Blood vessel organoids generated by base editing and harboring single nucleotide variation in Notch3 effectively recapitulate CADASIL-related pathogenesis.

Human blood vessel organoids (hBVOs) offer a promising platform for investigating vascular diseases and identifying therapeutic targets. In this study, we focused on in vitro modeling and therapeutic target finding of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common form of hereditary stroke disorder caused by mutations in the NOTCH3 gene. Despite the identification of these mutations, the underlying pathological mechanism is elusive, and effective therapeutic approaches are lacking. CADASIL primarily affects the blood vessels in the brain, leading to ischemic strokes, migraines, and dementia. By employing CRISPR/Cas9 base-editing technology, we generated human induced pluripotent stem cells (hiPSCs) carrying Notch3 mutations. These mutant hiPSCs were differentiated into hBVOs. The NOTCH3 mutated hBVOs exhibited CADASIL-like pathology, characterized by a reduced vessel diameter and degeneration of mural cells. Furthermore, we observed an accumulation of Notch3 extracellular domain (Notch3ECD), increased apoptosis, and cytoskeletal alterations in the NOTCH3 mutant hBVOs. Notably, treatment with ROCK inhibitors partially restored the disconnection between endothelial cells and mural cells in the mutant hBVOs. These findings shed light on the pathogenesis of CADASIL and highlight the potential of hBVOs for studying and developing therapeutic interventions for this debilitating human vascular disorder.

Differentially Expressed mRNA in Streptozotocin-Induced Diabetic Bladder Using RNA Sequencing Analysis.

PURPOSE: To detect elements governing the pathogenesis of diabetic cystopathy (DC), mRNA sequencing was carried out for bladder tissues from normal rats and those with induced diabetes mellitus (DM). This research therefore offers possible underlying molecular pathways for the advancement of DC in relation to differential mRNA expression, together with visceral functional and architectural alterations noted in individuals with this condition. METHODS: An intraperitoneal injection of streptozotocin (STZ) was utilized to provoke DM in male Sprague-Dawley rats. Dysregulation and significant variations between normal rats and those with induced DM were then identified by a fold change of ≥ 1.5 with a false discovery rate P < 0.05. Hierarchical clustering/heat map and Gene Ontology/DAVID reference sources were generated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and protein-protein interaction analysis were then performed. RESULTS: The diabetic rodent group exhibited a greater residual urine volume (4.0 ± 0.4 mL) than their control counterparts (0.7 ± 0.2 mL, P < 0.01) at 12 weeks after diagnosis of diabetes. Expression analysis revealed 16 upregulated and 4 downregulated genes in STZDM bladder samples. A notable increase in expression was seen in PTHLH, TNFAIP6, PRC1, MAPK10, LOC686120, CASQ2, ACTG2, PDLIM3, FCHSD1, DBN1, NKD2, PDLIM7, ATF4, RBPMS2, ITGB1 and HSPB8. A notable decrease in expression was seen in SREBLF1, PBGFR1, PBLD1 and CELF1. Major genetic themes associated with mRNA upregulation and downregulation ware identified via Gene Ontology analysis and KEGG pathways. Protein to protein interaction analysis detected PDLIM3, PDLIM7, ITGB1, ACTG2 as core high frequency nodes within the network. CONCLUSION: Changes in mRNA expression together with biological process and pathways that contribute to the etiologies underlying visceral impairment of the bladder in DM are evident. Our strategy is promising for recognizing mRNAs exclusive to the bladder in DM that might offer useful targets for diagnosis and treatment.

Anti-Cancer Effect of Neural Stem Cells Transfected with Carboxylesterase and sTRAIL Genes in Animals with Brain Lesions of Lung Cancer.

A metastatic brain tumor is the most common type of malignancy in the central nervous system, which is one of the leading causes of death in patients with lung cancer. The purpose of this study is to evaluate the efficacy of a novel treatment for metastatic brain tumors with lung cancer using neural stem cells (NSCs), which encode rabbit carboxylesterase (rCE) and the secretion form of tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). rCE and/or sTRAIL were transduced in immortalized human fetal NSCs, HB1.F3. The cytotoxic effects of the therapeutic cells on human lung cancer cells were evaluated in vitro with the ligands and decoy receptor expression for sTRAIL in the presence of CPT-11. Human NSCs encoding rCE (F3.CE and F3.CE.sTRAIL) significantly inhibited the growth of lung cancer cells in the presence of CPT-11 in vitro. Lung cancer cells were inoculated in immune-deficient mice, and therapeutic cells were transplanted systematically through intracardiac arterial injection and then treated with CPT-11. In resting state, DR4 expression in lung cancer cells and DcR1 in NSCs increased to 70% and 90% after CPT-11 addition, respectively. The volumes of the tumors in immune-deficient mice were reduced significantly in mice with F3.CE.sTRAIL transplantation and CPT-11 treatment. The survival was also significantly prolonged with treatment with F3.sTRAIL and F3.CE plus CPT-11 as well as F3.CE.sTRAIL plus CPT-11. NSCs transduced with rCE and sTRAIL genes showed a significant anti-cancer effect on brain metastatic lung cancer in vivo and in vitro, and the effect may be synergistic when rCE/CPT-11 and sTRAIL are combined. This stem-cell-based study using two therapeutic genes of different biological effects can be translatable to clinical application.

Established Immortalized Cavernous Endothelial Cells Improve Erectile Dysfunction in Rats with Cavernous Nerve Injury.

The main cause of erectile dysfunction (ED) is the damage in penile cavernous endothelial cells (EC). Murine primary ECs have a limited growth potential, and the easy availability of murine ECs will facilitate the study of cavernous endothelial dysfunction in rats. This study was performed to establish immortalized rat penile cavernous ECs (rEC) and investigate how they could repair erectile dysfunction in rats with cavernous nerve injury (CNI). rEC was isolated enzymatically by collagenase digestion and were cultured. An amphotropic replication-incompetent retroviral vector encoding v-myc oncogene was used to transfect rEC for immortalization (vREC). Morphological and immunohistochemical properties of vREC were examined. Eight-week-old male Sprague-Dawley rats were divided into three groups of five rats each, including group 1 = sham operation, group 2 = bilateral CN injury, group 3 = vREC (1 × 106 cells) treatment after CNI. Erectile response was assessed at 2, 4 weeks after transplantation of vREC., Penile tissue were harvested at 4 weeks after transplantation and immune−histochemical examination was performed. vREC showed the expression of CD31, vWF, cell type-specific markers for EC by RT-PCR and flowcytometry. At 2, 4 weeks after transplantation, rats with CNI had significantly lower erectile function than control group (p < 0.05). The group transplanted with vREC showed higher erectile function than the group without vRECs (p < 0.05). vREC was established and repaired erectile dysfunction in rats with CNI. This cell line may be useful for studying mechanisms and drug screening of erectile dysfunction of rats.

Changes of proapoptotic and antiapoptotic genes affect sensitivity to apoptotic stimuli in impaired contractility due to long term bladder outlet obstruction.

INTRODUCTION: The normal biological process that necessitates cell removal greatly depends on apoptosis. Long term bladder outlet obstruction (BOO) causes damaged smooth muscle cells to undergo apoptosis. However, smooth muscle cell apoptosis that BOO causes is not well known in impaired bladder contractility. Therefore, we designed this study to investigate whether long-term BOO could induce apoptosis activities and to obtain an expression profile of apoptosis related genes. MATERIALS AND METHODS: We used 10 Sprague-Dawley six-week-old female rats. We separated them equally into two groups: a sham intervention group (group 1) and an eight-week BOO group (group 2). We conducted cystometric evaluation eight weeks following BOO onset, with processing of bladder tissue for PCR array. Every array comprised 84 genes, which were established to contribute to an apoptosis response, cell differentiation and metabolism, and 12 sequences were established for the regulation of loading and the quality of cDNA. We performed real-time PCR. Changes in gene expression presented as a fold increase/decrease. Alterations of more than two-fold constituted the cut-off determining expression. RESULTS: Group 2 had a greater bladder weight and Impaired bladder contractility. Immunofluorescent staining with CAS3, TUNEL showed increased in the BOO group. In comparison to group 1, group 2 exhibited an at least two-fold upregulation in five genes, the Bcl-2 (15.1), Birc5 (5.8), Cd40lg (7.5), Il10 (16.2), and Naip2 (13.2). They also demonstrated at least a two-fold downregulation in the PRLR (-18.1) gene. Genes Bcl2ald, Circ5, Cd40lg, Il10, Naip2, and PRLR were among the genes with activity against apoptosis. TNF, STAT3 and TP53 mediated the effect that genes had on one another. CONCLUSION: This study demonstrated that the relative ratios of pro- and antiapoptotic genes determine bladder cell sensitivity cells to apoptotic stimuli in impaired contractility caused by long term BOO. Although we cannot confirm whether this finding is the result of the decompensated phase of the bladder or the process, the gene expression profiles could explain molecular mechanisms of apoptosis in impaired bladder contractility caused by long-term BOO with further studies.

Improvement of erectile dysfunction using endothelial progenitor cells from fetal cerebral vasculature in the cavernous nerve injury of rats.

BACKGROUND: Because of limited differentiation to endothelium from mesenchymal stem cells, it has been strongly recommended to use endothelial progenitor cells for the regeneration of the damaged endothelium of corpora cavernosa. This study was performed to investigate the immortalized human cerebral endothelial cells and their capability for repairing erectile dysfunction in a rat model of cavernous nerve injury. Circulating endothelial progenitor cells were isolated from human fetal brain vasculature at the periventricular region of telencephalic tissues. Over 95% of CD 31-positive cells were sorted and cultured for 10 days. Human cerebral endothelial progenitor cells were injected into the cavernosa of rats with cavernous nerve injury. Erectile response was then assessed. In in vivo assays, rats were divided into three groups: group 1, sham operation: group 2, bilateral cavernous nerve injury: and group 3, treatment with human cerebral endothelial cells after cavernous nerve injury. RESULTS: Established immortalized circulating endothelial progenitor cells showed expression of human telomerase reverse transcriptase transcript by RT-PCR. They also showed the expression of vascular endothelial growth factor, von Willebrand factor, vascular endothelial growth factor receptor, and CD31, cell type-specific markers for endothelial cells by RT-PCR. In in vitro angiogenesis assays, they demonstrated tube formation that suggested morphological properties of endothelial progenitor cells. In in vivo assays, impaired erectile function of rat with cavernous nerve injury recovered at 2, 4, and 12 weeks after transplantation of human cerebral endothelial cells into the cavernosa. CONCLUSIONS: Telomerase reverse transcriptase-circulating endothelial progenitor cells from fetal brain vasculature could repair erectile dysfunction of rats with cavernous nerve injury.

RéSUMé: CONTEXTE: En raison de la différenciation limitée de l'endothélium à partir de cellules souches mésenchymateuses, il a été fortement recommandé d'utiliser des cellules progénitrices endothéliales pour la régénération de l'endothélium endommagé des corps caverneux. Cette étude a été réalisée pour étudier les cellules endothéliales cérébrales humaines immortalisées, et leur capacité à réparer la dysfonction érectile dans un modèle de rat avec lésion du nerf caverneux. Les cellules progénitrices endothéliales circulantes ont été isolées du système vasculaire cérébral fœtal humain dans la région périventriculaire des tissus télencéphaliques. Plus de 95% des cellules CD31 positives ont été sélectionnées et cultivées pendant 10 jours. Des cellules progénitrices endothéliales cérébrales humaines ont été injectées dans les corps caverneux de rats présentant une lésion nerveuse des corps caverneux. La réponse érectile a ensuite été évaluée. Dans les essais in vivo, les rats ont été divisés en trois groupes: groupe 1, opération simulée; groupe 2, lésion bilatérale du nerf caverneux; et groupe 3, traitement par cellules endothéliales cérébrales humaines après lésion du nerf caverneux. RéSULTATS: Les cellules progénitrices endothéliales circulantes immortalisées établies ont montré l'expression de la transcription de la transcriptase inverse de la télomérase humaine par RT-PCR. Elles ont également montré l'expression du facteur de croissance de l'endothélium vasculaire, du facteur de von Willebrand, du récepteur du facteur de croissance de l'endothélium vasculaire, et de CD31, marqueurs spécifiques du type cellulaire par RT-PCR pour les cellules endothéliales. Dans les essais in vivo, la fonction érectile altérée des rats avec lésion du nerf caverneux s'est rétablie à 2, 4 et 12 semaines après transplantation de cellules endothéliales cérébrales humaines dans les corps caverneux. CONCLUSIONS: Les cellules progénitrices endothéliales circulantes exprimant la transcriptase inverse de la télomérase, provenant du système vasculaire cérébral fœtal humain, pourraient réparer la dysfonction érectile de rats atteints de lésions des nerfs caverneux. MOTS-CLéS: Dysfonction érectile; Cellules endothéliales humaines; Transcriptase inverse de la Télomérase humaine.

Human neural stem cell-derived extracellular vesicles protect against Parkinson's disease pathologies.

BACKGROUND: Neural stem cells (NSCs) have the ability to generate a variety of functional neural cell types and have a high potential for neuronal cell regeneration and recovery. Thus, they been recognized as the best source of cell therapy for neurodegenerative diseases, such as Parkinson's disease (PD). Owing to the possibility of paracrine effect-based therapeutic mechanisms and easier clinical accessibility, extracellular vesicles (EVs), which possess very similar bio-functional components from their cellular origin, have emerged as potential alternatives in regenerative medicine. MATERIAL AND METHODS: EVs were isolated from human fibroblast (HFF) and human NSC (F3 cells). The supernatant of the cells was concentrated by a tangential flow filtration (TFF) system. Then, the final EVs were isolated using a total EV isolation kit. RESULTS: In this study, we demonstrate the potential protective effect of human NSC-derived EVs, showing the prevention of PD pathologies in 6-hydroxydopamine (6-OHDA)-induced in vitro and in vivo mouse models. Human NSC and F3 cell (F3)-derived EVs reduced the intracellular reactive oxygen species (ROS) and associated apoptotic pathways. In addition, F3-derived EVs induced downregulation of pro-inflammatory factors and significantly decreased 6-OHDA-induced dopaminergic neuronal loss in vivo. F3 specific microRNAs (miRNAs) such as hsa-mir-182-5p, hsa-mir-183-5p, hsa-mir-9, and hsa-let-7, which are involved in cell differentiation, neurotrophic function, and immune modulation, were found in F3-derived EVs. CONCLUSIONS: We report that human NSC-derived EVs show an effective neuroprotective property in an in vitro transwell system and in a PD model. The EVs clearly decreased ROS and pro-inflammatory cytokines. Taken together, these results indicate that NSC-derived EVs could potentially help prevent the neuropathology and progression of PD.

Enhanced Hypoxia-Associated Genes in Impaired Contractility From Bladder Outlet Obstruction.

BACKGROUND: Hypoxia damages the bladder wall and contributes to the initiation of bladder dysfunction. The change of hypoxia is not well known in impaired bladder contractility caused by long-term bladder outlet obstruction (BOO). We aimed to find out whether hypoxia of bladder tissue is present and what signaling mechanisms are involved in the decompensated bladder in BOO. METHODS: Twenty 6-week-old female Sprague-Dawley rats were divided into 2 groups, 10 rats each: group 1, sham operation; group 2, BOO for 8 weeks. Eight weeks after the onset of BOO, we did cystometric evaluation and processed polymerase chain reaction (PCR) array for hypoxia pathway using bladder tissues. The PCR array consists of 84 genes known to be involved in the hypoxic response, cell differentiation, and metabolism. We did quantitative PCR (qPCR) and immunohistochemical staining of bladder tissue for hypoxia. RESULTS: Eight genes were at least 2-fold upregulated and 3 genes were at least 2-fold downregulated in BOO group, compared with the sham operation group. The up-regulated genes (fold change) belonging to the hypoxia-inducible factor (HIF) 1 interactor included Cdkn2a (11.0), and the down-regulated genes belonging to HIF and co-transcription factors included Hif3a (-39.6) and Per1 (-5.1) by BOO. Genes influenced each other by means of TGFß1, TNF, and TP53. CONCLUSION: Hypoxia genes were increased in impaired contractility because of long-term BOO. The gene expression profiles could explain the molecular mechanisms of hypoxia in impaired contractility because of long-term BOO.

Improved bladder contractility after transplantation of human mesenchymal stem cells overexpressing hepatocyte growth factor into underactive bladder from bladder outlet obstruction models of rats.

INTRODUCTION: An underactive bladder can lead to difficulty in voiding that causes incomplete emptying of the bladder, suggesting the need for a new strategy to increase bladder contractility in such patients. This study was performed to investigate whether human mesenchymal stem cells (hMSCs) were capable of restoring bladder contractility in rats with underactive bladder due to bladder outlet obstruction (BOO) and enhancing their effects by overexpressing hepatocyte growth factor (HGF) in hMSCs. MATERIALS AND METHODS: The hMSCs were transplanted into the bladder wall of rats. Fifty female Sprague-Dawley rats at six weeks of age were divided into five groups: group 1: control; group 2: sham intervention; group 3: eight-week BOO; group 4: BOO rats transplanted with hMSCs; and group 5: BOO rats transplanted with hMSCs overexpressing HGF. Two weeks after the onset of BOO in groups 4 and 5, hMSCs were injected into the bladder wall. Cystometry evaluation was followed by Masson's trichrome staining of bladder tissues. Realtime PCR and immunohistochemical staining were performed to determine for hypoxia, apoptosis, and angiogenesis. RESULTS: Collagen deposition of bladder increased in BOO but decreased after transplantation of hMSCs. The increased inter-contraction interval and residual urine volume after BOO was reversed after hMSCs transplantation. The decreased maximal voiding pressure after BOO was restored by hMSCs treatment. The mRNA expression of bladder collagen1 and TGF-ß1 increased in BOO but decreased after hMSCs transplantation. The decrease in vWF-positive cells in the bladder following BOO was increased after hMSCs transplantation. Caspase 3 and TUNEL-positive apoptosis of bladder cells increased in BOO but decreased after transplantation of hMSCs. These effects were enhanced by overexpressing HGF in hMSCs. CONCLUSION: Transplantation of hMSCs into bladder wall increased the number of micro-vessels, decreased collagen deposition and apoptosis of detrusor muscle, and improved bladder underactivity. The effects were enhanced by overexpressing HGF in hMSCs. Our findings suggest that the restoration of underactive bladder using hMSCs may be used to rectify micturition disorders in patients following resolution of BOO. Further studies are needed before hMSCs can be used in clinical applications.

L-myc Gene Expression in Canine Fetal Fibroblasts Promotes Self-Renewal Capacity but Not Tumor Formation.

Canines are useful in mammalian preclinical studies because they are larger than rodents and share many diseases with humans. Canine fetal fibroblast cells (CFFs) are an easily accessible source of somatic cells. However, they are easily driven to senescence and become unusable with continuous in vitro culture. Therefore, to overcome these deficiencies, we investigated whether tetracycline-inducible L-myc gene expression promotes self-renewal activity and tumorigenicity in the production of induced conditional self-renewing fibroblast cells (iCSFCs). Here, we describe the characterization of a new iCSFC line immortalized by transduction with L-myc that displays in vitro self-renewal ability without tumorigenic capacity. We established conditionally inducible self-renewing fibroblast cells by transducing CFF-3 cells with L-myc under the tetracycline-inducible gene expression system. In the absence of doxycycline, the cells did not express L-myc or undergo self-renewal. The iCSFCs had a fibroblast-like morphology, normal chromosome pattern, and expressed fibroblast-specific genes and markers. However, the iCSFCs did not form tumors in a soft agar colony-forming assay. We observed higher expression of three ES modules (core pluripotency genes, polycomb repressive complex genes (PRC), and MYC-related genes) in the iCSFCs than in the CFF-3 cells; in particular, the core pluripotency genes (OCT4, SOX2, and NANOG) were markedly up-regulated compared with the PRC and MYC module genes. These results demonstrated that, in canine fetal fibroblasts, L-myc tetracycline-inducible promoter-driven gene expression induces self-renewal capacity but not tumor formation. This study suggests that L-myc gene-induced conditional self-renewing fibroblast cells can be used as an in vitro tool in a variety of biomedical studies related to drug screening.

Human Blood Vessel Organoids Penetrate Human Cerebral Organoids and Form a Vessel-Like System.

Vascularization of tissues, organoids and organ-on-chip models has been attempted using endothelial cells. However, the cultured endothelial cells lack the capacity to interact with other somatic cell types, which is distinct from developing vascular cells in vivo. Recently, it was demonstrated that blood vessel organoids (BVOs) recreate the structure and functions of developing human blood vessels. However, the tissue-specific adaptability of BVOs had not been assessed in somatic tissues. Herein, we investigated whether BVOs infiltrate human cerebral organoids and form a blood-brain barrier. As a result, vascular cells arising from BVOs penetrated the cerebral organoids and developed a vessel-like architecture composed of CD31+ endothelial tubes coated with SMA+ or PDGFR+ mural cells. Molecular markers of the blood-brain barrier were detected in the vascularized cerebral organoids. We revealed that BVOs can form neural-specific blood-vessel networks that can be maintained for over 50 days.

Aripiprazole exerts a neuroprotective effect in mouse focal cerebral ischemia.

Previous studies have demonstrated that aripiprazole (APZ), a third-generation atypical antipsychotic drug, exhibits anti-depressant and neuroprotective effects by promoting dopaminergic neuronal cell recovery in stroke. To investigate the neuroprotective effects of APZ, behavioral and histopathological experiments were performed in the current study a mouse model of middle cerebral artery occlusion (MCAO)-induced ischemia following administration of APZ. The subacute phase of ischemic assaults was divided into 3 periods, each with a duration of 5 days, according to the start of APZ (3 mg/kg) administration (1-5, 5-9 or 10-14 days following MCAO). The beneficial effects of APZ on motor behavior demonstrated in the cylinder, rotarod and wire suspension tests were greatest when APZ was administered 1-5 days following MCAO, with clear improvements in motor function compared with vehicle-treated mice. Histopathological analysis revealed that prominent atrophic changes occurred in the striatum of MCAO mice and that these changes were reduced following APZ treatment. APZ also attenuated dopaminergic neuronal injury in the striatum. Cell death and microglial activation were decreased and the expression of Ca2+/calmodulin-dependent protein kinase II δ was enhanced following APZ treatment. These results indicate that the atypical antipsychotic drug, APZ, exhibits a neuroprotective effect in dopaminergic neuronal cells that may improve behavioral function following ischemic stroke.

Reconstruction of 12-lead ECG Using a Single-patch Device.

OBJECTIVES: The aim of this study is to develop an optimal electrode system in the form of a small and wearable single-patch ECG monitoring device that allows for the faithful reconstruction of the standard 12-lead ECG. METHODS: The optimized universal electrode positions on the chest and the personalized transformation matrix were determined using linear regression as well as artificial neural networks (ANNs). A total of 24 combinations of 4 neighboring electrodes on 35 channels were evaluated on 19 subjects. Moreover, we analyzed combinations of three electrodes within the four-electrode combination with the best performance. RESULTS: The mean correlation coefficients were all higher than 0.95 in the case of the ANN method for the combinations of four neighboring electrodes. The reconstructions obtained using the three and four sensing electrodes showed no significant differences. The reconstructed 12-lead ECG obtained using the ANN method is better than that using the MLR method. Therefore, three sensing electrodes and one ground electrode (forming a square) placed below the clavicle on the left were determined to be suitable for ensuring good reconstruction performance. CONCLUSIONS: Since the interelectrode distance was determined to be 5 cm, the suggested approach can be implemented in a single-patch device, which should allow for the continuous monitoring of the standard 12-lead ECG without requiring limb contact, both in daily life and in clinical practice.

Establishment and Characterization of Immortalized Minipig Neural Stem Cell Line.

Despite the increasing importance of minipigs in biomedical research, there has been relatively little research concerning minipig-derived adult stem cells as a promising research tool that could be used to develop stem cell-based therapies. We first generated immortalized neural stem cells (iNSCs) from primary minipig olfactory bulb cells (pmpOBCs) and defined the characteristics of the cell line. Primary neural cells were prepared from minipig neonate olfactory bulbs and immortalized by infection with retrovirus carrying the v-myc gene. The minipig iNSCs (mpiNSCs) had normal karyotypes and expressed NSC-specific markers, including nestin, vimentin, Musashi1, and SOX2, suggesting a similarity to human NSCs. On the basis of the global gene expression profiles from the microarray analysis, neurogenesis-associated transcript levels were predominantly altered in mpiNSCs compared with pmpOBCs. These findings increase our understanding of minipig stem cells and contribute to the utility of mpiNSCs as resources for immortalized stem cell experiments.

Tumor-specific gene therapy for pancreatic cancer using human neural stem cells encoding carboxylesterase.

Advanced pancreatic cancer is one of the most lethal malignant human diseases lacking effective treatment. Its extremely low survival rate necessitates development of novel therapeutic approach. Human neural stem cells (NSCs) are known to have tumor-tropic effect. We genetically engineered them to express rabbit carboxyl esterase (F3.CE), which activates prodrug CPT-11(irinotecan) into potent metabolite SN-38. We found significant inhibition of the growth of BxPC3 human pancreatic cancer cell line in vitro by F3.CE in presence of CPT-11. Apoptosis was also markedly increased in BxPC3 cells treated with F3.CE and CPT-11. The ligand VEGF and receptor VEGF-1(Flt1) were identified to be the relevant tumor-tropic chemoattractant. We confirmed in vivo that in mice injected with BxPC3 on their skin, there was significant reduction of tumor size in those treated with both F3.CE and BxPC3 adjacent to the cancer mass. Administration of F3.CE in conjunction with CPT-11 could be a new possibility as an effective treatment regimen for patients suffering from advanced pancreatic cancer.

Neurorestorative Role of Stem Cells in Alzheimer's Disease: Astrocyte Involvement.

Neurogenesis is maintained in both neonatal and adult brain, although it is dramatically reduced in aged neurogenic brain region such as the subgranular layer and subventricular zone of the dentate gyrus (DG). Astrocytes play important roles for survival and maintenance of neurons as well as maintenance of neurogenic niche in quiescent state. Aß can induce astrocyte activation which give rise to produce reactive oxygen species (ROS) and cytotoxic cytokines and chemokines, and subsequently induce neuronal death. Unfortunately, the current therapeutic medicines have been limited to reduce the symptoms and delay the pathogenesis of Alzheimer's disease (AD), but not to cure it. Stem cells enhance neurogenesis and Aß clearing as well as improved cognitive impairment. Neurotrophins and growth factors which are produced from both stem cells and astrocytes also have neuroprotective effects via neurogenesis. Secreted factors from both astrocytes and neural stem cells also are influenced in neurogenesis and neuron survival in neurodegenerative diseases. Transplanted stem cells overexpressing neurogenic factors may be an effective and therapeutic tool to enhance neurogenesis for AD.

Human Neural Stem Cells Overexpressing a Carboxylesterase Inhibit Bladder Tumor Growth.

Bladder cancer is a significant clinical and economic problem. Despite intravesical chemotherapy and immunotherapy, up to 80% of patients with non-muscle-invasive bladder cancer develop recurrent tumors, of which 20% to 30% evolve into more aggressive, potentially lethal tumors. Recently, bladder cancer cells are considered to be mediators of resistance to current therapies and therefore represent strong candidates as biologic targets. No effective chemotherapy has yet been developed for advanced bladder cancer. It is desirable that a drug can be delivered directly and specifically to bladder cancer cells. Stem cells have selective migration ability toward cancer cells, and therapeutic genes can be easily transduced into stem cells. In suicide gene therapy for cancer, stem cells carry a gene encoding a carboxylesterase (CE) enzyme that transforms an inert CPT-11 prodrug into a toxic SN-38 product, a topoisomerase 1 inhibitor. In immunodeficient mice, systemically transplanted HB1.F3.CE stem cells migrated toward the tumor implanted by the TCCSUP bladder cancer cell line, and, in combination with CPT-11, the volume of tumors was significantly reduced. These findings may contribute to the development of a new selective chemotherapeutic strategy against bladder cancer. Mol Cancer Ther; 15(6); 1201-7. ©2016 AACR.

Tracking Transplanted Stem Cells Using Magnetic Resonance Imaging and the Nanoparticle Labeling Method in Urology.

A reliable in vivo imaging method to localize transplanted cells and monitor their viability would enable a systematic investigation of cell therapy. Most stem cell transplantation studies have used immunohistological staining, which does not provide information about the migration of transplanted cells in vivo in the same host. Molecular imaging visualizes targeted cells in a living host, which enables determining the biological processes occurring in transplanted stem cells. Molecular imaging with labeled nanoparticles provides the opportunity to monitor transplanted cells noninvasively without sacrifice and to repeatedly evaluate them. Among several molecular imaging techniques, magnetic resonance imaging (MRI) provides high resolution and sensitivity of transplanted cells. MRI is a powerful noninvasive imaging modality with excellent image resolution for studying cellular dynamics. Several types of nanoparticles including superparamagnetic iron oxide nanoparticles and magnetic nanoparticles have been used to magnetically label stem cells and monitor viability by MRI in the urologic field. This review focuses on the current role and limitations of MRI with labeled nanoparticles for tracking transplanted stem cells in urology.

MRI for Crohn's Disease: Present and Future.

Crohn's disease (CD) is a chronic inflammatory condition with relapsing-remitting behavior, often causing strictures or penetrating bowel damage. Its lifelong clinical course necessitates frequent assessment of disease activity and complications. Computed tomography (CT) enterography has been used as primary imaging modality; however, the concern for radiation hazard limits its use especially in younger population. Magnetic resonance (MR) imaging has advantages of avoiding radiation exposure, lower incidence of adverse events, ability to obtain dynamic information, and good soft-tissue resolution. MR enterography (MRE) with oral contrast agent has been used as primary MR imaging modality of CD with high sensitivity, specificity, and interobserver agreement. The extent of inflammation as well as transmural ulcers and fibrostenotic diseases can be detected with MRE. Novel MR techniques such as diffusion-weighted MRI (DWI), motility study, PET-MRI, and molecular imaging are currently investigated for further improvement of diagnosis and management of CD. MR spectroscopy is a remarkable molecular imaging tool to analyze metabolic profile of CD with human samples such as plasma, urine, or feces, as well as colonic mucosa itself.

Long-term survival and differentiation of human neural stem cells in nonhuman primate brain with no immunosuppression.

Cellular fate of human neural stem cells (hNSCs) transplanted in the brain of nonhuman primates (NHPs) with no immunosuppression was determined at 22 and 24 months posttransplantation (PTx) regarding survival, differentiation, and tumorigenesis. Survival of hNSCs labeled with magnetic nanoparticles was successfully detected around injection sites in the brain at 22 months PTx by MRI. Histological examination of brain sections with H&E and Prussian blue staining at 24 months revealed that most of the grafted hNSCs were found located along the injection tract. Grafted hNSCs were found to differentiate into neurons at 24 months PTx. In addition, none of the grafted hNSCs were bromodeoxyuridine positive in the monkey brain, indicating that hNSCs did not replicate in the NHP brain and did not cause tumor formation. This study serves as a proof of principle and provides evidence that hNSCs transplanted in NHP brain could survive and differentiate into neurons in the absence of immunosuppression. It also serves as a preliminary study in our scheduled preclinical studies of hNSC transplantation in NHP stroke models.