scholarly journals CBMS-08 INVESTIGATION FOR NICOTINIC EFFECTS ON STEM CELL’S PROPERTY IN HSV-TK/GCV GENE THERAPY

2019 ◽  
Vol 1 (Supplement_2) ◽  
pp. ii6-ii6
Author(s):  
Hiroaki Kenmochi ◽  
Tomohiro Yamasaki ◽  
Tomoya Oishi ◽  
Makoto Horikawa ◽  
Taisuke Yamamoto ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Mtt Assay ◽  
Malignant Gliomas ◽  
Glioma Cells ◽  
Human Tumor ◽  
Cellular Migration ◽  
Matrigel Invasion ◽  
Tumor Tropism ◽  
Migration Capacity

Abstract BACKGROUND Herpes simplex virus-thymidine kinase/ganciclovir (HSV-tk/GCV) system is one of feasible therapeutic strategies for defeating malignant gliomas. Stem cells with intrinsic tumor tropism are used for suicide gene vehicles, which make this therapy further realistic. Nicotine is known to affect cellular migration capacity in variety types of cells but whether nicotine impacts on stem cells’ migration capacity to gliomas is not scrutinized. In this research, we investigated nicotinic impact on stem cells’ properties including tumor tropism and gap junctional intercellular communication (GJIC), which is crucial to this therapeutic strategy. METHODS Mouse induced pluripotent stem cell (iPSC)-derived neural stem cells (miPS-NSCs) and human dental pulp mesenchymal stem cells (hDPSCs) were used. Nicotine cytotoxicity for 24 hours was evaluated by MTT assay for stem cells and glioma cells; GS-9L and C6 (rat), GL261 (mouse), U251 and U87 (human). Tumor tropism to glioma-conditioned medium (CM) with or without non-toxic nicotine concentrations was assessed using Matrigel Invasion Chamber. Nicotine effect on GJIC was assessed with scrape loading/dye transfer assay (SL/DT assay) for co-culture of stem cells and glioma cells (stem cell/glioma cell) or parachute assay for glioma cells alone using high-content analysis. RESULTS MTT assay revealed 1 μM of nicotine, equivalent to serum nicotine concentration in habitual smoking, is the maximum safe concentration for stem cells and glioma cells. Tumor tropism (miPS-NSCs to GL261-CM, hDPSCs to U251- or U87-CM) and GJIC of co-culture of stem cells and glioma cells (miPS-NSC/GL261, hDPSC/U251) or glioma cells alone (GS-9L, C6, GL261 and U251) were not affected by 1 μM of nicotine. CONCLUSIONS Physiological nicotine presence did not affect (1) stem cell’s tumor tropism to gliomas and (2) GJIC between stem cells and glioma cells or within glioma cells. HSV-tk/GCV therapy may retain its therapeutic efficacy against gliomas even under physiological nicotine concentrations.

scholarly journals Improved biocompatibility and efficient labeling of neural stem cells with poly(L-lysine)-coated maghemite nanoparticles

2016 ◽  
Vol 7 ◽  
pp. 926-936 ◽  
Author(s):  
Igor M Pongrac ◽  
Marina Dobrivojević ◽  
Lada Brkić Ahmed ◽  
Michal Babič ◽  
Miroslav Šlouf ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Neural Stem Cells ◽  
Cellular Uptake ◽  
Cell Tracking ◽  
Cellular Migration ◽  
Cell Labeling ◽  
Blue Staining ◽  
Maghemite Nanoparticles ◽  
Acetoxymethyl Ester

Background: Cell tracking is a powerful tool to understand cellular migration, dynamics, homing and function of stem cell transplants. Nanoparticles represent possible stem cell tracers, but they differ in cellular uptake and side effects. Their properties can be modified by coating with different biocompatible polymers. To test if a coating polymer, poly(L-lysine), can improve the biocompatibility of nanoparticles applied to neural stem cells, poly(L-lysine)-coated maghemite nanoparticles were prepared and characterized. We evaluated their cellular uptake, the mechanism of internalization, cytotoxicity, viability and proliferation of neural stem cells, and compared them to the commercially available dextran-coated nanomag®-D-spio nanoparticles. Results: Light microscopy of Prussian blue staining revealed a concentration-dependent intracellular uptake of iron oxide in neural stem cells. The methyl thiazolyl tetrazolium assay and the calcein acetoxymethyl ester/propidium iodide assay demonstrated that poly(L-lysine)-coated maghemite nanoparticles scored better than nanomag®-D-spio in cell labeling efficiency, viability and proliferation of neural stem cells. Cytochalasine D blocked the cellular uptake of nanoparticles indicating an actin-dependent process, such as macropinocytosis, to be the internalization mechanism for both nanoparticle types. Finally, immunocytochemistry analysis of neural stem cells after treatment with poly(L-lysine)-coated maghemite and nanomag®-D-spio nanoparticles showed that they preserve their identity as neural stem cells and their potential to differentiate into all three major neural cell types (neurons, astrocytes and oligodendrocytes). Conclusion: Improved biocompatibility and efficient cell labeling makes poly(L-lysine)-coated maghemite nanoparticles appropriate candidates for future neural stem cell in vivo tracking studies.

Microcatheter delivery of neurotherapeutics: compatibility with mesenchymal stem cells

2020 ◽  
Vol 133 (4) ◽  
pp. 1182-1190 ◽  
Author(s):  
Visish M. Srinivasan ◽  
Joy Gumin ◽  
Kevin M. Camstra ◽  
Stephen R. Chen ◽  
Jeremiah N. Johnson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Cell Count ◽  
Glioma Cells ◽  
Injection Velocity ◽  
Viral Agent ◽  
Intraarterial Infusion ◽  
Significant Difference

OBJECTIVEBone marrow–derived human mesenchymal stem cells (BM-hMSCs) have been used in clinical trials for the treatment of several neurological disorders. MSCs have been explored as a delivery modality for targeted viral therapeutic agents in the treatment of intracranial pathologies. Delta-24-RGD, a tumor-selective oncolytic adenovirus designed to target malignant glioma cells, has been shown to be effective in animal models and in a recent clinical trial. However, the most efficient strategy for delivering oncolytic therapies remains unclear. BM-hMSCs have been shown to home toward glioma xenografts after intracarotid delivery. The feasibility of selective intraarterial infusion of BM-hMSCs loaded with Delta-24-RGD (BM-hMSC-Delta-24) to deliver the virus to the tumor is being investigated. To evaluate the feasibility of endovascular intraarterial delivery, the authors tested in vitro the compatibility of BM-hMSC-Delta-24 with a variety of commercially available, clinically common microcatheters.METHODSBM-hMSCs were cultured, transfected with Delta-24-RGD, and resuspended in 1% human serum albumin. The solution was then injected via 4 common neuroendovascular microcatheters of different inner diameters (Marathon, Echelon-14, Marksman, and SL-10). Cell count and viability after injection through the microcatheters were assessed, including tests of injection velocity and catheter configuration. Transwell assays were performed with the injected cells to test the efficacy of BM-hMSC-Delta-24 activity against U87 glioma cells. BM-hMSC-Delta-24 compatibility was also tested with common neuroendovascular medications: Omnipaque, verapamil, and heparin.RESULTSThe preinfusion BM-hMSC-Delta-24 cell count was 1.2 × 105 cells/ml, with 98.7% viability. There was no significant difference in postinfusion cell count or viability for any of the catheters. Increasing the injection velocity from 1.0 ml/min to 73.2 ml/min, or modifying the catheter shape from straight to tortuous, did not significantly reduce cell count or viability. Cell count and viability remained stable for up to 5 hours when the cell solution was stored on ice. Mixing BM-hMSC-Delta-24 with clinical concentrations of Omnipaque, verapamil, and heparin prior to infusion did not alter cell count or viability. Transwell experiments demonstrated that the antiglioma activity of BM-hMSC-Delta-24 was maintained after infusion.CONCLUSIONSBM-hMSC-Delta-24 is compatible with a wide variety of microcatheters and medications commonly used in neuroendovascular therapy. Stem cell viability and viral agent activity do not appear to be affected by catheter configuration or injection velocity. Commercially available microcatheters can be used to deliver stem cell neurotherapeutics via intraarterial routes.

scholarly journals Targeted Inhibition of the miR-199a/214 Cluster by CRISPR Interference Augments the Tumor Tropism of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Hypoxic Condition

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Yumei Luo ◽  
Xuehu Xu ◽  
Xiuli An ◽  
Xiaofang Sun ◽  
Shu Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Stem Cell ◽  
Cell Migration ◽  
Neural Stem Cells ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Tumor Tropism ◽  
Targeted Inhibition ◽  
Induced Pluripotent

The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However, the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration. We first developed a baculovirus-delivered CRISPR interference (CRISPRi) system that sterically blocked the E-box element in the promoter of the miR-199a/214 cluster with an RNA-guided catalytically dead Cas9 (dCas9). We then applied this CRISPRi system to hiPS-NSCs and successfully suppressed the expression of miR-199a-5p, miR-199a-3p, and miR-214 in the microRNA gene cluster. Meanwhile, the expression levels of their targets related to regulation of hypoxia-stimulated cell migration, such as HIF1A, MET, and MAPK1, were upregulated. Further migration assays demonstrated that the targeted inhibition of the miR-199a/214 cluster significantly enhanced the tumor tropism of hiPS-NSCs both in vitro and in vivo. These findings suggest a novel application of CRISPRi in NSC-based tumor-targeted gene therapy.

scholarly journals Mesenchymal stem cells promote glioma neovascularization in vivo by fusing with cancer stem cells

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Chao Sun ◽  
Xingliang Dai ◽  
Dongliang Zhao ◽  
Haiyang Wang ◽  
Xiaoci Rong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Tumor Growth ◽  
Glioma Cells ◽  
Stem Cell Markers ◽  
Xenograft Tumor ◽  
Angiogenic Effect

Abstract Background and objective Tumor angiogenesis is vital for tumor growth. Recent evidence indicated that bone marrow-derived mesenchymal stem cells (BMSCs) can migrate to tumor sites and exert critical effects on tumor growth through direct and/or indirect interactions with tumor cells. However, the effect of BMSCs on tumor neovascularization has not been fully elucidated. This study aimed to investigate whether fusion cells from glioma stem cells and BMSCs participated in angiogenesis. Methods SU3-RFP cells were injected into the right caudate nucleus of NC-C57Bl/6 J-GFP nude mice, and the RFP+/GFP+ cells were isolated and named fusion cells. The angiogenic effects of SU3-RFP, BMSCs and fusion cells were compared in vivo and in vitro. Results Fusion cells showed elevated levels of CD31, CD34 and VE-Cadherin (markers of VEC) as compared to SU3-RFP and BMSCs. The MVD-CD31 in RFP+/GFP+ cell xenograft tumor was significantly greater as compared to that in SU3-RFP xenograft tumor. In addition, the expression of CD133 and stem cell markers Nanog, Oct4 and Sox2 were increased in fusion cells as compared to the parental cells. Fusion cells exhibited enhanced angiogenic effect as compared to parental glioma cells in vivo and in vitro, which may be related to their stem cell properties. Conclusion Fusion cells exhibited enhanced angiogenic effect as compared to parental glioma cells in vivo and in vitro, which may be related to their stem cell properties. Hence, cell fusion may contribute to glioma angiogenesis.

scholarly journals Menaquinone-7 Supplementation Improves Osteogenesis in Pluripotent Stem Cell Derived Mesenchymal Stem Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Asim Cengiz Akbulut ◽  
Grzegorz B. Wasilewski ◽  
Nikolas Rapp ◽  
Francesco Forin ◽  
Heike Singer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Human Mesenchymal Stem Cells ◽  
Cellular Migration ◽  
Collagen Deposition ◽  
Runx2 Expression ◽  
Differentiated Cells ◽  
Cost Efficient ◽  
Species Production

Development of clinical stem cell interventions are hampered by immature cell progeny under current protocols. Human mesenchymal stem cells (hMSCs) are characterized by their ability to self-renew and differentiate into multiple lineages. Generating hMSCs from pluripotent stem cells (iPSCs) is an attractive avenue for cost-efficient and scalable production of cellular material. In this study we generate mature osteoblasts from iPSCs using a stable expandable MSC intermediate, refining established protocols. We investigated the timeframe and phenotype of cells under osteogenic conditions as well as the effect of menaquinone-7 (MK-7) on differentiation. From day 2 we noted a significant increase in RUNX2 expression under osteogenic conditions with MK-7, as well as decreases in ROS species production, increased cellular migration and changes to dynamics of collagen deposition when compared to differentiated cells that were not treated with MK-7. At day 21 OsteoMK-7 increased alkaline phosphatase activity and collagen deposition, as well as downregulated RUNX2 expression, suggesting to a mature cellular phenotype. Throughout we note no changes to expression of osteocalcin suggesting a non-canonical function of MK-7 in osteoblast differentiation. Together our data provide further mechanistic insight between basic and clinical studies on extrahepatic activity of MK-7. Our findings show that MK-7 promotes osteoblast maturation thereby increasing osteogenic differentiation.

scholarly journals Differential Responses of Transplanted Stem Cells to Diseased Environment Unveiled by a Molecular NIR-II Cell Tracker

Research ◽  
2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Hao Chen ◽  
Huaxiao Yang ◽  
Chen Zhang ◽  
Si Chen ◽  
Xin Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Therapy ◽  
Single Cell ◽  
Real Time ◽  
Stem Cell Therapy ◽  
Cellular Migration ◽  
Cell Cluster ◽  
Spatiotemporal Resolution

Stem cell therapy holds high promises in regenerative medicine. The major challenge of clinical translation is to precisely and quantitatively evaluate the in vivo cell distribution, migration, and engraftment, which cannot be easily achieved by current techniques. To address this issue, for the first time, we have developed a molecular cell tracker with a strong fluorescence signal in the second near-infrared (NIR-II) window (1,000-1,700 nm) for real-time monitoring of in vivo cell behaviors in both healthy and diseased animal models. The NIR-II tracker (CelTrac1000) has shown complete cell labeling with low cytotoxicity and profound long-term tracking ability for 30 days in high spatiotemporal resolution for semiquantification of the biodistribution of transplanted stem cells. Taking advantage of the unique merits of CelTrac1000, the responses of transplanted stem cells to different diseased environments have been discriminated and unveiled. Furthermore, we also demonstrate CelTrac1000 as a universal and effective technique for ultrafast real-time tracking of the cellular migration and distribution in a 100 μm single-cell cluster spatial resolution, along with the lung contraction and heart beating. As such, this NIR-II tracker will shift the optical cell tracking into a single-cell cluster and millisecond temporal resolution for better evaluating and understanding stem cell therapy, affording optimal doses and efficacy.

P04.19 A combined use of a G-quadruplex oligonucleotide (GQ) and neuro-inducing small molecules inhibit glioblastoma cell proliferation

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii22-ii22
Author(s):  
V Kolesnikova ◽  
N Samoylenkova ◽  
S Drozd ◽  
A Revishchin ◽  
D Y Usachev ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Cell Proliferation ◽  
Stem Cell ◽  
Cell Cultures ◽  
Mtt Assay ◽  
Proliferative Activity ◽  
Recombinant Dna ◽  
Glioblastoma Cell ◽  
G Quadruplex

Abstract BACKGROUND According to one of the theories, gliomas can occur as a result of dysregulation of stem cell division in the subventricular region of the brain. The CD133 membrane marker is a characteristic of both normal and tumor neural stem cells therefore it can be used to isolate a stem cell population from tumor tissue. Tumor cells actively proliferate which suggests that their possible differentiation may be achieved by inhibiting of their division as these two processes are mutually exclusive. For this purpose, G-quadruplex oligonucleotides together with neural-inducers such as a brain-derived neurotrophic factor (BDNF) may be used. MATERIAL AND METHODS Five cell cultures obtained from human glioblastoma tissues were analyzed for expression of CD133 using RT-qPCR. From cell culture with the highest level of CD133 using immunomagnetic separation CD133+ and CD133- cultures were received. CD133fr/peGFP-c1 recombinant DNA consisted of a CD133 second extracellular loop fragment and a peGFP-c1 vector was constructed to determine the localization of prominin-1, that is known as CD133 when found on cell membrane, using confocal microscopy. On chosen cell cultures an oligonucleotide bi-(AID-1-T) and its combination with BDNF were tested. The mechanism of GQ’s action is cytostatic and its non-toxicity properties were proved by flow cytometry. For evaluating the proliferative activity of cells MTT assay was performed on 10th and 20th days after exposure to the factors. RESULTS Cell culture G01 was chosen for further research as it had the highest level of the CD133. Colocalization of CD133 and GFP demonstrated a membrane localization of CD133 in cells with high expression level of this marker. MTT assay on 10th day after exposure to bi-(AID-1-T) as well as its combination with BDNF on cell culture G01 CD133- showed total inhibition of cell proliferation. The same combinations tested on G01 CD133+ cell culture demonstrated no difference in proliferative activity. After 20 days after exposure to bi-(AID-1-T) and combination of bi-(AID-1-T) with BDNF the significant decrease of G01 CD133+ cells’ proliferation was observed. When tested on whole glioblastoma cell culture G01 these combinations also showed significant inhibition of cell proliferation. CONCLUSION We showed that glioblastoma cells upon transfection with recombinant DNA, that contains a fragment of CD133, mainly have a membrane localization of this marker. It was observed that CD133+ cells are more stable to external influence that can be a proof of the fact that CD133 is charactered for glioblastoma stem cells. We tested the effect of an GQ bi-(AID-1-T) and its combination with BDNF and showed that BDNF is necessary for blocking proliferation of glioblastoma cells. Altogether, the results may be used for further research as it reveals a potential treatment for patients with glioblastoma. Grant №075-15-2020-809 (13.1902.21.0030).

scholarly journals Mesenchymal stem cells show little tropism for the resting and differentiated cancer stem cell-like glioma cells

2014 ◽  
Vol 44 (4) ◽  
pp. 1223-1232 ◽  
Author(s):  
ZHENLIN LIU ◽  
ZHONGMIN JIANG ◽  
JIANYONG HUANG ◽  
SHUQIANG HUANG ◽  
YANXIA LI ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Cancer Stem Cell ◽  
Glioma Cells

scholarly journals Identifying the Zika Virus Target Cell in Malignant Glioma

2019 ◽  
Vol 21 (Supplement_4) ◽  
pp. iv2-iv2
Author(s):  
Gemma Girdler ◽  
Tannia Gracia ◽  
Daniel Fountain ◽  
Ted Fajardo Jr ◽  
Jack Finlay ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Neural Stem Cell ◽  
Target Cell ◽  
Zika Virus ◽  
Primary Tumour ◽  
Malignant Gliomas ◽  
Stem Cell Population ◽  
Slice Culture ◽  
Normal Brain

Abstract The Zika Virus epidemic of 2015–2016 was associated with striking failure of forebrain development in infants born to infected mothers, resulting in microcephaly. Studies from numerous labs subsequently confirmed a selective effect of the virus on neural stem cell survival, self-renewal and differentiation. The glioma stem cells which drive glioblastoma and other malignant gliomas depend on neural stem cell transcription programs, so that understanding Zika infection in these cells promises valuable insights into future therapy. We describe here: 1) Study of low passage patient-derived glioblastoma cell lines and normal neural stem cells (CRUK Glioma Cellular Genetics Resource) in adherent culture to address the influence of glioma subtype on infectability 2) Application of genetically modified mCherry reporter Zika Virus strains to address the Zika target cell in glioblastoma. 3) Development of a cerebral organoid model to interrogate the differential effects of the virus on tumour cells and surrounding normal brain. 4) Demonstration of Zika infection on primary patient derived tissue in slice culture format Using these tools we find that Zika targets a common stem cell population across tumour subtypes and neural stem cell controls, and in embryonic and primary tumour explants, and that infection is influenced by activity of cholesterol biosynthesis pathways.

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