scholarly journals Prolonged Culturing of IPSC-derived Brain Endothelial-like Cells Is Associated With Quiescence, Downregulation of Glycolysis, and Resistance to Disruption by an Alzheimer’s Brain Milieu

2021 ◽  
Author(s):  
Lindsey M Williams ◽  
Takashi Fujimoto ◽  
Riley R Weaver ◽  
Aric F Logsdon ◽  
Miranda L Wilson ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Glucose Transporter ◽  
Adherens Junction ◽  
Induced Pluripotent Stem Cell ◽  
Proliferating Cells ◽  
Glucose Transporter 1 ◽  
Platelet Endothelial Cell Adhesion ◽  
Swedish Mutation ◽  
Tnf Α ◽  
Induced Pluripotent

Abstract Background: Human induced pluripotent stem cell (hiPSC)-derived brain endothelial-like cells (iBECs) are a robust, scalable, and translatable model of the human blood-brain barrier (BBB). Prior works have shown that high transendothelial electrical resistance (TEER) persists in iBECs for at least two weeks, emphasizing the utility of the model for longer term studies. However, most studies evaluate iBECs within the first few days of subculture, and little is known about their proliferative state, which could influence their functions. In this study, we characterized iBEC proliferative state in relation to key BBB properties at early (2 days) and late (9 days) post-subculture time points. Methods: hiPSCs were differentiated into iBECs using fully defined, serum-free medium. The proportion of proliferating cells was determined by BrdU assays. We evaluated TEER, expression of glycolysis enzymes and tight and adherens junction proteins (TJP and AJP), and glucose transporter-1 (GLUT1) function by immunoblotting, immunofluorescence, and quantifying radiolabeled tracer permeabilities. We also compared barrier disruption in response to TNF-α and conditioned medium (CM) from hiPSC-derived neurons harboring the Alzheimer’s disease (AD)-causing Swedish mutation (APPSwe/+).Results: A significant decline in iBEC proliferation over time in culture was accompanied by adoption of a more quiescent endothelial metabolic state, indicated by downregulation of glycolysis-related proteins and upregulation GLUT1. Interestingly, upregulation of GLUT1 was associated with reduced glucose transport rates in more quiescent iBECs. We also found significant decreases in claudin-5 (CLDN5) and vascular endothelial-cadherin (VE-Cad) and a trend toward a decrease in platelet endothelial cell adhesion molecule-1 (PECAM-1), whereas zona occludens-1 (ZO-1) increased and occludin (OCLN) remained unchanged. Despite differences in TJP and AJP expression, there was no difference in mean TEER on day 2 vs. day 9. TNF-α induced disruption irrespective of iBEC proliferative state. Conversely, APPSwe/+ CM disrupted only proliferating iBEC monolayers. Conclusion: iBECs can be used to study responses to disease-relevant stimuli in proliferating vs. more quiescent endothelial cell states, which may provide insight into BBB vulnerabilities in contexts of development, brain injury, and neurodegenerative disease.

scholarly journals Human induced pluripotent stem cell-based platform for modeling cardiac ischemia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Martta Häkli ◽  
Joose Kreutzer ◽  
Antti-Juhana Mäki ◽  
Hannu Välimäki ◽  
Henna Lappi ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Glucose Transporter ◽  
Ischemia Reperfusion ◽  
Field Potential ◽  
Induced Pluripotent Stem Cell ◽  
Cardiac Ischemia ◽  
Glucose Transporter 1 ◽  
Induced Pluripotent ◽  
Beating Frequency

AbstractIschemic heart disease is a major cause of death worldwide, and the only available therapy to salvage the tissue is reperfusion, which can initially cause further damage. Many therapeutics that have been promising in animal models have failed in human trials. Thus, functional human based cardiac ischemia models are required. In this study, a human induced pluripotent stem cell derived-cardiomyocyte (hiPSC-CM)-based platform for modeling ischemia–reperfusion was developed utilizing a system enabling precise control over oxygen concentration and real-time monitoring of the oxygen dynamics as well as iPS-CM functionality. In addition, morphology and expression of hypoxia-related genes and proteins were evaluated as hiPSC-CM response to 8 or 24 h hypoxia and 24 h reoxygenation. During hypoxia, initial decrease in hiPSC-CM beating frequency was observed, after which the CMs adapted to the conditions and the beating frequency gradually increased already before reoxygenation. During reoxygenation, the beating frequency typically first surpassed the baseline before settling down to the values close the baseline. Furthermore, slowing on the field potential propagation throughout the hiPSC-CM sheet as well as increase in depolarization time and decrease in overall field potential duration were observed during hypoxia. These changes were reversed during reoxygenation. Disorganization of sarcomere structures was observed after hypoxia and reoxygenation, supported by decrease in the expression of sarcomeric proteins. Furthermore, increase in the expression of gene encoding glucose transporter 1 was observed. These findings indicate, that despite their immature phenotype, hiPSC-CMs can be utilized in modeling ischemia–reperfusion injury.

Abstract 112: A Pathological Role of Endothelial p53 in the Regulation of Endothelial Function and Glucose Metabolism

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Masataka YOKOYAMA ◽  
Yoshio KOBAYASHI ◽  
Tohru MINAMINO
Keyword(s):  
Skeletal Muscle ◽  
Endothelial Cell ◽  
Glucose Metabolism ◽  
Endothelial Function ◽  
Mitochondrial Biogenesis ◽  
Glucose Transporter ◽  
Diabetic Patients ◽  
Glucose Transporter 1 ◽  
P53 Activation

Cellular senescence is a state of irreversible growth arrest induced by various stresses such as oncogenic stimuli. This response is controlled by negative regulators of the cell cycle like the p53 tumor suppressor protein. Accumulating evidence has suggested a role of p53 activation in various age-associated conditions including atherosclerosis, heart failure and diabetes. Here we show that endothelial p53 activation plays a pathological role in the regulation of endothelial function and glucose metabolism under diabetic conditions. Endothelial expression of p53 was markedly up-regulated in a streptozotocin-induced diabetes model. Endothelial function such as acetylcholine-dependent vasodilatation was markedly impaired in this model. Although hyperglycemia was not altered, impairment of endothelial function was significantly improved in mice with endothelial cell-specific p53 deficiency. In same way, p53 was markedly activated in ischemic vessels, and endothelial p53 deficiency enhanced ischemia-induced angiogenesis. Mechanistically, endothelial p53 up-regulated the expression of PTEN that negatively regulated the Akt-eNOS pathway, and therefore disruption of p53 improved endothelial dysfunction. We also found that endothelial p53 was markedly activated, and the Akt-eNOS pathway was attenuated in a diet-induced obesity model. Disruption of endothelial p53 activation improved dietary inactivation of eNOS that up-regulated the expression of PGC-1α in skeletal muscle, thereby increasing mitochondrial biogenesis and oxygen consumption. Inhibition of endothelial p53 also improved dietary impairment of glucose transport into skeletal muscle by up-regulating endothelial expression of glucose transporter 1. Consequently, mice with endothelial cell-specific p53 deficiency fed a high-calorie diet showed improvement of insulin sensitivity and less fat accumulation compared with control littermates. These results indicate that endothelial p53 negatively regulates endothelium-dependent vasodilatation, ischemia-induced angiogenesis, and mitochondrial biogenesis by inhibiting the Akt-eNOS pathway and suggest that inhibition of endothelial p53 could be a novel therapeutic target in diabetic patients.

Lithium chloride improves the efficiency of induced pluripotent stem cell-derived neurospheres

2015 ◽  
Vol 396 (8) ◽  
pp. 923-928 ◽  
Author(s):  
Azita Parvaneh Tafreshi ◽  
Aude Sylvain ◽  
Guizhi Sun ◽  
Daniella Herszfeld ◽  
Keith Schulze ◽  
...  
Keyword(s):  
Stem Cell ◽  
Lithium Chloride ◽  
Pluripotent Stem Cell ◽  
Lithium Treatment ◽  
Formation Rate ◽  
Induced Pluripotent Stem Cell ◽  
Neural Progenitors ◽  
Neural Cells ◽  
Proliferating Cells ◽  
Induced Pluripotent

Abstract Induced pluripotent stem cell (iPSC)-derived neurospheres, which consist mainly of neural progenitors, are considered to be a good source of neural cells for transplantation in regenerative medicine. In this study, we have used lithium chloride, which is known to be a neuroprotective agent, in an iPSC-derived neurosphere model, and examined both the formation rate and size of the neurospheres as well as the proliferative and apoptotic status of their contents. Our results showed that lithium enhanced the formation and the sizes of the iPSC-derived neurospheres, increased the number of Ki67-positive proliferating cells, but reduced the number of the TUNEL-positive apoptotic cells. This increased number of Ki67 proliferating cells was secondary to the decreased apoptosis and not to the stimulation of cell cycle entry, as the expression of the proliferation marker cyclin D1 mRNA did not change after lithium treatment. Altogether, we suggest that lithium enhances the survival of neural progenitors and thus the quality of the iPSC-derived neurospheres, which may strengthen the prospect of using lithium-treated pluripotent cells and their derivatives in a clinical setting.

Reactive oxygen species downregulate glucose transport system in retinal endothelial cells

2011 ◽  
Vol 300 (4) ◽  
pp. C927-C936 ◽  
Author(s):  
Rosa Fernandes ◽  
Ken-ichi Hosoya ◽  
Paulo Pereira
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Plasma Membrane ◽  
Endothelial Cell ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Cell Damage ◽  
Proteasome Inhibition ◽  
Glucose Transporter 1 ◽  
Retinal Endothelial Cells

Retinal endothelial cells are believed to play an important role in the pathogenesis of diabetic retinopathy. In previous studies, we and others demonstrated that glucose transporter 1 (GLUT1) is downregulated in response to hyperglycemia. Increased oxidative stress is likely to be the event whereby hyperglycemia is transduced into endothelial cell damage. However, the effects of sustained oxidative stress on GLUT1 regulation are not clearly established. The objective of this study is to evaluate the effect of increased oxidative stress on glucose transport and on GLUT1 subcellular distribution in a retinal endothelial cell line and to elucidate the signaling pathways associated with such regulation. Conditionally immortalized rat retinal endothelial cells (TR-iBRB) were incubated with glucose oxidase, which increases the intracellular hydrogen peroxide levels, and GLUT1 regulation was investigated. The data showed that oxidative stress did not alter the total levels of GLUT1 protein, although the levels of mRNA were decreased, and there was a subcellular redistribution of GLUT1, decreasing its content at the plasma membrane. Consistently, the half-life of the protein at the plasma membrane markedly decreased under oxidative stress. The proteasome appears to be involved in GLUT1 regulation in response to oxidative stress, as revealed by an increase in stabilization of the protein present at the plasma membrane and normalization of glucose transport following proteasome inhibition. Indeed, levels of ubiquitinated GLUT1 increase as revealed by immunoprecipitation assays. Furthermore, data indicate that protein kinase B activation is involved in the stabilization of GLUT1 at the plasma membrane. Thus subcellular redistribution of GLUT1 under conditions of oxidative stress is likely to contribute to the disruption of glucose homeostasis in diabetes.

P5746Human induced pluripotent stem cell-derived mesenchymal stem cell therapy effectively reduced brain infarct volume and preserved neurological function in rat after acute intracranial hemorrhage

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
Y C Li ◽  
F Y Lee ◽  
S Chua ◽  
H K Yip
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Neurological Function ◽  
Opposite Pattern ◽  
Group 4 ◽  
Tnf Α ◽  
Group 3 ◽  
Group 2 ◽  
Induced Pluripotent

Abstract Intracerebral hemorrhage (ICH) causes 10%-20% of all strokes and results in higher morbidity compared to other subtypes of cerebral stroke. Although early surgical intervention can clear the expanding hematoma, clinical outcomes following ICH have not significantly improved over the decades. Since ICH elicits neuroinflammation to exacerbate brain edema, damage the blood-brain barrier (BBB), lead to secondary neuronal injury, anti-inflammation may be a critical therapeutic strategy. Mesenchymal stem cell (MSC) therapy processes anti-inflammatory, immunomodulatory and tissue regenerative properties, suggesting that MSC therapy could be an effective therapy for ICH. Therefore, this study tested the hypothesis that human induced pluripotent stem cell-derived mesenchymal stem cell (iPSC-MSC) therapy could effectively reduce brain-infract volume (BIV) and improve neurological function in rat after acute ICH induced by a weight-drop device. Adult-male SD rats (n=40) were equally divided into group 1 (sham-operated control), group 2 (ICH), group 3 (ICH + hyaluronic acid (HA)/intracranial injection/3h after ICH), group 4 [ICH + HA + iPSC-MSC (1.2x106 cells/intracranial injection/3h after ICH)] and euthanized by day 28 after ICH procedure. In vitro study showed that hemorrhagic-brain tissue augmented protein expressions of inflammation (HMGB1/MyD88/TLR-4/TLR-2/NF-κB/TNF-α/iNOS/IL-1β) in cultured neurons that were significantly inhibited by iPSC-MSC treatment (all p<0.001). By days 7/14 after ICH procedure, circulating inflammatory levels of TNF-α/IL-6/MPO expressed were lowest in group 1, highest in group 2 and significantly lower in group 4 than in group 3 (all p<0.0001). By day 14 after ICH procedure, neurological function and BIV expressed an opposite pattern, whereas protein expressions of inflammation (HMGB1/MyD88/TLR-4/TLR-2/NF-κB/I-kB/TNF-α/iNOS/IL-1β/MMP-9), oxidative stress (NOX-1/NOX-2/oxidized protein) and apoptosis (mitochondrial-Bax/cleaved-caspase-2/PARP) in brain exhibited an identical pattern to circulating inflammation among the four groups (all p<0.001). Microscopy demonstrated that the number of vascular remodeling/GFAP+/53BP1+/γ-H2AX+ cells displayed an identical pattern of inflammation, whereas the NeuN+ cells displayed an opposite pattern of inflammation among the four groups (all p<0.001). In conclusion, iPSC-MSC therapy markedly reduced BIV and preserved neurological function mainly by inhibiting inflammatory/oxidative-stress generation. Acknowledgement/Funding Kaohsiung Chang Gung Memorial Hospital, Taiwan Society of Stem Cell Research

scholarly journals Development of Physiologically Responsive Human iPSC-Derived Intestinal Epithelium to Study Barrier Dysfunction in IBD

2020 ◽  
Vol 21 (4) ◽  
pp. 1438 ◽  
Author(s):  
John P. Gleeson ◽  
Hannah Q. Estrada ◽  
Michifumi Yamashita ◽  
Clive N. Svendsen ◽  
Stephan R. Targan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Intestinal Permeability ◽  
Intestinal Epithelium ◽  
Adherens Junction ◽  
Induced Pluripotent Stem Cell ◽  
Barrier Dysfunction ◽  
Junction Protein ◽  
Vitro Model ◽  
Induced Pluripotent

In inflammatory bowel disease (IBD), the intestinal epithelium is characterized by increased permeability both in active disease and remission states. The genetic underpinnings of this increased intestinal permeability are largely unstudied, in part due to a lack of appropriate modelling systems. Our aim is to develop an in vitro model of intestinal permeability using induced pluripotent stem cell (iPSC)-derived human intestinal organoids (HIOs) and human colonic organoids (HCOs) to study barrier dysfunction. iPSCs were generated from healthy controls, adult onset IBD, and very early onset IBD (VEO-IBD) patients and differentiated into HIOs and HCOs. EpCAM+ selected cells were seeded onto Transwell inserts and barrier integrity studies were carried out in the presence or absence of pro-inflammatory cytokines TNFα and IFNγ. Quantitative real-time PCR (qRT-PCR), transmission electron microscopy (TEM), and immunofluorescence were used to determine altered tight and adherens junction protein expression or localization. Differentiation to HCO indicated an increased gene expression of CDX2, CD147, and CA2, and increased basal transepithelial electrical resistance compared to HIO. Permeability studies were carried out in HIO- and HCO-derived epithelium, and permeability of FD4 was significantly increased when exposed to TNFα and IFNγ. TEM and immunofluorescence imaging indicated a mislocalization of E-cadherin and ZO-1 in TNFα and IFNγ challenged organoids with a corresponding decrease in mRNA expression. Comparisons between HIO- and HCO-epithelium show a difference in gene expression, electrophysiology, and morphology: both are responsive to TNFα and IFNγ stimulation resulting in enhanced permeability, and changes in tight and adherens junction architecture. This data indicate that iPSC-derived HIOs and HCOs constitute an appropriate physiologically responsive model to study barrier dysfunction and the role of the epithelium in IBD and VEO-IBD.

scholarly journals Modeling Intestinal Epithelial Response to Interferon-γ in Induced Pluripotent Stem Cell-Derived Human Intestinal Organoids

2020 ◽  
Vol 22 (1) ◽  
pp. 288
Author(s):  
Michael J. Workman ◽  
Elissa Troisi ◽  
Stephan R. Targan ◽  
Clive N. Svendsen ◽  
Robert J. Barrett
Keyword(s):  
Interferon Gamma ◽  
Adherens Junction ◽  
Induced Pluripotent Stem Cell ◽  
Enrichment Analysis ◽  
Cell Types ◽  
Gene Set Enrichment Analysis ◽  
Intestinal Organoids ◽  
Ifn Γ ◽  
Induced Pluripotent

Human intestinal organoids (HIOs) are increasingly being used to model intestinal responses to various stimuli, yet few studies have confirmed the fidelity of this modeling system. Given that the interferon-gamma (IFN-γ) response has been well characterized in various other cell types, our goal was to characterize the response to IFN-γ in HIOs derived from induced pluripotent stem cells (iPSCs). To achieve this, iPSCs were directed to form HIOs and subsequently treated with IFN-γ. Our results demonstrate that IFN-γ phosphorylates STAT1 but has little effect on the expression or localization of tight and adherens junction proteins in HIOs. However, transcriptomic profiling by microarray revealed numerous upregulated genes such as IDO1, GBP1, CXCL9, CXCL10 and CXCL11, which have previously been shown to be upregulated in other cell types in response to IFN-γ. Notably, “Response to Interferon Gamma” was determined to be one of the most significantly upregulated gene sets in IFN-γ-treated HIOs using gene set enrichment analysis. Interestingly, similar genes and pathways were upregulated in publicly available datasets contrasting the gene expression of in vivo biopsy tissue from patients with IBD against healthy controls. These data confirm that the iPSC-derived HIO modeling system represents an appropriate platform to evaluate the effects of various stimuli and specific environmental factors responsible for the alterations in the intestinal epithelium seen in various gastrointestinal conditions such as inflammatory bowel disease.

scholarly journals A Pulmonary Vascular Model From Endothelialized Whole Organ Scaffolds

2021 ◽  
Vol 9 ◽  
Author(s):  
Yifan Yuan ◽  
Katherine L. Leiby ◽  
Allison M. Greaney ◽  
Micha Sam Brickman Raredon ◽  
Hong Qian ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Vascular Diseases ◽  
Induced Pluripotent Stem Cell ◽  
Sequencing Analysis ◽  
Conventional Culture ◽  
Bioreactor System ◽  
Whole Organ Engineering ◽  
Induced Pluripotent ◽  
The Impact

The development of an in vitro system for the study of lung vascular disease is critical to understanding human pathologies. Conventional culture systems fail to fully recapitulate native microenvironmental conditions and are typically limited in their ability to represent human pathophysiology for the study of disease and drug mechanisms. Whole organ decellularization provides a means to developing a construct that recapitulates structural, mechanical, and biological features of a complete vascular structure. Here, we developed a culture protocol to improve endothelial cell coverage in whole lung scaffolds and used single-cell RNA-sequencing analysis to explore the impact of decellularized whole lung scaffolds on endothelial phenotypes and functions in a biomimetic bioreactor system. Intriguingly, we found that the phenotype and functional signals of primary pulmonary microvascular revert back—at least partially—toward native lung endothelium. Additionally, human induced pluripotent stem cell-derived endothelium cultured in decellularized lung systems start to gain various native human endothelial phenotypes. Vascular barrier function was partially restored, while small capillaries remained patent in endothelial cell-repopulated lungs. To evaluate the ability of the engineered endothelium to modulate permeability in response to exogenous stimuli, lipopolysaccharide (LPS) was introduced into repopulated lungs to simulate acute lung injury. After LPS treatment, proinflammatory signals were significantly increased and the vascular barrier was impaired. Taken together, these results demonstrate a novel platform that recapitulates some pulmonary microvascular functions and phenotypes at a whole organ level. This development may help pave the way for using the whole organ engineering approach to model vascular diseases.

scholarly journals C5a Activates a Pro-Inflammatory Gene Expression Profile in Human Gaucher iPSC-Derived Macrophages

2021 ◽  
Vol 22 (18) ◽  
pp. 9912
Author(s):  
Jacquelyn C. Serfecz ◽  
Afsoon Saadin ◽  
Clayton P. Santiago ◽  
Yuji Zhang ◽  
Søren M. Bentzen ◽  
...  
Keyword(s):  
Immune Response ◽  
Inflammatory Responses ◽  
Gene Array ◽  
Induced Pluripotent Stem Cell ◽  
Autosomal Recessive Disorder ◽  
Novel Therapy ◽  
Complement Inhibitors ◽  
Expression Of Genes ◽  
Tnf Α ◽  
Induced Pluripotent

Gaucher disease (GD) is an autosomal recessive disorder caused by bi-allelic GBA1 mutations that reduce the activity of the lysosomal enzyme β-glucocerebrosidase (GCase). GCase catalyzes the conversion of glucosylceramide (GluCer), a ubiquitous glycosphingolipid, to glucose and ceramide. GCase deficiency causes the accumulation of GluCer and its metabolite glucosylsphingosine (GluSph) in a number of tissues and organs. In the immune system, GCase deficiency deregulates signal transduction events, resulting in an inflammatory environment. It is known that the complement system promotes inflammation, and complement inhibitors are currently being considered as a novel therapy for GD; however, the mechanism by which complement drives systemic macrophage-mediated inflammation remains incompletely understood. To help understand the mechanisms involved, we used human GD-induced pluripotent stem cell (iPSC)-derived macrophages. We found that GD macrophages exhibit exacerbated production of inflammatory cytokines via an innate immune response mediated by receptor 1 for complement component C5a (C5aR1). Quantitative RT-PCR and ELISA assays showed that in the presence of recombinant C5a (rC5a), GD macrophages secreted 8–10-fold higher levels of TNF-α compared to rC5a-stimulated control macrophages. PMX53, a C5aR1 blocker, reversed the enhanced GD macrophage TNF-α production, indicating that the observed effect was predominantly C5aR1-mediated. To further analyze the extent of changes induced by rC5a stimulation, we performed gene array analysis of the rC5a-treated macrophage transcriptomes. We found that rC5a-stimulated GD macrophages exhibit increased expression of genes involved in TNF-α inflammatory responses compared to rC5a-stimulated controls. Our results suggest that rC5a-induced inflammation in GD macrophages activates a unique immune response, supporting the potential use of inhibitors of the C5a-C5aR1 receptor axis to mitigate the chronic inflammatory abnormalities associated with GD.

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