Human adipose tissue-derived multilineage progenitor cells (hADMPCs) are attractive for cell therapy and tissue engineering because of their multipotency and ease of isolation without serial ethical issues. However, their limited in vitro lifespan in culture systems hinders their therapeutic application. Some somatic stem cells, including hADMPCs, are known to be localized in hypoxic regions; thus, hypoxia may be beneficial for ex vivo culture of these stem cells. These cells exhibit a high level of glycolytic metabolism in the presence of high oxygen levels and further increase their glycolysis rate under hypoxia. However, the physiological role of glycolytic activation and its regulatory mechanisms are still incompletely understood. Here, we show that Notch signaling is required for glycolysis regulation under hypoxic conditions. Our results demonstrate that 5% O2 dramatically increased the glycolysis rate, improved the proliferation efficiency, prevented senescence, and maintained the multipotency of hADMPCs. Intriguingly, these effects were not mediated by hypoxia-inducible factor (HIF), but rather by the Notch signaling pathway. Five percent O2 significantly increased the level of activated Notch1 and expression of its downstream gene, HES1. Furthermore, 5% O2 markedly increased glucose consumption and lactate production of hADMPCs, which decreased back to normoxic levels on treatment with a ?-secretase inhibitor. We also found that HES1 was involved in induction of GLUT3, TPI, and PGK1 in addition to reduction of TIGAR and SCO2 expression. These results clearly suggest that Notch signaling regulates glycolysis under hypoxic conditions and, thus, likely affects the cell lifespan via glycolysis.
Lymphoepithelioma-like carcinoma is an undifferentiated carcinoma with histological features similar to undifferentiated, non-keratinizing carcinoma of the nasopharynx. Lymphoepithelioma-like carcinoma of the urinary bladder is uncommon with a reported incidence of 0.3%- 1.3% of all bladder cancer. We report a Japanese case of predominant lymphoepithelioma-like carcinoma of the urinary bladder and review all of the English literature after performing a pooled analysis of the cases including the present one.
Transcriptome analysis of the epidermis of Hes1(-/-) mouse revealed the direct relationship between Hes1 (hairy and enhancer of split-1) and BNIP3 (BCL2 and adenovirus E1B 19-kDa-interacting protein 3), a potent inducer of autophagy. Keratinocyte differentiation is going along with activation of lysosomal enzymes and organelle clearance, expecting the contribution of autophagy in this process. We found that BNIP3 was expressed in the suprabasal layer of the epidermis, where autophagosome formation is normally observed. Forced expression of BNIP3 in human primary epidermal keratinocytes (HPEKs) resulted in autophagy induction and keratinocyte differentiation, whereas knockdown of BNIP3 had the opposite effect. Intriguingly, addition of an autophagy inhibitor significantly suppressed the BNIP3-stimulated differentiation of keratinocytes, suggesting that BNIP3 plays a crucial role in keratinocyte differentiation by inducing autophagy. Furthermore, the number of dead cells increased in the human epidermal equivalent of BNIP3 knockdown keratinocytes, which suggests that BNIP3 is important for maintenance of skin epidermis. Interestingly, although UVB irradiation stimulated BNIP3 expression and cleavage of caspase3, suppression of UVB-induced BNIP3 expression led to further increase in cleaved caspase3 levels. This suggests that BNIP3 has a protective effect against UVB-induced apoptosis in keratinocytes. Overall, our data provide valuable insights into the role of BNIP3 in the differentiation and maintenance of epidermal keratinocytes.
Accumulating evidence indicates that a small population of cancer stem cells (CSCs) is involved in intrinsic resistance to cancer treatment. The hypoxic microenvironment is an important stem cell niche that promotes the persistence of CSCs in tumors. Our aim here was to elucidate the role of hypoxia and CSCs in the resistance to gefitinib in non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9 and HCC827, which express the EGFR exon 19 deletion mutations, were exposed to high concentration of gefitinib under normoxic or hypoxic conditions. Seven days after gefitinib exposure, a small fraction of viable cells were detected, and these were referred to as "gefitinib-resistant persisters" (GRPs). CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1-all genes involved in stemness-were highly expressed in GRPs in PC9 and HCC827 cells, and PC9 GRPs exhibited a high potential for tumorigenicity in vivo. The expression of insulin-like growth factor 1 (IGF1) was also upregulated and IGF1 receptor (IGF1R) was activated on GRPs. Importantly, hypoxic exposure significantly increased sphere formation, reflecting the self-renewal capability, and the population of CD133- and Oct4-positive GRPs. Additionally, hypoxia upregulated IGF1 expression through hypoxia-inducible factor 1? (HIF1?), and markedly promoted the activation of IGF1R on GRPs. Knockdown of IGF1 expression significantly reduced phosphorylated IGF1R-expressing GRPs under hypoxic conditions. Finally, inhibition of HIF1? or IGF1R by specific inhibitors significantly decreased the population of CD133- and Oct4-positive GRPs, which were increased by hypoxia in PC9 and HCC827 cells. Collectively, these findings suggest that hypoxia increased the population of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC by activating IGF1R. Targeting the IGF1R pathway may be a promising strategy for overcoming gefitinib resistance in EGFR mutation-positive NSCLC induced by lung CSCs and microenvironment factors such as tumor hypoxia.
Emphysematous cystitis is a rare clinically entity, more commonly seen in diabetic, immunocompromised patients, which was characterized by air within the bladder wall and lumen. A 83-year-old woman was introduced to our department with fever elevation and abnormal findings of computed tomography (CT). She took orally prednisolone for autoimmune hepatitis. Pelvic CT revealed diffuse air throughout the bladder wall. Urinalysis showed combined hematuria and pyuria. Escherichia coli was detected in blood culture. Abnormal findings of complete blood count and laboratory examination included an elevated WBC count (12,200/?L), C-reactive protein (11.7?mg/dL), and creatinine (1.07?mg/dL). Cystoscopy confirmed diffuse submucosal emphysema throughout. On the basis of diagnosis with emphysematous cystitis, she was treated with antibiotics based on the results of blood culture and indwelling Foley catheter. After treatment, the improvement of inflammatory findings and submucosal emphysema on cystoscopy and CT were achieved.
Amyloidosis is characterized by extracellular deposition of abnormal insoluble fibrils, which cause structural and functional disorders. Amyloidosis is classified into primary and secondary disease. We report a case of localized amyloidosis of the urinary bladder. In the English literature, this is the first case effectively treated with occlusive dressing therapy using dimethyl sulfoxide.
We report a case of micropapillary variant of urothelial carcinoma in the ureter. A 62-year-old man was referred to our hospital under the diagnosis of left ureteral cancer. We performed retroperitoneoscopic nephroureterectomy. Histological examination of the resected specimen revealed a micropapillary variant of urothelial carcinoma of the ureter. In this case, micropapillary carcinoma was found in 10% of the tumor histologically. Two months after the operation, magnetic resonance imaging (MRI) showed liver and paraaortic lymph node metastases. After 3 cycles of gemcitabine-cisplatin therapy, these metastases disappeared on computed tomography (CT).
Xanthogranulomatous pyelonephritis (XGP) is a rare entity and constitutes less than 1% of chronic pyelonephritis. A 71-year-old male was introduced to our department with general malaise and abnormal findings of computed tomography (CT). Abnormal findings of complete blood count and laboratory examination included an elevated WBC count and C-reactive protein. Urinalysis showed combined hematuria and pyuria, and Escherichia coli was detected in urine culture. Abdominal CT revealed left hydronephrosis with staghorn renal calculi and thin cortex of the left kidney. Because of poor condition, the patient underwent construction of the left nephrostomy. After that, the remission of the inflammation was achieved. Several months after the construction, frequent obstructions of nephrostomy catheter because of turbid urine and intermittent fever elevation were observed. The patient and his family desired left nephrectomy despite his poor condition in general. Surgical dissection was very difficult due to fixed mass. Not long after that the patient died due to sepsis and cardiovascular failure. Microscopic findings of the left kidney revealed infiltration of lymphocytes and lipid-laden macrophages (xanthoma cells) corresponding to XGP.
Genetic modification of human adipose tissue-derived multilineage progenitor cells (hADMPCs) is highly valuable for their exploitation in therapeutic applications. Here, we have developed a novel single tet-off lentiviral vector platform. This vector combines (1) a modified tetracycline (tet)-response element composite promoter, (2) a multi-cistronic strategy to express an improved version of the tet-controlled transactivator and the blasticidin resistance gene under the control of a ubiquitous promoter, and (3) acceptor sites for easy recombination cloning of the gene of interest. In the present study, we used the cytomegalovirus (CMV) or the elongation factor 1 ? (EF-1?) promoter as the ubiquitous promoter, and EGFP was introduced as the gene of interest. hADMPCs transduced with a lentiviral vector carrying either the CMV promoter or the EF-1? promoter were effectively selected by blasticidin without affecting their stem cell properties, and EGFP expression was strictly regulated by doxycycline (Dox) treatment in these cells. However, the single tet-off lentiviral vector carrying the EF-1? promoter provided more homogenous expression of EGFP in hADMPCs. Intriguingly, differentiated cells from these Dox-responsive cell lines constitutively expressed EGFP only in the absence of Dox. This single tet-off lentiviral vector thus provides an important tool for applied research on hADMPCs.
Familial hypercholesterolemia (FH) is an autosomal codominant disease characterized by high concentrations of proatherogenic lipoproteins secondary to deficiency in low-density lipoprotein (LDL) receptor. We reported recently the use of in situ stem cell therapy of human adipose tissue-derived multilineage progenitor cells (hADMPCs) in lowering serum total cholesterol in the homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model of homozygous FH. Here we demonstrate that pravastatin, an HMG-CoA reductase inhibitor, augmented the cholesterol-lowering effect of transplanted hADMPCs and enhanced LDL clearance in homozygous WHHL rabbit. The results suggest the potential beneficial effects of in situ stem cell therapy in concert with appropriately selected pharmaceutical agents, in regenerative medicine.
Familial hypercholesterolemia (FH) is an autosomal codominant disease characterized by high concentrations of proatherogenic lipoproteins and premature atherosclerosis secondary to low-density lipoprotein (LDL) receptor deficiency. We examined a novel cell therapy strategy for the treatment of FH in the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for homozygous FH. We delivered human adipose tissue-derived multilineage progenitor cells (hADMPCs) via portal vein and followed by immunosuppressive regimen to avoid xenogenic rejection. Transplantation of hADMPCs resulted in significant reductions in total cholesterol, and the reductions were observed within 4 weeks and maintained for 12 weeks. (125)I-LDL turnover study showed that the rate of LDL clearance was significantly higher in the WHHL rabbits with transplanted hADMPCs than those without transplanted. After transplantation hADMPCs were localized in the portal triad, subsequently integrated into the hepatic parenchyma. The integrated cells expressed human albumin, human alpha-1-antitrypsin, human Factor IX, human LDL receptors, and human bile salt export pump, indicating that the transplanted hADMPCs resided, survived, and showed hepatocytic differentiation in vivo and lowered serum cholesterol in the WHHL rabbits. These results suggested that hADMPC transplantation could correct the metabolic defects and be a novel therapy for inherited liver diseases.
We report a case of metachronous bilateral testicular tumors. A 36-year-old man was admitted to our hospital with the chief complaint of painless left scrotal swelling. He had undergone right high orchiectomy for testicular seminoma, stage I, one year earlier. This time, ultrasonography demonstrated two hypoechoic mass and microlithiasis of the left testis. Abdominal and chest computed tomography revealed no lymph adenopathy and no metastasis. The preoperative diagnosis was stage I testicular tumor and subsequently left high orchiectomy was performed. Histopathological examination revealed typical seminoma. To our knowledge, including the present case, 191 cases of metachronous bilateral testicular tumors have been reported in Japan. Contralateral testicular tumor was observed at a mean age of 37.3 years and the mean interval of time between the initial testicular tumor and contralateral one was 73.0 months Approximately fifty percent of metachronous bilateral testicular tumors previously reported have recurred after five years or more from the initial surgery. In the testicular tumor, long-term follow-up and self examination of the contralateral testis are of great importance.
Eosinophilic cystitis is a rare inflammatory lesion of the bladder, characterized by massive eosinophilic infiltration of the bladder wall. Its cause is not known definitely. A 49-year-old man consulted our department with a miction pain, gross hematuria, and frequent micturition. Urinalysis showed combined hematuria and pyuria, but urine culture was sterile. Abnormal findings of laboratory examination included an elevated white blood cell (WBC) count (15,700/?L) and the proportion of eosinophils in the peripheral blood was 12% of the WBCs (normal 0-5%). Cystoscopy revealed a solid mass with severe edematous mucosa. Magnetic resonance imaging (MRI) also indicated marked bladder wall thickening, which was suspected for invasive bladder cancer. Transurethral biopsy of the bladder mass was performed with pathological examination revealing features of eosinophilic cystitis. After administration of a combination of prednisolone and suplatast tosilate, followed by monotherapy with suplatast tosilate, regression of the bladder mass, and normalization of the count of peripheral eosinophils were achieved. Fourteen months after steroid therapy, under treatment with suplatast tosilate, there was no relapse of urinary symptoms and the bladder mass.
IgG4-related sclerosing disease is a novel clinicopathological entity characterized by fibrosis, extensive infiltration of IgG4-positive plasma cells, and serum IgG4 elevation. This disorder includes a variety of diseases, such as autoimmune pancreatitis, retroperitoneal fibrosis, sialadenitis, thyroiditis, inflammatory abdominal aneurysm, tubulointerstitial nephritis, and inflammatory pseudotumor [World J Gastroenterol 2008;14:3948-3955]. A 71-year-old man visited our hospital with the complaint of left flank pain and gross hematuria. Computed tomography (CT) revealed left hydronephrosis and a thick retroperitoneal soft tissue mass around the ureteropelvic junction, suspicious of renal pelvic cancer. Urine cytology using a urine sample from the left renal pelvis was negative. On laboratory examination, serum levels of IgG and IgG4 were found to be elevated. The patient refused tumor biopsy. Therefore, he was treated with corticosteroid therapy on the basis of a clinical diagnosis with IgG4-related retroperitoneal fibrosis. Regression of the retroperitoneal mass as well as improvement of left hydronephrosis and decrease in serum IgG4 levels were accomplished. These effects strongly suggested that the present case was an IgG4-related retroperitoneal fibrosis. However, in this instance, since we could not completely rule out malignancies by biopsy, careful follow-up was necessary with these points in mind.
Adipose tissues contain populations of pluripotent mesenchymal stem cells that also secrete various cytokines and growth factors to support repair of damaged tissues. In this study, we examined the role of oxidative stress on human adipose-derived multilineage progenitor cells (hADMPCs) in neurite outgrowth in cells of the rat pheochromocytoma cell line (PC12).
Somatic mutations in the epidermal growth factor receptor (EGFR) gene, such as exon 19 deletion mutations, are important factors in determining therapeutic responses to gefitinib in non-small-cell lung cancer (NSCLC). However, some patients have activating mutations in EGFR and show poor responses to gefitinib. In this study, we examined three NSCLC cell lines, HCC827, PC9, and HCC2935, that expressed an EGFR exon 19 deletion mutation. All cells expressed mutant EGFR, but the PC9 and HCC2935 cells also expressed wild-type EGFR. The HCC827 cells were highly sensitive to gefitinib under both normoxia and hypoxia. However, the PC9 and HCC2935 cells were more resistant to gefitinib under hypoxic conditions compared to normoxia. Phosphorylation of EGFR and ERK was suppressed with gefitinib treatment to a lesser extent under hypoxia. The expression of transforming growth factor-? (TGF?) was dramatically upregulated under hypoxia, and the knockdown of TGF? or hypoxia-inducible factor-1? (HIF1?) reversed the resistance to gefitinib in hypoxic PC9 and HCC2935 cells. Finally, introduction of the wild-type EGFR gene into the HCC827 cells caused resistance to gefitinib under hypoxia. This phenomenon was also reversed by the knockdown of TGF? or HIF1?. Our results indicate that hypoxia causes gefitinib resistance in EGFR-mutant NSCLC through the activation of wild-type EGFR mediated by the upregulation of TGF?. The presence of wild-type and mutant EGFR along with tumor hypoxia are important factors that should be considered when treating NSCLC patients with gefitinib.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.