scholarly journals TAMI-24. BEHAVIOR OF GLIOBLASTOMA STEM-LIKE CELLS WITH KNOWN IDH1 STATUS IN CEREBRAL ORGANOIDS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii218-ii218
Author(s):  
Sanjay Singh ◽  
Maxime Munyeshyaka ◽  
Joy Gumin ◽  
Jing Yang ◽  
Daniel Ledbetter ◽  
...  
Keyword(s):  
Stem Cells ◽  
Fluorescent Proteins ◽  
Clinical History ◽  
Idh1 Mutation ◽  
Model Systems ◽  
Low Grade ◽  
Normal Brain ◽  
Orthotopic Xenografts

Abstract Glioblastomas (GBM) exhibit high proliferative index, areas of necrosis, high vascularization, and are highly invasive to normal brain tissues. The most common and lethal form of GBMs are primary GBMs, with no prior clinical history. Whereas, secondary GBMs arise from low-grade gliomas and are associated with IDH1 mutation. Pre-clinical studies of GBM largely depend on patient-derived GBM stem-like cells (GSCs) in vitro and in vivo as orthotopic xenografts. Cerebral organoids (COs) derived from induced pluripotent stem cells can serve as allogenic in vitro model systems to study interactions between normal brain and GSCs. COs have been shown to harbor neural stem cells and their differentiated progenies as well as microglia within distinct niches. Here, we co-cultured 45 day-old COs and MDA-GSCs lines representing mesenchymal sub-group (M-MDA-GSC), classical sub-group (C-MDA-GSC), and IDH1 mutant (IDH1R132H-MDA-GSC). MDA-GSCs stably express fluorescent proteins and is used to track GSCs within COs. These GSC bearing COs were fixed, embedded, sectioned, immuno-stained, and imaged by confocal microscope. There was a positive correlation between GSC numbers in allografted niche and invasion into COs as measured from the edge of organoid, M-MDA-GSC (R2=0.99; 0.89μm/cell), C-MDA-GSC (R2=0.92; 0.66μm/cell), and IDH1R132H-MDA-GSC (R2=0.89; 0.5μm/cell). Additionally, M-MDA-GSCs had significantly high percentage of Ki67+ve invasive cells (24%) in comparison to C-MDA-GSCs (5.1%; p=0.0057). As a measure of interaction of MDA-GSC with normal cells, we assessed proximity of IBA1+ve microglia in GSC niche within organoids and show that M-MDA-GSC and IDH1R132H-MDA-GSC highly co-localized with IBA1+ve microglia on day12 of co-culture. In conclusion, our cerebral organoid-based allograft study shows that mesenchymal GSCs (M-MDA-GSC) are most invasive whereas IDH1 mutant GSCs (IDH1R132H-MDA-GSC) are least invasive. C-MDA-GSCs are least proliferative while invading into normal COs. Uniqueness of CO based allograft system is highlighted by observed similarity between M-MDA-GSC and IDH1R132H-MDA-GSC for their potential to attract IBA1+ve microglia.

Demonstration of subpopulations with differing cancer stem cell phenotypes in xenograft and in vitro models of colorectal liver metastases.

2013 ◽  
Vol 31 (4_suppl) ◽  
pp. 394-394
Author(s):  
Dominic E. Sanford ◽  
Andrew Giorgi ◽  
Brian D. Goetz ◽  
Roheena Z. Panni ◽  
William G. Hawkins ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Liver Metastases ◽  
In Vitro Models ◽  
Model Systems ◽  
Cell Populations ◽  
Tumor Initiating Cells ◽  
Distinct Cell

394 Background: Tumors are composed of heterogeneous cell populations, some of which demonstrate enhanced tumor-forming capabilities (so-called tumor initiating cells [TIC] or cancer stem cells). In colorectal cancer (CRC), CD133, 44, and 24 are cell surface markers that identify TIC. Therefore, we sought to determine if CRC liver metastases (CRC-LM) form xenografts (in vivo) and cell cultures (in vitro) with TIC markers. Methods: CRC-LM were grafted in NOD/SCID mice and passaged serially. Xenografts were mechanically dissociated and cultured under sphere forming conditions. Flow cytometry was performed for TIC phenotype. Results: 16 of 18 (89%) CRC-LM specimens formed tumors in mice. Xenografts formed EpCAM+ tumors and spheres. The frequency of CD133+, CD44+, and CD133+/CD44+ tumor cells were 55%, 33%, and 23%, respectively. There was a subpopulation of CD133+/CD44+ cells with elevated CD44 expression(CD44hi). This CD133+/CD44hi population was also CD24+; representing 5% of cells. Eight of eleven (73%) xenografts formed spheres in vitro. The frequency of CD133+, CD44+, and CD133+/CD44+ cells were 63%, 47%, and 26%, respectively. CD133+/CD44+/CD24+ cells made up 8% of sphere-forming cells. There was a non-significant trend towards increased frequency of CD133+, CD44+, and CD133/CD44 positive cells in the spheres compared to the xenografts. However, the percentage of CD133+/CD44+/CD24+ cells was significantly increased in spheres relative to xenografts (8% vs. 5%, respectively; p<0.05) (see Table). Conclusions: CRC-LM derived xenografts and spheres are composed of distinct cell populations with differing levels of TIC/cancer stem cells. Sphere cultures may enhance for the most enriched TIC population. Thus, xenografts and sphere cultures are important model systems to further study the importance of cancer stem cells in CRC progression and metastases. [Table: see text]

Abstract P482: Elucidating And Characterizing The Molecular Mechanistic Role Of Phospholamban L39 Stop In The Pathophysiology Of Cardiomyopathy Using Patient-derived Human Induced Pluripotent Stem Cells And Humanized Knock-in Mouse Model Systems

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Anichavezhi Devendran ◽  
Rasheed Bailey ◽  
Sumanta Kar ◽  
Francesca Stillitano ◽  
Irene Turnbull ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Mononuclear Cells ◽  
Embryonic Stem ◽  
Model Systems ◽  
Patient Specific ◽  
Induced Pluripotent

Background: Heart failure (HF) is a complex clinical condition associated with substantial morbidity and mortality worldwide. The contractile dysfunction and arrhythmogenesis related to HF has been linked to the remodelling of calcium (Ca ++ ) handling. Phospholamban (PLN) has emerged as a key regulator of intracellular Ca ++ concentration. Of the PLN mutations, L39X is intriguing as it has not been fully characterized. This mutation is believed to be functionally equivalent to PLN null (KO) but contrary to PLN KO mice, L39X carriers develop a lethal cardiomyopathy (CMP). Our study aims at using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from homozygous L39X carriers to elucidate the role of L39X in human pathophysiology. Our plan also involves the characterization of humanized L39X knock-in mice (KM), which we hypothesize will develop a CMP from mis-localization of PLN and disruption of Ca ++ signalling. Methodology and Results: Mononuclear cells from Hom L39X carriers were obtained to generate 11 integration-free patient-specific iPSC clones. The iPSC-CMs were derived using established protocols. Compared to the WT iPSC-CMs, the Hom L39X derived-CMs PLN had an abnormal cytoplasmic distribution and formed intracellular aggregates, with the loss of perinuclear localization. There was also a 70% and 50% reduction of mRNA and protein expression of PLN respectively in L39X compared to WT iPSC-CMs. These findings indicated that L39X PLN is both under-expressed and mis-localized within the cell. To validate this observation in-vivo, we genetically modified FVB mice to harbour the human L39X. Following electroporation, positively transfected mouse embryonic stem cells were injected into host blastocysts to make humanized KM that were subsequently used to generate either a protamine-Cre (endogenous PLN driven expression) or a cardiac TNT mouse (i.e., CMP specific). Conclusion: Our data confirm an abnormal intracellular distribution of PLN, with the loss of perinuclear accumulation and mis-localization, suggestive of ineffective targeting to or retention of L39X. The mouse model will be critically important to validate the in-vitro observations and provides an ideal platform for future studies centred on the development of novel therapeutic strategies including virally delivered CRISPR/Cas9 for in-vivo gene editing and testing of biochemical signalling pathways.

scholarly journals Kinases of the Focal Adhesion Complex Contribute to Cardiomyocyte Specification

2021 ◽  
Vol 22 (19) ◽  
pp. 10430
Author(s):  
Sacha Robert ◽  
Marcus Flowers ◽  
Brenda M. Ogle
Keyword(s):  
Stem Cells ◽  
Focal Adhesion ◽  
Pluripotent Stem Cells ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Culture Conditions ◽  
Model Systems ◽  
Critical Components

Differentiation of pluripotent stem cells to cardiomyocytes is influenced by culture conditions including the extracellular matrices or similar synthetic scaffolds on which they are grown. However, the molecular mechanisms that link the scaffold with differentiation outcomes are not fully known. Here, we determined by immunofluorescence staining and mass spectrometry approaches that extracellular matrix (ECM) engagement by mouse pluripotent stem cells activates critical components of canonical wingless/integrated (Wnt) signaling pathways via kinases of the focal adhesion to drive cardiomyogenesis. These kinases were found to be differentially activated depending on type of ECM engaged. These outcomes begin to explain how varied ECM composition of in vivo tissues with development and in vitro model systems gives rise to different mature cell types, having broad practical applicability for the design of engineered tissues.

scholarly journals Orthotopic Patient-Derived Xenografts of Gastric Cancer to Decipher Drugs Effects on Cancer Stem Cells and Metastatic Dissemination

Cancers ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 560 ◽  
Author(s):  
Julie Giraud ◽  
Damien Bouriez ◽  
Lornella Seeneevassen ◽  
Benoit Rousseau ◽  
Elodie Sifré ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Mouse Models ◽  
Distant Metastases ◽  
Preclinical Models ◽  
Immunocompromised Mice ◽  
Orthotopic Xenografts

Gastric cancer is the third leading cause of cancer mortality worldwide. Cancer stem cells (CSC) are at the origin of tumor initiation, chemoresistance, and the formation of metastases. However, there is a lack of mouse models enabling the study of the metastatic process in gastric adenocarcinoma (GC). The aims of this study were to develop original mouse models of patient-derived primary GC orthotopic xenografts (PDOX) allowing the development of distant metastases as preclinical models to study the anti-metastatic efficiency of drugs such as the phosphatidylinositol 3-kinase (PI3K) inhibitor Buparlisib (BKM120). Luciferase-encoding cells generated from primary GC were injected into the stomach wall of immunocompromised mice; gastric tumor and metastases development were followed by bioluminescence imaging. The anti-CSC properties of BKM120 were evaluated on the GC cells’ phenotype (CD44 expression) and tumorigenic properties in vitro and in vivo on BKM120-treated mice. After eight weeks, PDOX mice formed tumors in the stomach as well as distant metastases, that were enriched in CSC, in the liver, the lung, and the peritoneal cavity. BKM120 treatment significantly inhibited the CSC properties in vitro and reduced the number of distant metastases in mice. These new preclinical models offer the opportunity to study the anti-metastatic efficiency of new CSC-based therapeutic strategies.

scholarly journals Cystathionine-γ-lyase drives antioxidant defense in cysteine-restricted IDH1 mutant astrocytomas

2021 ◽  
Author(s):  
Andrés Cano-Galiano ◽  
Anais Oudin ◽  
Fred Fack ◽  
Maria-Francesca Allega ◽  
David Sumpton ◽  
...  
Keyword(s):  
Antioxidant Defense ◽  
De Novo ◽  
Clinical Specimen ◽  
Idh1 Mutation ◽  
Isocitrate Dehydrogenase 1 ◽  
Astrocytic Gliomas ◽  
Orthotopic Xenografts ◽  
The Impact

Abstract Background Mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) define glioma subtypes and are considered primary events in gliomagenesis, impacting tumor epigenetics and metabolism. IDH enzyme activity is crucial for the generation of reducing potential in normal cells, yet the impact of the mutation on the cellular antioxidant system in glioma is not understood. The aim of this study was to determine how glutathione (GSH), the main antioxidant in the brain, is maintained in IDH1 mutant gliomas, despite an altered NADPH/NADP balance. Methods Proteomics, metabolomics, metabolic tracer studies, genetic silencing and drug targeting approaches in vitro and in vivo were applied. Analyses were done in clinical specimen of different glioma subtypes, in glioma patient-derived cell lines carrying the endogenous IDH1 mutation and corresponding orthotopic xenografts in mice. Results We find that cystathionine-γ-lyase (CSE), the enzyme responsible for cysteine production upstream of GSH biosynthesis, is specifically upregulated in IDH1 mutant astrocytomas. CSE inhibition sensitized these cells to cysteine depletion, an effect not observed in IDH1 wild-type gliomas. This correlated with an increase in reactive oxygen species and reduced GSH synthesis. Propargylglycine (PAG), a brain-penetrant drug specifically targeting CSE, led to delayed tumor growth in mice. Conclusions We show that IDH1 mutant astrocytic gliomas critically rely on NADPH-independent de novo GSH synthesis via CSE to maintain the antioxidant defense, which highlights a novel metabolic vulnerability that may be therapeutically exploited.

Panel Discussion II: Usefulness of in vitro and in vivo Model Systems for Predicting the Adverse Public Health Outcome for Antimicrobial Drug Residues in Food

2000 ◽  
Vol 12 (3) ◽  
pp. 45-53
Author(s):  
Andrew Beaulieu ◽  
Jacques Boisseau ◽  
Carl Cerniglia ◽  
Denis Corpet ◽  
A. Haydée Fernández ◽  
...  
Keyword(s):  
Public Health ◽  
Health Outcome ◽  
Panel Discussion ◽  
Antimicrobial Drug ◽  
Model Systems ◽  
In Vivo Model ◽  
Drug Residues ◽  
Public Health Outcome

Autologous transplantation of mesenchymal stem cells into the portal vein of the miniature pig; a preliminary experiment for NOTES approach

2019 ◽  
Vol 98 (9) ◽  
pp. 350-355
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Therapy ◽  
Portal Vein ◽  
Liver Parenchyma ◽  
Miniature Pig ◽  
Hepatic Diseases ◽  
Study Results

Introduction: There is evidence that mesenchymal stem cells (MSCs) could trans-differentiate into the liver cells in vitro and in vivo and thus may be used as an unfailing source for stem cell therapy of liver disease. Combination of MSCs (with or without their differentiation in vitro) and minimally invasive procedures as laparoscopy or Natural Orifice Transluminal Endoscopic Surgery (NOTES) represents a chance for many patients waiting for liver transplantation in vain. Methods: Over 30 millions of autologous MSCs at passage 3 were transplanted via the portal vein in an eight months old miniature pig. The deposition of transplanted cells in liver parenchyma was evaluated histologically and the trans-differential potential of CM-DiI labeled cells was assessed by expression of pig albumin using immunofluorescence. Results: Three weeks after transplantation we detected the labeled cells (solitary, small clusters) in all 10 samples (2 samples from each lobe) but no diffuse distribution in the samples. The localization of CM-DiI+ cells was predominantly observed around the portal triads. We also detected the localization of albumin signal in CM-DiI labeled cells. Conclusion: The study results showed that the autologous MSCs (without additional hepatic differentiation in vitro) transplantation through the portal vein led to successful infiltration of intact miniature pig liver parenchyma with detectable in vivo trans-differentiation. NOTES as well as other newly developed surgical approaches in combination with cell therapy seem to be very promising for the treatment of hepatic diseases in near future.

Differentiation of mesenchymal stem cells from humans and animals into insulin-producing cells: An overview in vitro induction forms

2020 ◽  
Vol 16 ◽  
Author(s):  
Bruna O. S. Câmara ◽  
Bruno M. Bertassoli ◽  
Natália M. Ocarino ◽  
Rogéria Serakides
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture Medium ◽  
Adipogenic Differentiation ◽  
Medium Composition ◽  
Cell Therapies ◽  
Insulin Producing Cells ◽  
Msc Differentiation

The use of stem cells in cell therapies has shown promising results in the treatment of several diseases, including diabetes mellitus, in both humans and animals. Mesenchymal stem cells (MSCs) can be isolated from various locations, including bone marrow, adipose tissues, synovia, muscles, dental pulp, umbilical cords, and the placenta. In vitro, by manipulating the composition of the culture medium or transfection, MSCs can differentiate into several cell lineages, including insulin-producing cells (IPCs). Unlike osteogenic, chondrogenic, and adipogenic differentiation, for which the culture medium and time are similar between studies, studies involving the induction of MSC differentiation in IPCs differ greatly. This divergence is usually evident in relation to the differentiation technique used, the composition of the culture medium, the cultivation time, which can vary from a few hours to several months, and the number of steps to complete differentiation. However, although there is no “gold standard” differentiation medium composition, most prominent studies mention the use of nicotinamide, exedin-4, ß-mercaptoethanol, fibroblast growth factor b (FGFb), and glucose in the culture medium to promote the differentiation of MSCs into IPCs. Therefore, the purpose of this review is to investigate the stages of MSC differentiation into IPCs both in vivo and in vitro, as well as address differentiation techniques and molecular actions and mechanisms by which some substances, such as nicotinamide, exedin-4, ßmercaptoethanol, FGFb, and glucose, participate in the differentiation process.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals LPTO-06. A NOVEL BRAIN-PERMEANT CHEMOTHERAPEUTIC AGENT FOR THE TREATMENT OF BREAST CANCER LEPTOMENINGEAL METASTASIS

2019 ◽  
Vol 1 (Supplement_1) ◽  
pp. i7-i7
Author(s):  
Jiaojiao Deng ◽  
Sophia Chernikova ◽  
Wolf-Nicolas Fischer ◽  
Kerry Koller ◽  
Bernd Jandeleit ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Brain Tumors ◽  
Cell Lines ◽  
Chemotherapeutic Agent ◽  
Leptomeningeal Metastasis ◽  
Breast Cancer Cell Lines ◽  
Normal Brain ◽  
Breast Cancers

Abstract Leptomeningeal metastasis (LM), a spread of cancer to the cerebrospinal fluid and meninges, is universally and rapidly fatal due to poor detection and no effective treatment. Breast cancers account for a majority of LMs from solid tumors, with triple-negative breast cancers (TNBCs) having the highest propensity to metastasize to LM. The treatment of LM is challenged by poor drug penetration into CNS and high neurotoxicity. Therefore, there is an urgent need for new modalities and targeted therapies able to overcome the limitations of current treatment options. Quadriga has discovered a novel, brain-permeant chemotherapeutic agent that is currently in development as a potential treatment for glioblastoma (GBM). The compound is active in suppressing the growth of GBM tumor cell lines implanted into the brain. Radiolabel distribution studies have shown significant tumor accumulation in intracranial brain tumors while sparing the adjacent normal brain tissue. Recently, we have demonstrated dose-dependent in vitro and in vivo anti-tumor activity with various breast cancer cell lines including the human TNBC cell line MDA-MB-231. To evaluate the in vivo antitumor activity of the compound on LM, we used the mouse model of LM based on the internal carotid injection of luciferase-expressing MDA-MB-231-BR3 cells. Once the bioluminescence signal intensity from the metastatic spread reached (0.2 - 0.5) x 106 photons/sec, mice were dosed i.p. twice a week with either 4 or 8 mg/kg for nine weeks. Tumor growth was monitored by bioluminescence. The compound was well tolerated and caused a significant delay in metastatic growth resulting in significant extension of survival. Tumors regressed completely in ~ 28 % of treated animals. Given that current treatments for LM are palliative with only few studies reporting a survival benefit, Quadriga’s new agent could be effective as a therapeutic for both primary and metastatic brain tumors such as LM. REF: https://onlinelibrary.wiley.com/doi/full/10.1002/pro6.43

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