Contrast-induced nephropathy (CIN) is a major clinical problem associated with acute kidney injury during hospitalization. However, effective treatments for CIN are currently lacking. Mesenchymal ...stem cells (MSCs) have protective effects against kidney injury by suppressing inflammation and fibrosis. We previously showed that MSCs cultured in serum-free medium (SF-MSCs) enhance their anti-inflammatory and anti-fibrotic effects. However, whether SF-MSCs potentiate their anti-apoptotic effects is unknown. Here, we investigated the effects of SF-MSCs on a CIN mouse model.
To create CIN model mice, we removed right kidney at first. One week later, the left renal artery was clamped for 30 min to cause ischemia-reperfusion injury, and mice were injected with iohexol. Then the kidney received 10 Gy of irradiation, and MSCs or SF-MSCs were injected immediately. At 24 h post-injection, mice were sacrificed, and their blood and kidneys were collected to evaluate renal function, DNA damage, and apoptosis. In addition, apoptosis was induced in HEK-293 cells by irradiation and cells were treated with conditioned medium from MSCs or SF-MSCs.
Treatment of CIN model mice with SF-MSCs markedly improved renal function compared with MSCs treatment. Cleaved caspase-3 levels and TUNEL-positive cell numbers were strongly suppressed in CIN model mice treated with SF-MSCs compared with the findings in those treated with MSCs. γH2AX levels, a chromosome damage marker, were reduced by MSCs and further reduced by SF-MSCs. In addition, cleaved caspase-3 in irradiated HEK-293 cells was more strongly suppressed by conditioned medium from SF-MSCs than by that from MSCs. Secretion of epidermal growth factor (EGF) was enhanced by culturing MSCs in serum-free medium. Knockdown of EGF by siRNA attenuated the inhibitory effects of SF-MSCs on CIN-induced renal dysfunction and tubular apoptosis.
These findings strongly suggest that SF-MSCs improve CIN in model mice by exerting anti-apoptotic effects in a paracrine manner. Thus, SF-MSCs represent a potential novel therapy for CIN.
Fibrosis is a common histological feature in the process from chronic organ injury to organ failure. Chronic tissue injury causes inflammatory cell infiltration into the injured tissue. The ...persistence of this inflammatory cell infiltration leads to fibrosis and organ failure. Adipose-derived mesenchymal stem cells (ASCs) have received much attention as a regenerative therapeutic tool to prevent progression from organ injury to failure. Subcutaneous abdominal adipose tissue is divided into superficial and deep layers by a superficial fascia. Adipose tissue easily collected by liposuction is usually obtained from a deep layer, so ASCs derived from a deep layer are generally used for regenerative medicine. However, no research has been conducted to investigate differences in the therapeutic effects of ASCs from the superficial and deep layers (Sup-ASCs and Deep-ASCs, respectively). Therefore, we compared the therapeutic potencies of Sup-ASCs and Deep-ASCs.
ASCs were isolated from superficial and deep subcutaneous abdominal adipose tissues collected from patients who underwent breast reconstruction. We first compared cell characteristics, such as morphology, cell proliferation, cell surface markers, adipogenic and osteogenic differentiation, cell senescence markers, and expression of coagulation and anticoagulant factors between Sup-ASCs and Deep-ASCs. Furthermore, we compared their ability to promote polarization of M2 macrophages and to inhibit transforming growth factor (TGF)-β/Smad signaling using THP-1 cells and TGF-β1 stimulated HK-2 cells incubated with conditioned media from Sup-ASCs or Deep-ASCs. In in vivo experiments, after renal ischemia-reperfusion injury (IRI) procedure, Sup-ASCs or Deep-ASCs were injected through the abdominal aorta. At 21 days post-injection, the rats were sacrificed and their left kidneys were collected to evaluate fibrosis. Finally, we performed RNA-sequencing analysis of Sup-ASCs and Deep-ASCs.
Sup-ASCs had greater proliferation and adipogenic differentiation compared with Deep-ASCs, whereas both ASC types had similar morphology, cell surface markers, senescence markers, and expression of coagulation and anticoagulant factors. Conditioned media from Sup-ASCs and Deep-ASCs equally promoted polarization of M2 macrophages and suppressed TGF-β/Smad signaling. Moreover, administration of Sup-ASCs and Deep-ASCs equally ameliorated renal fibrosis induced by IRI in rats. RNA-sequencing analysis revealed no significant difference in the expression of genes involved in anti-inflammatory and anti-fibrotic effects between Sup-ASCs and Deep-ASCs.
These results indicate that both Sup-ASCs and Deep-ASCs can be used effectively and safely as an intravascular ASC therapy for organ injury.
Dietary carbon sources in headwater stream food webs are divided into allochthonous and autochthonous organic matters. We hypothesized that: 1) the dietary allochthonous contribution for fish in ...headwater stream food webs positively relate with canopy cover; and 2) essential fatty acids originate from autochthonous organic matter regardless of canopy covers, because essential fatty acids, such as 20:5ω3 and 22:6ω3, are normally absent in allochthonous organic matters. We investigated predatory fish
stomach contents in four headwater stream systems, which are located in subarctic region in northern Japan. In addition, stable carbon and nitrogen isotope ratios, fatty acid profile, and stable carbon isotope ratios of essential fatty acids were analyzed. Bulk stable carbon analysis showed the major contribution of autochthonous sources to assimilated carbon in
Surface baits in the stomach had intermediate stable carbon isotope ratios between autochthonous and allochthonous organic matter, indicating aquatic carbon was partly assimilated by surface baits. Stable carbon isotope ratios of essential fatty acids showed a positive relationship between autochthonous sources and
across four study sites. This study demonstrated that the main supplier of dietary carbon and essential fatty acids was autochthonous organic matter even in headwater stream ecosystems under high canopy cover.
Induced pluripotent stem cells (iPSCs) can be generated from patients with specific diseases by the transduction of reprogramming factors and can be useful as a cell source for cell transplantation ...therapy for various diseases with impaired organs. However, the low efficiency of iPSC derived from somatic cells (0.01–0.1%) is one of the major problems in the field. The phosphoinositide 3-kinase (PI3K) pathway is thought to be important for self-renewal, proliferation, and maintenance of embryonic stem cells (ESCs), but the contribution of this pathway or its well-known negative regulator, phosphatase, and tensin homolog deleted on chromosome ten (Pten), to somatic cell reprogramming remains largely unknown. Here, we show that activation of the PI3K pathway by the Pten inhibitor, dipotassium bisperoxo(5-hydroxypyridine-2-carboxyl)oxovanadate, improves the efficiency of germline-competent iPSC derivation from mouse somatic cells. This simple method provides a new approach for efficient generation of iPSCs.
RhoBTB proteins are atypical members of the Rho family of small GTPases. Two of the three RhoBTB proteins, RhoBTB1 and RhoBTB2, have been proposed as tumor suppressors and might function as adaptors ...of Cul3-dependent ubiquitin ligase complexes. Using yeast two-hybrid analysis and co-immunoprecipitation we show that all three RhoBTB proteins interact with Cul3. The interaction requires the N-terminal region of Cul3 and the first BTB domain of RhoBTB. RhoBTB3, the only RhoBTB with a prenylation motif, associates with vesicles that are frequently found in the vicinity of microtubules, suggesting a participation in some aspects of vesicle trafficking. We also show that RhoBTB2 and RhoBTB3 are capable of homo and heterodimerizing through the BTB domain region. The GTPase domain, which does not bind GTP, is able to interact with the BTB domain region, thus preventing proteasomal degradation of RhoBTB. This fits into a model in which an intramolecular interaction maintains RhoBTB in an inactive state, preventing the formation or the functionality of Cul3-dependent complexes. We also report a significantly decreased expression of RHOBTB and CUL3 genes in kidney and breast tumor samples and a very good correlation in the expression changes between RHOBTB and CUL3 that suggests that these genes are subject to a common inactivation mechanism in tumors.
Oncolytic virotherapies have emerged as new modalities for cancer treatment. We previously reported that coxsackievirus B3 (CVB3) is a novel oncolytic virus (OV) with a strong ability to lyse human ...non-small cell lung cancer cells; however, its non-specific toxicity against normal cells remains to be resolved. To improve its safety profile, microRNA target sequences complementary to miR-34a/c, which is expressed preferentially in normal cells, were inserted into the 5′ UTR or 3′ UTR of the CVB3 genome. In the presence of miR-34a/c, the gene-modified CVB3 could not replicate in normal cells. We also found that the pathogenicity of CVB3 was reduced to a greater extent by targeting miR-34a than miR-34c; in addition, it was more effective to insert the target sequences into the 3′ UTR rather than the 5′ UTR of the viral genome. Ultimately, we developed a double-miR-34a targeting virus (53a-CVB) by inserting miR-34a targets in both the 5′ UTR and 3′ UTR of the virus. 53a-CVB was minimally toxic to cells in normal tissue, but maintained nearly its full oncolytic activity in mice xenografted with human lung cancer. 53a-CVB is the first miR-34-regulated OV and represents a promising platform for the development of safe and effective anti-cancer therapies.
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Recent generation of induced pluripotent stem (iPSCs) has made a significant impact on the field of human regenerative medicine. Prior to the clinical application of iPSCs, testing of their safety ...and usefulness must be carried out using reliable animal models of various diseases. In order to generate iPSCs from common marmoset (CM; Callithrix jacchus), one of the most useful experimental animals, we have lentivirally transduced reprogramming factors, including POU5F1 (also known as OCT3/4), SOX2, KLF4, and c‐MYC into CM fibroblasts. The cells formed round colonies expressing embryonic stem cell markers, however, they showed an abnormal karyotype denoted as 46, X, del(4q), +mar, and formed human dysgerminoma‐like tumors in SCID mice, indicating that the transduction of reprogramming factors caused unexpected tumorigenesis of CM cells. Moreover, CM dysgerminoma‐like tumors were highly sensitive to DNA‐damaging agents, irradiation, and fibroblast growth factor receptor inhibitor, and their growth was dependent on c‐MYC expression. These results indicate that DNA‐damaging agents, irradiation, fibroblast growth factor receptor inhibitor, and c‐MYC‐targeted therapies might represent effective treatment strategies for unexpected tumors in patients receiving iPSC‐based therapy.
Reprogramming factors for iPSC generation can be oncogenic, and thus reprogramming factor transduced cells might have tumorigenic potential. Here we characterized common marmoset dysgerminoma like tumor, CM DGs, generated by the lentivirally transduced reprogramming factors into fibroblasts. The growth of CM DGs was dependent on c‐MYC expression and bFGF signaling. Moreover CM DGs were highly sensitive to DNA damaging agents, irradiation and FGFR inhibitors. Therefore irradiation, DNA damaging agents and FGFR inhibitors might be effective for controlling reprogramming factor related tumors that may be found in patients treated with iPSC‐based medicine.
Vaccination with irradiated granulocyte macrophage colony-stimulating factor (GM-CSF)-transduced autologous tumor cells (GVAX) has been shown to induce therapeutic antitumor immunity. However, its ...effectiveness is limited. We therefore attempted to improve the antitumor effect by identifying little-known key pathways in GM-CSF-sensitized dendritic cells (GM-DC) in tumor-draining lymph nodes (TDLN). We initially confirmed that syngeneic mice subcutaneously injected with poorly immunogenic Lewis lung carcinoma (LLC) cells transduced with Sendai virus encoding GM-CSF (LLC/SeV/GM) remarkably rejected the tumor growth. Using cDNA microarrays, we found that expression levels of type I interferon (IFN)-related genes, predominantly expressed in plasmacytoid DCs (pDC), were significantly upregulated in TDLN-derived GM-DCs and focused on pDCs. Indeed, mouse experiments demonstrated that the effective induction of GM-CSF-induced antitumor immunity observed in immunocompetent mice treated with LLC/SeV/GM cells was significantly attenuated when pDC-depleted or IFNα receptor knockout (IFNAR(-/-)) mice were used. Importantly, in both LLC and CT26 colon cancer-bearing mice, the combinational use of imiquimod with autologous GVAX therapy overcame the refractoriness to GVAX monotherapy accompanied by tolerability. Mechanistically, mice treated with the combined vaccination displayed increased expression levels of CD86, CD9, and Siglec-H, which correlate with an antitumor phenotype, in pDCs, but decreased the ratio of CD4(+)CD25(+)FoxP3(+) regulatory T cells in TDLNs. Collectively, these findings indicate that the additional use of imiquimod to activate pDCs with type I IFN production, as a positive regulator of T-cell priming, could enhance the immunologic antitumor effects of GVAX therapy, shedding promising light on the understanding and treatment of GM-CSF-based cancer immunotherapy.
The cullin-RING family of ubiquitin ligases regulates diverse cellular functions, such as cell cycle control, via ubiquitylation of specific substrates. CUL3 targets its substrates through BTB ...proteins. Here we show that depletion of CUL3 and the BTB protein KLHL18 causes a delay in mitotic entry. Centrosomal activation of Aurora-A, a kinase whose activity is required for entry into mitosis, is also delayed in depleted cells. Moreover, we identify Aurora-A as a KLHL18-interacting partner. Overexpression of KLHL18 and CUL3 promotes Aurora-A ubiquitylation in vivo, and the CUL3-KLHL18-ROC1 ligase ubiquitylates Aurora-A in vitro. Our study reveals that the CUL3-KLHL18 ligase is required for timely entry into mitosis, as well as for the activation of Aurora-A at centrosomes. We propose that the CUL3-KLHL18 ligase regulates mitotic entry through an Aurora-A-dependent pathway.
•Self-renewal of common marmoset embryonic stem cells (CM ESCs) is promoted by bFGF.•bFGF activates the PI3K-AKT pathway in CM ESCs on feeder cells.•bFGF and TGFβ in combination support culture of CM ...ESCs without feeder cells.•CM ESCs show phenotypes similar to those of human ESCs and mouse epiblast SCs.
Common marmoset (CM) is widely recognized as a useful non-human primate for disease modeling and preclinical studies. Thus, embryonic stem cells (ESCs) derived from CM have potential as an appropriate cell source to test human regenerative medicine using human ESCs. CM ESCs have been established by us and other groups, and can be cultured in vitro. However, the growth factors and downstream pathways for self-renewal of CM ESCs are largely unknown. In this study, we found that basic fibroblast growth factor (bFGF) rather than leukemia inhibitory factor (LIF) promoted CM ESC self-renewal via the activation of phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT) pathway on mouse embryonic fibroblast (MEF) feeders. Moreover, bFGF and transforming growth factor β (TGFβ) signaling pathways cooperatively maintained the undifferentiated state of CM ESCs under feeder-free condition. Our findings may improve the culture techniques of CM ESCs and facilitate their use as a preclinical experimental resource for human regenerative medicine.
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