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Purpose:Treatment with KRASG12C inhibitors such as sotorasib can produce substantial regression of tumors in some patients with non–small cell lung cancer (NSCLC). These patients require alternative treatment after acquiring resistance to the inhibitor. The mechanisms underlying this acquired resistance are unclear. The purpose of this study was to identify the mechanisms underlying acquired sotorasib resistance, and to explore potential treatments for rescuing patients with sotorasib-resistant KRASG12C NSCLC cells.Experimental Design:Clones of sotorasib-sensitive KRASG12C NSCLC H23 cells exposed to different concentrations of sotorasib were examined using whole-genomic transcriptome analysis, multiple receptor kinase phosphorylation analysis, and gene copy-number evaluation. The underlying mechanisms of resistance were investigated using immunologic examination, and a treatment aimed at overcoming resistance was tested in vitro and in vivo.Results:Unbiased screening detected subclonal evolution of MET amplification in KRASG12C NSCLC cells that had developed resistance to sotorasib in vitro. MET knockdown using small interfering RNA (siRNA) restored susceptibility to sotorasib in these resistant cells. MET activation by its amplification reinforced RAS cycling from its inactive form to its active form. In addition to RAS-mediated MEK–ERK induction, MET induced AKT activation independently of RAS. Crizotinib, a MET inhibitor, restored sensitivity to sotorasib by eliminating RAS–MEK–ERK as well as AKT signaling. MET/KRASG12C dual inhibition led to tumor shrinkage in sotorasib-resistant xenograft mice.Conclusions:MET amplification leads to the development of resistance to KRASG12C inhibitors in NSCLC. Dual blockade of MET and KRASG12C could be a treatment option for MET-amplified, KRASG12C-mutated NSCLC.

Purpose: Treatment with KRASG12C inhibitors such as sotorasib can produce substantial regression of tumors in some patients with non–small cell lung cancer (NSCLC). These patients require alternative treatment after acquiring resistance to the inhibitor. The mechanisms underlying this acquired resistance are unclear. The purpose of this study was to identify the mechanisms underlying acquired sotorasib resistance, and to explore potential treatments for rescuing patients with sotorasib-resistant KRASG12C NSCLC cells. Experimental Design: Clones of sotorasib-sensitive KRASG12C NSCLC H23 cells exposed to different concentrations of sotorasib were examined using whole-genomic transcriptome analysis, multiple receptor kinase phosphorylation analysis, and gene copy-number evaluation. The underlying mechanisms of resistance were investigated using immunologic examination, and a treatment aimed at overcoming resistance was tested in vitro and in vivo. Results: Unbiased screening detected subclonal evolution of MET amplification in KRASG12C NSCLC cells that had developed resistance to sotorasib in vitro. MET knockdown using small interfering RNA (siRNA) restored susceptibility to sotorasib in these resistant cells. MET activation by its amplification reinforced RAS cycling from its inactive form to its active form. In addition to RAS-mediated MEK–ERK induction, MET induced AKT activation independently of RAS. Crizotinib, a MET inhibitor, restored sensitivity to sotorasib by eliminating RAS–MEK–ERK as well as AKT signaling. MET/KRASG12C dual inhibition led to tumor shrinkage in sotorasib-resistant xenograft mice. Conclusions: MET amplification leads to the development of resistance to KRASG12C inhibitors in NSCLC. Dual blockade of MET and KRASG12C could be a treatment option for MET-amplified, KRASG12C-mutated NSCLC.

Removal of dysfunctional mitochondria is essential step to maintain normal cell physiology, and selective autophagy in mitochondria, called mitophagy, plays a critical role in quality control of mitochondria. While in several diseases and aging, disturbed mitophagy has been observed. In stem cells, accumulation of damaged mitochondria can lead to deterioration of stem cell properties. Here, we focused on miR-155-5p (miR-155), one of the most prominent miRNAs in inflammatory and aged tissues, and found that miR-155 disturbed mitophagy in mesenchymal stem cells (MSCs). As a molecular mechanism of miR-155-mediated mitophagy suppression, we found that BCL2 associated athanogene 5 (BAG5) is a direct target of miR-155. Reduction of BAG5 resulted in destabilization of PTEN-induced kinase (PINK1) and consequently disrupted mitophagy. Our study suggests a novel mechanism connecting aging and aging-associated inflammation with mitochondrial dysfunction in stem cells through a miRNA-mediated mechanism.

Frailty of the locomotory organs has become a widespread problem in the geriatric population. The major factor leading to frailty is an age-associated decrease in muscular mass and a reduced number of muscular cells and myofibers. In aged muscular tissues, muscular satellite cells (MuSCs) are reduced due to abnormalities in their self-renewal and the induction of apoptosis. However, the molecular mechanisms connecting aging-associated physiological changes and the reduction of MuSCs are largely unknown. NIMA-related kinase 2 (Nek2), a member of the Nek family of serine/threonine kinases, was found to be downregulated in aged MuSCs/progenitors. Further, Nek2 downregulation was found to inhibit self-renewal and apoptotic cell death by activating the p53-dependent checkpoint. Attenuated NEK2 expression was also observed in the muscular tissues of elderly donors, and its function was confirmed to be conserved in humans. Overall, this study proposes a novel mechanism for inducing muscular atrophy to understand aging-associated muscular diseases.

Sarcopenia is an aging-associated attenuation of muscular volume and strength and is the major cause of frailty and falls in elderly individuals. The number of individuals with sarcopenia is rapidly increasing worldwide; however, little is known about the underlying mechanisms of the disease. Sarcopenia often copresents with obesity, and some patients with sarcopenia exhibit accumulation of peri-organ or intra-organ adipose tissue as ectopic fat deposition, including atrophied skeletal muscle. In this study, we showed that transplantation of the perimuscular adipose tissue (PMAT) to the hindlimb thigh muscles of young mice decreased the number of integrin α7/CD29-double positive muscular stem/progenitor cells and that the reaction was mediated by PMAT-derived exosomes. We also found that the inhibition of cell proliferation was induced by Let-7d-3p miRNA that targets HMGA2, which is an important transcription factor for stem cell self-renewal, in muscular stem/progenitor cells and the composite molecular reaction in aged adipocytes. Reduction of Let-7 miRNA repressor Lin28 A/B and activation of nuclear factor-kappa B signaling can lead to the accumulation of Let-7d-3p in the exosomes of aged PMAT. These findings suggest a novel crosstalk between adipose tissue and skeletal muscle in the development of aging-associated muscular atrophy and indicate that adipose tissue-derived miRNAs may play a key role in sarcopenia.

Treatment withClones of sotorasib-sensitiveUnbiased screening detected subclonal evolution of

Summary Inflammation-induced reactive oxygen species (ROS) are implicated in cellular dysfunction and an important trigger for aging- or disease-related tissue degeneration. Inflammation-induced ROS in stem cells lead to deterioration of their properties, altering tissue renewal or regeneration. Pathological ROS generation can be induced by multiple steps, and dysfunction of antioxidant systems is a major cause. The identification of the central molecule mediating the above-mentioned processes would pave the way for the development of novel therapeutics for aging, aging-related diseases, or stem cell therapies. In recent years, microRNAs (miRNAs) have been shown to play important roles in many biological reactions, including inflammation and stem cell functions. In inflammatory conditions, certain miRNAs are highly expressed and mediate some cytotoxic actions. Here, we focused on miR-155, which is one of the most prominent miRNAs in inflammation and hypothesized that miR-155 participates to inflammation-induced ROS generation in stem cells. We observed mesenchymal stem cells (MSCs) from 1.5-year-old aged mice and determined that antioxidants, Nfe2l2, Sod1, and Hmox1, were suppressed, while miR-155-5p was highly expressed. Subsequent in vitro studies demonstrated that miR-155-5p induces ROS generation by suppression of the antioxidant genes by targeting the common transcription factor C/ebpβ. Moreover, this mechanism occurred during the cell transplantation process, in which ROS generation is triggering loss of transplanted stem cells. Finally, attenuation of antioxidants and ROS accumulation were partially prevented in miR-155 knockout MSCs. In conclusion, our study suggests that miR-155 is an important mediator connecting aging, inflammation, and ROS generation in stem cells.

Elevation of the levels of reactive oxygen species (ROS) is a major tissue-degenerative phenomenon involved in aging and aging-related diseases. The detailed mechanisms underlying aging-related ROS generation remain unclear. Presently, the expression of microRNA (miR)-142-5p was significantly upregulated in bone marrow mesenchymal stem cells (BMMSCs) of aged mice. Overexpression of miR-142 and subsequent observation revealed that miR-142 involved ROS accumulation through the disruption of selective autophagy for peroxisomes (pexophagy). Mechanistically, attenuation of acetyltransferase Ep300 triggered the upregulation of miR-142 in aged BMMSCs, and miR-142 targeted endothelial PAS domain protein 1 (Epas1) was identified as a regulatory protein of pexophagy. These findings support a novel molecular mechanism relating aging-associated ROS generation and organelle degradation in BMMSCs, and suggest a potential therapeutic target for aging-associated disorders that are accompanied by stem cell degeneration.

Purification of undifferentiated cells by removing differentiated parts is an essential step in pluripotent stem cell culture. This process has been traditionally performed manually using a fine glass capillary or plastic tip under a microscope, or by culturing in a selective medium supplemented with anti-differentiation inhibitors. However, there are several inevitable problems associated with these methods, such as contamination or biological side-effects. Here, we developed a laser-assisted cell removing (LACR) technology that enables precise, fast, and contact-less cell removal. Using LACR combined with computational image recognition/identification-discriminating technology, we achieved automatic cell purification (A-LACR). Practicability of A-LACR was evaluated by two demonstrations: selective removal of trophoblast stem (TS) cells from human iPS and TS cell co-cultures, and purification of undifferentiated iPS cells by targeting differentiated cells that spontaneously developed. Our results suggested that LACR technology is a novel approach for stem cell processing in regenerative medicine.

Tissue renewal and muscle regeneration largely rely on the proliferation and differentiation of muscle stem cells called muscular satellite cells (MuSCs). MuSCs are normally quiescent, but they are activated in response to various stimuli, such as inflammation. Activated MuSCs proliferate, migrate, differentiate, and fuse to form multinucleate myofibers. Meanwhile, inappropriate cues for MuSC activation induce premature differentiation and bring about stem cell loss. Recent studies revealed that stem cell regulation is disrupted in various aged tissues. We found that the expression of microRNA (miR)-155, which is an inflammation-associated miR, is upregulated in MuSCs of aged muscles, and this upregulation activates the differentiation process through suppression of C/ebpβ, which is an important molecule for maintaining MuSC self-renewal. We also found that Notch1 considerably repressed miR-155 expression, and loss of Notch1 induced miR-155 overexpression. Our findings suggest that miR-155 can act as an activator of muscular differentiation and might be responsible for accelerating aging-associated premature differentiation of MuSCs.

A high-throughput test of cell growth inhibition was performed using mouse embryonic stem (ES) cells to assess chemical toxicities. We herein demonstrated using a 96-well culture plate approach and the MTT assay that this method was suitable for prioritization of chemicals for their cytotoxic properties. In order to categorize chemicals, we used p53 gene-modified mouse ES cells as well as wild-type ES cells. The p53 gene is a well-known tumor suppressor and controls programmed cell death (apoptosis) and cellular senescence that is triggered by DNA-damaging agents such as alkylating agents and radiation. In the present study, p53-deficient ES cells were found to be more resistant to a tumor initiator, diethylnitrosamine (DEN), than wild-type ES cells, suggesting the inhibition of apoptosis or senescence by a dysfunction in p53. Chromosome aberrations were more frequently detected in p53-deficient ES cells than in wild-type cells, indicating genomic instability due to the deletion of p53. Other tumor initiators, methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (NMU), did not reveal apparent differences in cytotoxicity between wild-type and p53-deficient ES cells. Thus, ES test system using gene-modified ES cells may be used to categorize chemicals by detecting their characteristic effects on apoptosis, genotoxic potentials as well as general cytotoxicity.

Highlights • Hyaluronic acid (HA) has a pharmacological role for reduction of cellular superoxide. • In HA-treated chondrocytes, expression of Nrf2 and its downstream genes was upregulated. • Inhibition of Akt or suppression of HA receptors prevented HA-mediated Nrf2 accumulation. • Nrf2 siRNA inhibited the HA effect on antioxidant enzymes. • HA might contribute to ROS reduction through Nrf2 regulation by activating Akt., One important pharmacological function of hyaluronic acid (HA) in chondrocytes is reduction of cellular superoxide generation and accumulation. Here we demonstrated a relationship between HA supplementation and accumulation of Nuclear factor-erythroid-2-related factor 2 (Nrf2), which is a master transcription factor in cellular redox reactions, in cultured chondrocytes derived from bovine joint cartilage. In HA-treated chondrocytes, expression of Nrf2 and its downstream genes was upregulated. In HA-treated chondrocytes, Akt was phosphorylated, and inhibition of Akt activity or suppression of HA receptors CD44 and/or RHAMM with siRNAs prevented HA-mediated Nrf2 accumulation. Furthermore, Nrf2 siRNA inhibited the HA effect on antioxidant enzymes. These results show that HA might contribute to ROS reduction through Nrf2 regulation by activating Akt. Our study suggests a new mechanism for extracellular matrix (ECM)-mediated redox systems in chondrocytes.

The objective was to investigate the levels of TWIST1 in normal and OA cartilage and examine its role in regulating gene expression in chondrocytes. Human cartilage tissues and chondrocytes were obtained at autopsy from normal knee joints and from OA-affected joints at the time of total knee arthroplasty. TWIST1 expression was increased in human OA knee cartilage compared to normal knee cartilage. TWIST1 induced matrix metalloproteinase 3 (MMP3) expression without direct binding to MMP3 promoter and increased the 5-hydroxymethylcytosine (5hmC) level at the MMP3 promoter. The effect of TWIST1 on expression of TET family (TET1, 2 and 3) was measured in stable TWIST1 transfected TC28 cells, and TET1 expression was up-regulated. TWIST1 dependent upregulation of Mmp3 expression was suppressed in Tet triple KO fibroblast derived from mouse ES cells. Increased TWIST1 expression is a feature of OA-affected cartilage. We identified a novel mechanism of catabolic reaction where TWIST1 up-regulates MMP3 expression by enriching 5hmC levels at the MMP3 promoter via TET1 induction. These findings implicate TWIST1 as an important factor regulating OA related gene expression. Clarifying epigenetic mechanisms of 5hmC induced by TWIST1 is a critical molecule to understanding OA pathogenesis.

In mammalian ovaries, many immature follicles remain after the dominant follicles undergo ovulation. Here we report the successful production of rabbit embryonic stem cells (ESCs) from oocytes produced by in vitro culture of immature follicles and subsequent in vitro maturation treatment. In total, we obtained 53 blastocysts from oocytes that received intracytoplasmic sperm injection followed by in vitro culture. Although only weak expression of POU5f1 was observed in the inner cell masses of in-vitro-cultured follicle-derived embryos, repeated careful cloning enabled establishment of 3 stable ESC lines. These ESC lines displayed the morphological characteristics of primed pluripotent stem cells. The ESC lines also expressed the pluripotent markers Nanog, POU5f1, and Sox2. Further, these ESCs could be differentiated into each of the 3 different germ layers both in vitro and in vivo. These results demonstrate that immature follicles from rabbits can be used to generate ESCs. Moreover, the use of rabbit oocytes as a cell source provides an experimental system that closely matches human reproductive and stem cell physiology.

Embryonic stem cells (ESCs) have the potential to be used as an unlimited cell source for cell transplantation therapy, as well as for studying mechanisms of disease and early mammalian development. However, applications involving ESCs have been limited by the lack of reliable differentiation methods in many cases. Mesenchymal stem cells (MSCs) have also emerged as a promising cell source, but as suggested in recent studies, these cells display limited potential for proliferation and differentiation, thereby limiting their usefulness in the clinic and in the laboratory. Unfortunately, effective methods for induction of MSCs from pluripotent stem cells have not been established, and the development of such methods remains a major challenge facing stem cell biologists. Oxygen concentration is one of the most important factors regulating tissue development. It has profound effects on cell metabolism and physiology and can strongly influence stem cell fate. Here we demonstrate that severe low O2concentrations (1%) can function as a selective pressure for removing undifferentiated pluripotent cells during the induction of MSCs from rabbit ESCs (rESCs) and that MSCs induced under severe hypoxic conditions function as normal MSCs; that is, they repopulate after cloning, express specific markers (vimentin, CD29, CD90, CD105, and CD140a) and differentiate into adipocytes, osteoblasts, and chondrocytes. Furthermore, we demonstrate that these cells can contribute to cartilage regeneration in an in vivo rabbit model for joint cartilage injury. These results support the notion that exposing ESCs to severe hypoxic conditions during differentiation can be used as a strategy for the preparation of functional MSCs from ESCs.

Intracerebral inflammation resulting from injury or disease is implicated in disruption of neural regeneration and may lead to irreversible neuronal dysfunction. Analysis of inflammation-related microRNA profiles in various tissues, including the brain, has identified miR-155 among the most prominent miRNAs linked to inflammation. Here, we hypothesize that miR-155 mediates inflammation-induced suppression of neural stem cell (NSC) self-renewal. Using primary mouse NSCs and human NSCs derived from induced pluripotent stem (iPS) cells, we demonstrate that three important genes involved in NSC self-renewal (Msi1, Hes1 and Bmi1) are suppressed by miR-155. We also demonstrate that suppression of self-renewal genes is mediated by the common transcription factor C/EBPβ, which is a direct target of miR-155. Our study describes an axis linking inflammation and miR-155 to expression of genes related to NSC self-renewal, suggesting that regulation of miR-155 may hold potential as a novel therapeutic strategy for treating neuroinflammatory diseases.

Recently, an additional type of pluripotent stem cell-line derived from mouse embryos has been established and termed epiblast stem cell (EpiSC), and is expected to be an important tool for studying the mechanisms of maintenance of pluripotency since they depend on basic fibroblast growth factor-MAPK and Activin A-Smad2/3 signaling to maintain pluripotency, unlike mouse embryonic stem cells (ESCs). Further, because of the similarities between mouse EpiSCs and human ESCs, EpiSCs are expected to be effective experimental models for human stem cell therapy. Recently, study for conversion from ESC state to EpiSC state or reversion from EpiSC state to ESC state has attracted interest since these techniques may lead to increasing the potential of pluripotent stem cells and our knowledge about their developmental status. In the present study, we find that a low oxygen concentration in culture environment accelerated, improved, and stabilized the EpiSC state of the converted cells from the ESC state using Oct4ΔPE-GFP transgenic ESCs. Induced EpiSCs (iEpiSCs) in hypoxia possess closer gene expression patterns to native EpiSCs, and bisulfite sequences for the promoter regions of Stella and Oct4 genes have elucidated that the iEpiSC gain EpiSC-specific methylation patterns in hypoxia. Our data provide evidence that oxygen concentration is an important factor for establishment of the EpiSC-specific state.

Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the stemness or related molecular mechanisms have not been well determined. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, AKT phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the AKT phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator.

The embryos of some rodents and primates can precede early development without the process of fertilization; however, they cease to develop after implantation because of restricted expressions of imprinting genes. Asexually developed embryos are classified into parthenote/gynogenote and androgenote by their genomic origins. Embryonic stem cells (ESCs) derived from asexual origins have also been reported. To date, ESCs derived from parthenogenetic embryos (PgESCs) have been established in some species, including humans, and the possibility to be alternative sources for autologous cell transplantation in regenerative medicine has been proposed. However, some developmental characteristics, which might be important for therapeutic applications, such as multiple differentiation capacity and transplantability of the ESCs of androgenetic origin (AgESCs) are uncertain. Here, we induced differentiation of mouse AgESCs and observed derivation of neural cells, cardiomyocytes and hepatocytes in vitro. Following differentiated embryoid body (EB) transplantation in various mouse strains including the strain of origin, we found that the EBs could engraft in theoretically MHC-matched strains. Our results indicate that AgESCs possess at least two important characteristics, multiple differentiation properties in vitro and transplantability after differentiation, and suggest that they can also serve as a source of histocompatible tissues for transplantation.

Non-human primates are suitable models for preclinical research aimed at cell-replacement therapies. Recently, it has been reported that Rho-associated kinase inhibitor Y-27632 markedly reduced dissociation-induced apoptosis of human embryonic stem (hES) cells, and is expected as a novel supplement for hES cell maintenance or differentiation inductions; however, the effects of the chemical are still to be determined in model animals. Here, we demonstrated the effect of Y-27632 on cynomolgus monkey ES (cyES) cells. Also, in cyES cells, Y-27632 treatment dramatically improved the efficiency of colony formation from single cells without affecting the pluripotent state and karyotype. Y-27632 supplementation was also effective for feeder-free culture and differentiation induction. Neural stem cells directly induced from cyES cells could give rise to neurons, astrocytes and dopamine producing cells. The present result not only suggests that the chemical was effective for improving the culture system of primate ES cells, but also the similarity between cyES and hES cells regarding the reactions to the chemical, which might be further evidence that cyES cells are superior models for hES cells.

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.

Embryonic stem cells (ESCs) are a good material for the study of mammalian development, production of genetically modified animals, and drug discovery because they proliferate infinitely while maintaining a multilineage differentiation potency and a normal karyotype. However, ethical considerations limit the use of human embryos for the establishment of ESCs. Recently, ESCs have been produced from blastomeres divided by biopsy in mice and humans. The method is expected to be less controversial because it does not destroy the embryo. However, no one has yet produced both a pup and an ESC from a single embryo. Here, we describe the production of individual/ESC pairs from each of three embryos out of 20 attempts, and is thus considered efficient. Blastomere-derived ESC could differentiate some types of tissues and contribute to chimera mouse. These results show that each blastomere at two-cell stage possesses pluripotency and separated blastomeres maintain viability to develop to a pup or pluripotent ESC.

Embryonic stem cells (ESCs) of nonhuman primates are important for research into human gametogenesis because of similarities between the embryos and fetuses of nonhuman primates and those of humans. Recently, the formation of germ cells from mouse ESCs in vitro has been reported. In this study, we established cynomolgus monkey ES cell lines (cyESCs) and attempted to induce their differentiation into germ cells to obtain further information on the development of primate germ cells by observing the markers specific to germ cells. Three cyESCs were newly established and confirmed to be pluripotent. When the cells are induced to differentiate, the transcripts of Vasa and some meiotic markers were expressed. VASA protein accumulated in differentiated cell clumps and VASA-positive cells gathered in clumps as the number of differentiation days increased. In the later stages, VASA-positive clumps coexpressed OCT-4, suggesting that these cells might correspond to early gonocytes at the postmigration stage. Furthermore, meiosis-specific gene expression was also observed. These results demonstrate that cyESCs can differentiate to developing germ cells such as primordial germ cells (PGCs) or more developed gonocytes in our differentiation systems, and may be a suitable model for studying the mechanisms of primate germ cell development.

The cell adhesion molecule Cadherin 2 (Cdh2) plays important roles in somatic cell adhesion, proliferation and migration. Cdh2 is also highly expressed in mouse epiblast stem cells (mEpiSCs), but its function in these cells is unknown. To understand the function of Cdh2 in mEpiSCs, we compared the expression of pluripotency-related genes in mEpiSCs and mouse embryonic stem cells (mESCs) after either Cdh2 knockdown or Cdh2 over-expression. Introduction of specific siRNA against Cdh2 led to attenuation of pluripotency-related genes. Pluripotent gene expression was not recovered by over-expression of Cdh1 following Cdh2 knockdown. Western blot analysis and co-immunoprecipitation assays revealed that Cdh2 stabilizes FGFR1 in mEpiSCs. Furthermore, stable transfection of mESCs with Cdh2 cDNA followed by FGF2 supplementation accelerated cell differentiation. Thus, Cdh2 contributes to the establishment and maintenance of FGF signaling-dependent self-renewal in mEpiSCs through stabilization of FGFR1.

Background: The risk factors for surgical site infection (SSI) following urological operations have not been clearly identified, although the presence of a preoperative urinary tract infection (UTI) is thought to be one risk factor. We studied potential risk factors to clarify when and how bacteria contaminate wounds and SSI develop. Methods: Objects of the present study were patients with SSI after open urological operations that were performed at the Department of Urology, Miyazaki Medical College Hospital, University of Miyazaki, Kiyotake, Miyazaki, Japan, during the period between June 1999 and December 2000. Endourological operations, operations on children and short operations of less than 2 h duration were excluded. Patients were screened for the presence of UTI before the operation and subcutaneous swabs for culture were collected at the end of the operation by brushing with a sterile cotton-swab just before skin closure. Results: Surgical site infections occurred in 20 of 134 patients. Bacteria from the subcutaneous swabs were detected in 15 (75.0%) of the patients with SSI. All patients received antimicrobial prophylaxis (AMP), but bacteria from the subcutaneous swabs of patients with SSI were less susceptible to the agents (20.0%). Preoperative UTI were observed in 11 (55.0%) of the patients with SSI. In these patients, four had the same species of bacteria detected from urine, swab and wound, three had the same species from swab and wound and one had the same species from urine and wound. Conclusions: Preoperative UTI was the most important risk factor for SSI following urological operations. It is most likely that the bacteria in the urine contaminated the surgical fields and the AMP resistant strains produced SSI.

A total of 68 ureteroneocystostomies performed between 1980 and 1992 in 63 patients with vesicoureteral reflux (UVR) secondary to neurogenic bladder were reviewed to elucidate factors of postoperative hydronephrosis. Urogram, cystogram, and urodynamics were analyzed, and the cases were classified into two types (areflexia, hyperreflexia) of detrusor muscle response. Occurrence or progression of hydronephrosis was demonstrated in 26 operations (complicated group). Compared with the rest (uncomplicated group), significant difference was not observed concerning proportions of vesical deformity, VUR grades, preoperative hydronephrosis, cystometric types and operative factors. In cases of hyperreflexia maximum vesical volume, maximum vesical pressure or maximum urethral pressure was not different between both groups. However, in those of areflexia maximum vesical volume of the complicated group was significantly smaller than that of the uncomplicated group (290 +/- 35 ml vs. 370 +/- 35 ml, P = 0.03), and the proportion of bladders with compliance less than 10 ml/cmH2O was significantly higher in the complicated group than in the uncomplicated group (P = 0.05) though maximum vesical pressure or maximum urethral pressure was not different. In areflex bladder deformity was observed more in the complicated group than in the uncomplicated group (P = 0.06). Relative inactiveness of detrusor muscle in areflex bladder might render such preoperative findings as vesical deformity, contracted bladder and low compliance more prognosticative of postoperative hydronephrosis than in hyperreflex bladder. During the follow up (6 to 132, mean 42 months) no case showed progression of hydronephrosis or renal deterioration, nor did any case require revisional surgery.(ABSTRACT TRUNCATED AT 250 WORDS)

A very rare case of primary adenocarcinoma arising from a paraurethral cyst in a 63-year-old woman is reported. Initially she was diagnosed as having a simple paraurethral cyst because of absent communication with the urethra. The resected paraurethral cyst was histologically associated with adenocarcinoma. We also performed chemotherapy composed of methotrexate, vinblastine, Adriamycin and cisplatin because of lymph node metastasis. Our treatment, however, was not effective and the patient died of systemic metastases.

Excessive mechanical stress on the cartilage causes the degradation of the matrix, leading to the osteoarthritis (OA). Matrix metalloproteinases 13 (MMP13) is a major catalytic enzyme in OA and p38 plays an important role in its induction. However, precise pathway inducing p38 activation has not been elucidated. We hypothesized here that the small GTPase Rho and its effector ROCK might function in upper part of the mechanical stress-induced matrix degeneration pathway. Bovine metacarpal phalangeal articular cartilage explants were loaded with 1 MPa dynamic compression for 6 h with or without a ROCK specific inhibitor Y27632 or/and a p38 specific inhibitor SB202190. Then p38 phosphorylation and MMP13 expression were assessed by western blot or/and quantitative RT-PCR. Rho-activity was measured by pull-down assay using glutathione S-transferase fusion protein of Rho binding domain. Cyclic compression caused Rho activation, p38 phosphorylation and MMP13 expression. Both Y27632 and SB202190 were found to block the mechanical stress-enhanced p38 phosphorylation and subsequent MMP13 expression. The present results show that p38 phosphorylation and MMP13 expression are regulated by Rho/ROCK activation, and support the potential novel pathway that Rho/ROCK is in the upper part of the mechanical stress-induced matrix degeneration cascade in cartilage comprised of p38 and MMP13.

Recently, several research groups have shown that germ cells can be produced in vitro from pluripotent embryonic stem cells (ESCs). In the mouse, live births of offspring using germ cells induced from ESCs in vitro have been reported. Furthermore, some efficient methods for inducing the useful number of germ cells from ESCs have also been developed. On the other hand, in primates, despite the appearances of germ cell-like cells including meiotic cells were observed by spontaneous differentiation or introducing transgenes, it has not been determined whether fully functional germ cells can be derived from ESCs. To elucidate the property for the germ cells induced from primate ESCs, specification of the promoting factors for the germ cell development and improving the efficiency of germ cell derivation are essential. Leukemia inhibitory factor (LIF) has been reported as one of the important factors for mouse primordial germ cell (PGC) survival in vitro. However, the effects of LIF on germ cell formation from pluripotent cells of primates have not been examined. The aim of this study is to determine whether LIF addition can improve in vitro germ cell production from cynomolgus monkey ESCs (cyESCs). After 8 days of differentiation, LIF added culture induced dome-shaped germ cell colonies as indicated by the intense expression of alkaline phosphatase activity (ALP). These cells also demonstrate high-level expression of the germ cell-marker VASA, OCT-4, and BLIMP-1, and show SSEA-1 expression that supports their early stage germ cell identity. Finally, we observed that adding LIF to differentiating cultures inhibited meiotic gene expressions and increased the percentage of ALP-positive cells, and demonstrate that the addition of LIF to differentiation media increases differentiation of early germ cells from the cyESCs.

Recent studies have illustrated multiple differentiation potentials of embryonic stem cells (ESCs), derived from parthenogenetic embryos, to various kinds of cells (all three embryonic germ layers). However, differentiation diversity of the parthenogenetic ESCs (PgESCs) in vivo remains to be elucidated. In the present study, we established mouse PgESC-lines and observed their contribution diversity in vivo by producing chimeric mice using embryos possessing single nucleotide polymorphisms of mitochondrial DNA (mtDNA) as hosts. Based on southern blot analysis using specific probes to detect the SNPs on mtDNA, PgESC-derived mtDNA were contained in many organs such as brain, lung, and heart of the chimeric mouse. We concluded that PgESCs contributed to various internal organs in vivo, and that they were also stably maintained in adult animals.

Neural stem cells (NSCs) are tissue-specific stem cells with self-renewal potential in brain, and are committed cells of the central nervous system. Recently, some reports have suggested the possibility of the NSCs to differentiate into non-CNS mesodermal derivatives, such as blood cells and skeletal muscle cells. Here we isolated NSCs as neurospheres from a neonatal mouse brain using serum replacement medium, and demonstrated that the stem cell population expressing pluripotent-related genes such as Oct-4, Sox-2, and Nanog possess multiple differentiation potentials to ectodermal, mesodermal, and endodermal lineages, that is, some neural cells, beating cardiomyocytes, adipocytes, and insulin-producing cells. The results of the present study partly provide further evidence for multiple differentiation properties of NSCs and suggest common characteristics between NSCs and other pluripotent stem cells.

The embryos of some rodents and primates can precede early development without the process of fertilization; however, they cease to develop after implantation because of restricted expressions of imprinting genes. Asexually developed embryos are classified into parthenote/gynogenote and androgenote by their genomic origins. Embryonic stem cells (ESCs) derived from asexual origins have also been reported. To date, ESCs derived from parthenogenetic embryos (PgESCs) have been established in some species, including humans, and the possibility to be alternative sources for autologous cell transplantation in regenerative medicine has been proposed. However, some developmental characteristics, which might be important for therapeutic applications, such as multiple differentiation capacity and transplantability of the ESCs of androgenetic origin (AgESCs) are uncertain. Here, we induced differentiation of mouse AgESCs and observed derivation of neural cells, cardiomyocytes and hepatocytes in vitro. Following differentiated embryoid body (EB) transplantation in various mouse strains including the strain of origin, we found that the EBs could engraft in theoretically MHC-matched strains. Our results indicate that AgESCs possess at least two important characteristics, multiple differentiation properties in vitro and transplantability after differentiation, and suggest that they can also serve as a source of histocompatible tissues for transplantation.

The majority of somatic cell nuclear transferred (SCNT) embryos die before or after implantation. Many studies have focused on morphological remodeling of the donor nucleus and its associated cytoskeletal structures in the early events of nuclear transfer. However, little is known about the 2-cell stage of SCNT embryos after the first division. In this study, we compared the morphological status of chromosomal division during the 1-cell stage to the 2-cell stage in SCNT embryos with that in intracytoplasmic sperm injection (ICSI) embryos. The microtubules and cytoplasmic asters, which are related to chromatin segregation, disappeared at the pronuclear stage, although formation of the first mitotic spindle was normal in both the SCNT and ICSI embryos. However, nuclear fragmentation was observed in 30% of the 2-cell SCNT embryos and 12% of the 2-cell ICSI embryos. Nuclear fragmentation was present in both blastomeres of these embryos. No apoptotic DNA fragmentation was observed in TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) assays for either the SCNT or ICSI embryos. In both the SCNT and ICSI embryos, the distribution of chromosomes in the first mitotic spindle was disturbed during the process of division from the 1-cell stage to the 2-cell stage. These results suggest that loss of SCNT embryos just before or after implantation may be due to an abnormal chromosome distribution at the 2-cell stage.

The risk factors for surgical site infection (SSI) following urological operations have not been clearly identified, although the presence of a preoperative urinary tract infection (UTI) is thought to be one risk factor. We studied potential risk factors to clarify when and how bacteria contaminate wounds and SSI develop.Objects of the present study were patients with SSI after open urological operations that were performed at the Department of Urology, Miyazaki Medical College Hospital, University of Miyazaki, Kiyotake, Miyazaki, Japan, during the period between June 1999 and December 2000. Endourological operations, operations on children and short operations of less than 2 h duration were excluded. Patients were screened for the presence of UTI before the operation and subcutaneous swabs for culture were collected at the end of the operation by brushing with a sterile cotton-swab just before skin closure.Surgical site infections occurred in 20 of 134 patients. Bacteria from the subcutaneous swabs were detected in 15 (75.0%) of the patients with SSI. All patients received antimicrobial prophylaxis (AMP), but bacteria from the subcutaneous swabs of patients with SSI were less susceptible to the agents (20.0%). Preoperative UTI were observed in 11 (55.0%) of the patients with SSI. In these patients, four had the same species of bacteria detected from urine, swab and wound, three had the same species from swab and wound and one had the same species from urine and wound.Preoperative UTI was the most important risk factor for SSI following urological operations. It is most likely that the bacteria in the urine contaminated the surgical fields and the AMP resistant strains produced SSI.

Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) are well known as specific tumor markers of prostate cancer, but carcinoembryonic antigen (CEA)- and carbohydrate antigen 19-9 (CA19-9)-producing adenocarcinoma originating in the prostate is rare. We report here a case of prostatic adenocarcinoma positive for these 4 tumor markers in a 50-year-old man who had initially complained about chest pain due to metastatic bone tumor. In spite of the extensive treatment involving hormone and radiation therapy, the patient died of rapid tumor extension only 4 months after initial diagnosis. Autopsy revealed multiple metastases to the bone, liver, lungs and lymph nodes. Histologically, two types of adenocarcinoma were involved in both primary prostate and metastatic sites: one was a poorly differentiated adenocarcinoma positive for PSA and PAP but not CEA or CA19-9, and the other one was a less differentiated adenocarcinoma partially positive for CEA and CA19-9 but not for PSA or PAP. Based on this case and previous cases by review of the literature, CEA- and CA19-9-producing adenocarcinoma of the prostate was suggested to rapidly progress with multiple metastases and to show poor prognosis with strong resistance to any treatment.

s / Osteoarthritis and Cartilage 21 (2013) S63–S312 S130 ADAM and ADAMTS families of metalloproteinases their inhibitors the TIMPs, all CCN growth factor family members, certain FGFs and neuropeptide precursors and receptors in immortalised human chondrocytes, C28/I2. Methods: We compared the response of C28/I2 cells cultured in monolayer (2D) or on polyhema (3D; preventing cell adhesion to the culture plastic) which is known to maintain chondrocyte phenotype, exploring expression of 95 genes analysed by Taqman Low Density Array. Ten genes showed significant changes (p< 0.05) of at least 2-fold. Results: In 2D cultures Interleukin 6 (IL6) was up-regulated 3-fold by both bradykinin and Substance P. IL6 is up-regulated in OA joint fluid and elevated IL6 is associated with enhanced sensitivity to pain. Although in 3D culture no significant modulation of IL6 was observed by neuropeptides we did observe up-regulation of IL6 expression in response to IL1/Oncostatin M (used as a positive control). In 3D culture expression of six genes was significantly repressed (at least 2-fold) by both bradykinin and substance P and no genes were significantly induced. Amongst these genes it was interesting to note suppression of inhibitors TIMP2 (inhibitor of all MMPs) and TIMP3 (inhibits all MMPs, certain ADAMs and ADAMTS proteinases). Expression of three metalloproteinases, ADAMTS9 (an aggrecanase), MMP2 and MMP15 was also significantly suppressed although expression of these enzymes was still high. FGF18 was also suppressed by both neuropeptides in 3D only and this growth factor has been shown to promote cartilage repair. WISP3/ CCN6 was repressed 2-fold in 3D culture by both neuropeptides. We have previously demonstrated that WISP3/CCN6 is up-regulated in end stage OA cartilage and regulates MMP and ADAMTS4 and 5 in chondrocytes. Whilst neurotrophin BDNF expression was repressed by both neuropeptides, tachykinin 3 (the precursor to neurokinin B) was also repressed fold by bradykinin but interestingly not by the tachykinin substance P. We also explored expression of two of the genes regulated in C28/I2 cells by qPCR analysis in primary human chondrocytes and confirmed that expression of both WISP3/CCN6 and TIMP3 was also repressed by the two neuropeptides. Conclusions: Overall our data suggest that the effects of substance P and bradykinin on chondrocyte gene expression and matrix metabolism are environment-dependent. 235 LOCAL RAS IN THE HYPERTROPHIC DIFFERENTIATION OF CHONDROCYTES I. Tsukamoto y, M. Akagi y, S. Inoue y, Y. Oda y, T. Teramura z, T. Takehara z, Y. Onodera z. yDept. of Orthopaedic Surgery, Faculty of Med., Kinki Univ., Osaka-Sayama City, Japan; zDiv. of Cell Biology for Regenerative Med., Kinki Univ., Osaka-Sayama City, Japan Purpose: Recently, the local renin-angiotensin system (RAS) has attracted many researchers in many pathophysiological issues. Also in orthopedics, expression of the local RAS was found in fracture callus, bone tissues and arthritic synovium. In the last year, we reported that the local RAS expressed in chondrocytes of the epiphyseal plates of mice. The purpose of this study is to reveal immunohistological localization of the RAS components in the limb buds of mice where another physiological hypertrophic differetiation occurs and to analyze function of the local RAS in the processes of hypertrophic differentiation using ATDC5 cell line. Methods: The limb buds of 15-day-viviparous mice were immunostained with antibodies to angiotensinogen, angiotensinogen converting enzyme 1 (ACE1), angiotensinII type 1 receptor (AT1R) and angiotensinII type 2 receptor (AT2R). We cultured ATDC5 cell line and evaluated expression of angiotensinogen, ACE1, AT1R and AT2R during the hypertrophic term using quantitative real-time PCR and Western blot analysis. Then, we separately stimulated AT1R and AT2R in hypertrophic term using angiotensin II, olmesartan and PD123319 and evaluated expression of type X collagen using quantitative real-time PCR and Western blot analysis. Results: In the limb buds of mice, angiotensinogen and AT1R expressed in the resting chondrocytes, the proliferative chondrocytes and hypertrophic chondrocytes; however, ACE1 and AT2R expressed only in the hypertrophic chondrocytes. In ATDC5 cell line, angiotensinogen and AT1R expressed mainly in the proliferative term and slightly in the hypertrophic term; conversely, ACE1 and AT2R expressed mainly in the hypertrophic term and slightly in the proliferating term. In the hypertrophic term, expression of type X collagen were downregulated activating AT1R and were conversely upregulated activating AT2R. Conclusions: The local RAS also expresses in the limb buds of mice. In the hypertrophic term of the chondrocyte differentiation, activating AT1R decelerates the hypertrophic differentiation; conversely, activating AT2R accelerates the hypertrophic differentiation. Therefore, the local RAS might regulate the chondrocyte hypertrophic differentiation. 236 PRONOUNCED DECREASE IN EXPRESSION OF CHONDROPROTECTIVE SMAD2/3 PHOSPHORYLATING TGFb-SIGNALING RECEPTORS DURING AGEING IN HEALTHY CARTILAGE A. van Caam, E. Blaney Davidson, W. Madej, W. van den Berg, P. van der Kraan. Radboud Univ. Nijmegen Med. Ctr., Nijmegen, The Netherlands Purpose: Osteoarthritis (OA) is a multi-factorial disease involving bone, synovium and cartilage, with ageing as its key risk factor. Signaling by the Transforming Growth Factor b (TGFb)-superfamily of ligands via Smad phosphorylation (SmadP) plays a crucial role in cartilage homeostasis, as Smad1/5/8p induces while Smad2/3p protects against chondrocyte hypertrophy. In cartilage, age-related changes in expression of TGFb-superfamily members, like TGFb1 and BMP7, have been observed. Furthermore, age-related changes in expression of the TGFbsuperfamily receptors ACVRL1 (ALK1) and TGFBRI (ALK5) are thought to be involved in onset of OA. Based on these findings, we wanted to investigate if gene expression of other members of the TGFb-superfamily signaling pathway changes during ageing in healthy cartilage. Therefore, in this study we set out to identify age-related changes in expression of TGFb-superfamily members and their receptors in normal cartilage. Methods: From the metacarpophalangeal joint of freshly butchered (< 3 h post mortem), cows (Bos Taurus) (n1⁄4 32, 7 male, 25 female), 28 mm2 full thickness cartilage explants were collected with a biopsy punch. Age of the animals ranged from 1 up to 10 years, and samples were evenly spread over this range. Macroscopically, none of the animals showed any signs of OA. Samples were taken from the apical side of the metacarpal chondyle medial to the intertrochlear notch, quickly washed in saline to remove synovial fluid, and flash frozen in liquid nitrogen. Cartilage was pulverized using a dismembrator and RNA was extracted using the RNAeasy method. Gene expression was measured by quantitative real time PCR, using cDNA specific primers and SYBRgreen. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. Regression analyses were done using SPSS. Results: To assess the validity of the dataset, Collagen type 2 alpha 1 (Col2A1), and Tgfb1 , expression was measured, as expression of both genes should decrease during ageing. Col2A1 expression decreased 4000-fold (R2 1⁄4 0.85, p < 0.0001) fold, and Tgfb1 over 2 fold (R2 1⁄4 0.35, p1⁄4 0.0007) in a linear fashion between young and old animals, showing that our dataset is valid for studying ageing of healthy cartilage . Subsequently expression of TGFb-superfamily type I and type II receptors was measured. Most notably, expression of all three Smad2/3 phosphorylating receptors, Acvr1b (ALK4) (R2 1⁄4 0.36, p 1⁄4 0.0043), Tgfbr1 (ALK5) (R2 1⁄4 0.21, p 1⁄4 0.0077), and Acvr1c (ALK7) (R2 1⁄4 0.26, p 1⁄4 0.0317), was lowered by, on average, 4-fold during ageing, while of the four Smad1/5/8 phosphorylating receptors only expression of Bmpr1a (ALK3) (R2 1⁄4 0.19, p 1⁄4 0.012) was lowered by 2-fold. Of the type II receptors, only a decrease of approximately 3-fold in Bmpr2 (R2 1⁄4 0.42, p 1⁄4 0.0002) expression could be observed. No change in expression of the two type III receptors Eng and Tgfbr3 was found. Furthermore no changes in expression of the following members of the Transforming Growth Factor b (TGFb)-superfamily: Tgfb2, Tgfb3, Bmp2, Bmp4, Gdf5 and Bmp7 were observed. Conclusions: Age-related changes previously observed in human and murine cartilage could also be observed in bovine cartilage, showing the validity of our dataset. We observed more changes in receptor expression than in growth factor (ligand) expression, suggesting a more important role for change in receptor expression during the ageing process compared to change in ligand expression. Strikingly, in our dataset, expression of all three Smad2/3p phosphorylating receptors was lowered during ageing, while expression of Smad1/5/8 phosphorylating receptors remained largely unaffected. Given the importance of Smad2/3p in maintaining articular cartilage phenotype, this can underlie the increased responsiveness of aged chondrocytes to processes that induce terminal differentiation.

A local tissue-specific renin–angiotensin system (local RAS) has been identified in many organs. However, no report has described the role of a local RAS in the hypertrophic differentiation of chondrocytes. To examine the role of a local RAS in the hypertrophic differentiation, we activated angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) separately in the cell line ATDC5, which involves differentiation from mesenchymal stem cells to hypertrophic chondrocytes. Activation of AT1R suppressed and activation of AT2R enhanced the expression of markers of hypertrophic differentiation, including type X collagen, matrix metalloproteinase 13 and runt-related transcription factor 2., Highlights • Renin–angiotensin system components are expressed during hypertrophic differentiation. • AT1R is expressed intensely during chondrocyte proliferation. • AT2R is expressed intensely in the hypertrophic phase. • The expression of Col.X, MMP13 and Runx2 are repressed by activating AT1R. • The expression of Col.X, MMP13 and Runx2 are enhanced by activating AT2R.