression induced by activated NRF2. Transcriptionally up-regulated PINK1 diminished oxidative stress-associated cell death. The results indicate that PINK1 expression is positively regulated by NRF2 and that the NRF2-PINK1 signaling axis is deeply involved in cell survival.

Expression pattern of Dkk-3, a secreted Wnt pathway inhibitor, in mouse intestinal tissue and three-dimensional cultured Caco-2 spheroids.

We investigated the expression pattern of Dkk-3, a secreted Wnt pathway inhibitor, in mouse intestinal tissue and three-dimensional cultured Caco-2 spheroids. Dkk-3 was expressed at the bottom of crypts from the mouse small intestine. Human colon adenocarcinoma Caco-2 cells expressed Dkk-3 under a semi-confluent condition, but Dkk-3 expression was seen in only some of the Caco-2 cells when the cells were sparse. Caco-2 cells formed hollow spheroids in Matrigel, and the layer-forming cells in the hollow spheroids expressed Dkk-3. Our results demonstrated that Dkk-3 might affect intestinal cells when the fate of stem cells changes.

DNAX-activating protein 10 (DAP10) membrane adaptor associates with receptor for advanced glycation end products (RAGE) and modulates the RAGE-triggered signaling pathway in human keratinocytes.

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.

Inhibition of RAGE signaling through the intracellular delivery of inhibitor peptides by PEI cationization.

The receptor for advanced glycation end products (RAGE) is a multi-ligand cell surface receptor and a member of the immunoglobulin superfamily. RAGE is involved in a wide range of inflammatory, degenerative and hyper-proliferative disorders which span over different organs by engaging diverse ligands, including advanced glycation end products, S100 family proteins, high-mobility group protein B1 (HMGB1) and amyloid ß. We previously demonstrated that the cytoplasmic domain of RAGE is phosphorylated upon the binding of ligands, enabling the recruitment of two distinct pairs of adaptor proteins, Toll-interleukin 1 receptor domain-containing adaptor protein (TIRAP) and myeloid differentiation protein 88 (MyD88). This engagement allows the activation of downstream effector molecules, and thereby mediates a wide variety of cellular processes, such as inflammatory responses, apoptotic cell death, migration and cell growth. Therefore, inhibition of the binding of TIRAP to RAGE may abrogate intracellular signaling from ligand-activated RAGE. In the present study, we developed inhibitor peptides for RAGE signaling (RAGE-I) by mimicking the phosphorylatable cytosolic domain of RAGE. RAGE-I was efficiently delivered into the cells by polyethylenimine (PEI) cationization. We demonstrated that RAGE-I specifically bound to TIRAP and abrogated the activation of Cdc42 induced by ligand-activated RAGE. Furthermore, we were able to reduce neuronal cell death induced by an excess amount of S100B and to inhibit the migration and invasion of glioma cells in vitro. Our results indicate that RAGE-I provides a powerful tool for therapeutics to block RAGE-mediated multiple signaling.

SARM1 and TRAF6 bind to and stabilize PINK1 on depolarized mitochondria.

Mutations in PTEN-induced putative kinase 1 (PINK1) or parkin cause autosomal recessive forms of Parkinson's disease. Recent work suggests that loss of mitochondrial membrane potential stabilizes PINK1 and that accumulated PINK1 recruits parkin from the cytoplasm to mitochondria for elimination of depolarized mitochondria, which is known as mitophagy. In this study, we find that PINK1 forms a complex with sterile α and TIR motif containing 1 (SARM1) and tumor necrosis factor receptor-associated factor 6 (TRAF6), which is important for import of PINK1 in the outer membrane and stabilization of PINK1 on depolarized mitochondria. SARM1, which is known to be an adaptor protein for Toll-like receptor, binds to PINK1 and promotes TRAF6-mediated lysine 63 chain ubiquitination of PINK1 at lysine 433. Down-regulation of SARM1 and TRAF6 abrogates accumulation of PINK1, followed by recruitment of parkin to damaged mitochondria. Some pathogenic mutations of PINK1 reduce the complex formation and ubiquitination. These results indicate that association of PINK1 with SARM1 and TRAF6 is an important step for mitophagy.

Design and synthesis of a series of α-benzyl phenylpropanoic acid-type peroxisome proliferator-activated receptor (PPAR) gamma partial agonists with improved aqueous solubility.

In the continuing study directed toward the development of peroxisome proliferator-activated receptor gamma (hPPARγ) agonist, we attempted to improve the water solubility of our previously developed hPPARγ-selective agonist 3, which is insufficiently soluble for practical use, by employing two strategies: introducing substituents to reduce its molecular planarity and decreasing its hydrophobicity via replacement of the adamantyl group with a heteroaromatic ring. The first approach proved ineffective, but the second was productive. Here, we report the design and synthesis of a series of α-benzyl phenylpropanoic acid-type hPPARγ partial agonists with improved aqueous solubility. Among them, we selected (R)-7j, which activates hPPARγ to the extent of about 65% of the maximum observed with a full agonist, for further evaluation. The ligand-binding mode and the reason for the partial-agonistic activity are discussed based on X-ray-determined structure of the complex of hPPARγ ligand-binding domain (LBD) and (R)-7j with previously reported ligand-LDB structures. Preliminal apoptotic effect of (R)-7j against human scirrhous gastric cancer cell line OCUM-2MD3 is also described.

DOCK7 is a critical regulator of the RAGE-Cdc42 signaling axis that induces formation of dendritic pseudopodia in human cancer cells.

Cellular migration is a fundamental process linked to cancer metastasis. Growing evidence indicates that the receptor for advanced glycation end products (RAGE) plays a pivotal role in this process. With regard to downstream signal transducers of RAGE, diaphanous-1 and activated small guanine nucleotide triphosphatases, Rac1 and Cdc42, have been identified. To obtain precise insight into the direct downstream signaling mechanism of RAGE, we screened for proteins interacting with the cytoplasmic domain of RAGE employing an immunoprecipitation-liquid chromatography coupled with an electrospray tandem mass spectrometry system. In the present study, we found that the cytoplasmic domain of RAGE interacted with an atypical DOCK180-related guanine nucleotide exchange factor, dedicator of cytokinesis protein 7 (DOCK7). DOCK7 bound to the RAGE cytoplasmic domain and transduced a signal to Cdc42, resulting in the formation of abundant highly branched filopodia-like protrusions, dendritic pseudopodia. Blocking of the function of DOCK7 greatly abrogated the formation of dendritic pseudopodia and suppressed cellular migration. These results indicate that DOCK7 functions as an essential and downstream regulator of RAGE-mediated cellular migration through the formation of dendritic pseudopodia.

Mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2) as an antiinflammatory target: discovery and in vivo activity of selective pyrazolo[1,5-a]pyrimidine inhibitors using a focused library and structure-based optimization approach.

A novel class of mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2) inhibitors was discovered through screening a kinase-focused library. A homology model of MAPKAP-K2 was generated and used to guide the initial SAR studies and to rationalize the observed selectivity over CDK2. An X-ray crystal structure of a compound from the active series bound to crystalline MAPKAP-K2 confirmed the predicted binding mode. This has enabled the discovery of a series of pyrazolo[1,5-a]pyrimidine derivatives showing good in vitro cellular potency as anti-TNF-α agents and in vivo efficacy in a mouse model of endotoxin shock.

S100A7 promotes the migration and invasion of osteosarcoma cells via the receptor for advanced glycation end products.

Osteosarcoma is the most common malignant tumor of bone in childhood and adolescence. Despite intensive research for new therapies, the outcome in patients with metastasis remains extremely poor. S100 proteins are involved in the proliferation, cell cycle progression and metastasis of numerous malignant tumors, including osteosarcoma. In the present study, we identified S100A7 as a candidate to promote the migration of osteosarcoma cells. S100A7 promoted the migration and invasion of osteosarcoma cells as assayed in vitro. An in vitro pull-down assay revealed the binding of the recombinant S100A7 protein with its putative receptor, the receptor for advanced glycation end products (RAGE). The downregulation of RAGE by a specific siRNA markedly suppressed the migration and invasion of osteosarcoma cells. Furthermore, the matrix metalloproteinase activity of osteosarcoma cells was enhanced by S100A7 and suppressed by the downregulation of RAGE. These results indicate that S100A7 promotes the migration and invasion of osteosarcoma cells through RAGE. The S100A7-RAGE axis may thus be a new target for preventing the invasion and/or metastasis of osteosarcoma.

Partial sensitization of human bladder cancer cells to a gene-therapeutic adenovirus carrying REIC/Dkk-3 by downregulation of BRPK/PINK1.

REIC/Dkk-3 is a tumor suppressor gene that was first identified as a gene downregulated in association with immortalization of normal human fibroblasts. We have demonstrated that an adenovirus carrying REIC/Dkk-3 (Ad-REIC) showed a tumor-specific killing effect on a wide range of cancers. However, some human cancers, bladder cancers in particular, are resistant to Ad-REIC. In this study, we investigated the combination effect of downregulation of BRPK/PINK1 (PINK1) and Ad-REIC on bladder cancer cells. Five bladder cancer cell lines among six cell lines examined were resistant to Ad-REIC. Among the cell lines, the resistance of two cell lines was probably due to low infection efficiency of the adenovirus. PINK1-specific siRNA remarkably downregulated Bcl-xL and TRAP1 proteins and upregulated BAX protein expression. Finally, downregulation of PINK1 partially sensitized the other three cell lines that were resistant to Ad-REIC. This sensitization was associated with increasing production of reactive oxygen species (ROS). These results indicate that PINK1 is one of the key molecules for the mitochondrial protection system and that PINK1 can be a new target molecule to sensitize bladder cancer cells that are resistant to Ad-REIC.

TIRAP, an adaptor protein for TLR2/4, transduces a signal from RAGE phosphorylated upon ligand binding.

The receptor for advanced glycation end products (RAGE) is thought to be involved in the pathogenesis of a broad range of inflammatory, degenerative and hyperproliferative diseases. It binds to diverse ligands and activates multiple intracellular signaling pathways. Despite these pivotal functions, molecular events just downstream of ligand-activated RAGE have been surprisingly unknown. Here we show that the cytoplasmic domain of RAGE is phosphorylated at Ser391 by PKCζ upon binding of ligands. TIRAP and MyD88, which are known to be adaptor proteins for Toll-like receptor-2 and -4 (TLR2/4), bound to the phosphorylated RAGE and transduced a signal to downstream molecules. Blocking of the function of TIRAP and MyD88 largely abrogated intracellular signaling from ligand-activated RAGE. Our findings indicate that functional interaction between RAGE and TLRs coordinately regulates inflammation, immune response and other cellular functions.

Expression of REIC/Dkk-3 in normal and hyperproliferative epidermis.

Dickkopf (Dkk) family members are known as Wnt modulators involved in the development, cell growth/differentiation and cancer. REIC/Dkk-3, which does not interfere with Wnt signalling, has been proposed to be a tumor suppressor gene, but its physiological function has remained unclear. In this study, we analysed the expression of REIC/Dkk-3 in normal interfollicular epidermis (IFE) and hyperproliferative epidermis. REIC/Dkk-3 was expressed in human and mouse IFE, being localized at the interface of upper spinous layer and granular layer. Skin cancer cell lines lost REIC/Dkk-3 expression as reported previously. When we analysed patient samples, REIC/Dkk-3 expression was down-regulated in the hyperproliferative epidermis including skin cancers and non-cancerous proliferative diseases. REIC/Dkk-3 expression was also suppressed in the regenerative and inflammative epidermis of model mice. These findings will certainly contribute to the extension of studies on REIC/Dkk-3.

Intraperitoneal administration of an adenovirus vector carrying REIC/Dkk-3 suppresses peritoneal dissemination of scirrhous gastric carcinoma.

Expression levels of the novel tumor suppressor gene REIC/Dkk-3 are reduced in many human cancers. We have previously showed that an adenovirus vector carrying REIC/Dkk-3 (Ad-REIC) induced apoptosis of cancer cells selectively and exerted bystander antitumor effects via ER stress. We examined possible effects of Ad-REIC in a peritoneal dissemination model of scirrhous gastric carcinoma (SGC). Among various types of gastric cancer, SGC continues to be associated with the worst prognosis due to a high incidence of metastases in the peritoneal cavity. We found that a single intraperitoneal injection of Ad-REIC suppressed tumor dissemination and disease progression. Immunomodulation by Ad-REIC led to recruitment of natural killer cells inside tumor nodules. We conclude that Ad-REIC gene therapy may be a potential tool in combinatorial approaches to achieve curative effects in SGC.

A new cytosolic pathway from a Parkinson disease-associated kinase, BRPK/PINK1: activation of AKT via mTORC2.

Accumulating evidence indicates that dysfunction of mitochondria is a common feature of Parkinson disease. Functional loss of a familial Parkinson disease-linked gene, BRPK/PINK1 (PINK1), results in deterioration of mitochondrial functions and eventual neuronal cell death. A mitochondrial chaperone protein has been shown to be a substrate of PINK1 kinase activity. In this study, we demonstrated that PINK1 has another action point in the cytoplasm. Phosphorylation of Akt at Ser-473 was enhanced by overexpression of PINK1, and the Akt activation was crucial for protection of SH-SY5Y cells from various cytotoxic agents, including oxidative stress. Enhanced Akt phosphorylation was not due to activation of phosphatidylinositol 3-kinase but due to activation of mammalian target of rapamycin complex 2 (mTORC2) by PINK1. Rictor, a specific component of mTORC2, was phosphorylated by overexpression of PINK1. Furthermore, overexpression of PINK1 enhanced cell motility. These results indicate that PINK1 exerts its cytoprotective function not only in mitochondria but also in the cytoplasm through activation of mTORC2.

Internalization of REIC/Dkk-3 protein by induced pluripotent stem cell-derived embryoid bodies and extra-embryonic tissues.

REIC/Dkk-3 was first identified as a down-regulated gene in a number of human immortalized cells and human tumor-derived cell lines. Overexpression of the REIC/Dkk-3 gene using an adenovirus vector (Ad-REIC) has showed a potent selective therapeutic effect on various human cancers through induction of ER stress. Furthermore, we recently showed that Ad-REIC has an indirect host-mediated anti-tumor activity by induction of IL-7. However, the physiological function of REIC/Dkk-3 is still unclear. As a first step to study the possible receptor(s) for secreted REIC/Dkk-3, we analyzed the internalization of Cy3-labeled recombinant REIC/Dkk-3 protein. Among the cell lines screened, mouse induced pluripotent stem (iPS) cells showed a unique pattern of internalization. The internalization was observed in peripheral cells of spherical colonies formed spontaneously, but not in undifferentiated iPS cells. When we analyzed embryoid bodies (EBs) derived from iPS cells, REIC/Dkk-3 protein was internalized specifically by differentiated cells located at the periphery of EBs. Interestingly, Dkk-1 was internalized by undifferentiated cells at the center of the EBs. When developmental tissue was analyzed, internalization of REIC/Dkk-3 protein was strictly limited to extra-embryonic tissue, such as the trophectoderm layer of 4.5 days post-coitus (dpc) blastocysts and the chorionic membrane at 16.5 dpc. The mechanism of the internalization was confirmed to be endocytosis. These findings will contribute to knowledge on the interaction of REIC/Dkk-3 with a possible receptor(s).