scholarly journals 2518. Development And Characterization Of Human Microglial Models To Elucidate HIV Transmission Events And Pathogenesis

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S874-S875
Author(s):  
Mohammad Ali Rai ◽  
Jason E Hammonds ◽  
Paul Spearman ◽  
Paul Spearman
Keyword(s):  
Flow Cytometry ◽  
Hiv Infection ◽  
Cell Lines ◽  
Microglial Cell ◽  
Hiv Transmission ◽  
Cell Types ◽  
Model Systems ◽  
Cellular Events ◽  
The Central Nervous System ◽  
Induced Pluripotent

Abstract Background HIV-associated neurocognitive disorders cause significant morbidity and mortality despite the advent of antiretroviral therapy. An understanding of fundamental mechanisms underlying HIV infection and transmission events in the central nervous system (CNS) is needed. Microglia are resident myeloid cells that are readily infected by HIV and may constitute a CNS reservoir. We evaluated and compared existing microglial cell lines and primary cell-derived microglia as potential model systems for studying HIV-microglia interactions. Methods We cultured two immortalized human microglial lines (HMC3, C20) and developed two primary microglial models: induced microglia (iMG) derived from primary human monocytes; and microglial-like cells (iMGL) differentiated from induced pluripotent stem cells (iPSCs). We compared these four microglial cell types to commercially available fetal microglia (PM) for a microglial comparator, and monocyte-derived macrophages as a non-microglial comparator cell. Each cell type was evaluated for the presence of typical myeloid and microglia-specific markers by flow cytometry and immunofluorescence microscopy. HIV infection was performed using macrophage-tropic HIV or VSV-G-pseudotyped HIV. Results After differentiation, the iMG and iMGL displayed characteristic microglial morphology: a spindle shape and a reduction in the central body, along with ramified cell processes. Flow cytometry revealed significant differences in surface markers among the cell types. iMG and iMGL displayed CD11b, CD45, CXCR4, CCR5 and lack of expression of CD4 and CX3CR1. In contrast, HMC3, C20, and PM were negative for CD11b, CD45, CX3CR1, CD4, CXCR4. Immunostaining showed that iMG and iMGL were positive for microglial markers TMEM119 and P2RY12. RNA Seq analysis is currently underway to determine gene expression differences between the microglial cell lines and our microglia models. In preliminary results, iMG and iMGL were both readily infected with HIV, and comparison with other lines is ongoing. Conclusion There is no standard model available for defining the molecular and cellular events involved in HIV infection of microglia. Significant differences in microgial markers and in HIV receptor and coreceptor levels were noted in this study. iMG and iMGL appear to be viable microglial models susceptible to HIV infection. Disclosures All authors: No reported disclosures.

scholarly journals CellMap: Characterizing the types and composition of iPSC-derived cells from RNA-seq data

2021 ◽  
Author(s):  
Zhengyu Ouyang ◽  
Nathanael Bourgeois ◽  
Eugenia Lyashenko ◽  
Paige Cundiff ◽  
Patrick F Cullen ◽  
...  
Keyword(s):  
Single Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Model Systems ◽  
Rna Seq ◽  
Cell Type ◽  
Fine Grained ◽  
Single Nucleus ◽  
Induced Pluripotent

Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.

Investigation of the Stem Cells Used in Modeling Human Placental Trophoblast

2021 ◽  
Author(s):  
Lulu Ji ◽  
Lin Wang
Keyword(s):  
Stem Cells ◽  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Cell Types ◽  
Model Systems ◽  
Alternative Methods ◽  
Laboratory Animals ◽  
Placental Development ◽  
Induced Pluripotent

Human placenta is vital for fetal development, and act as an interface between the fetus and the expecting mother. Abnormal placentati on underpins various pregnancy complications such as miscarriage, pre-eclampsia and intrauterine growth restriction. Despite the important role of placenta, the molecular mechanisms governing placental formation and trophoblast cell lineage specification is poorly understand. It is mostly due to the lack of appropriate model system. The great various in placental types across mammals make it limit for the use of laboratory animals in studying human placental development. However, over the past few years, alternative methods have been employed, including human embryonic stem cells, induced pluripotent stem cells, human trophoblast stem cell, and 3-dimensional organoids. Herein, we summarize the present knowledge about human development, differentiated cell types in the trophoblast epithelium and current human placental trophoblast model systems.

scholarly journals Neuroinflammation and EIF2 signaling persist in an HiPSC tri-culture model of HIV infection despite antiretroviral treatment

2019 ◽  
Author(s):  
Sean K. Ryan ◽  
Michael V. Gonzalez ◽  
James P. Garifallou ◽  
Frederick C. Bennett ◽  
Kimberly S. Williams ◽  
...  
Keyword(s):  
Hiv Infection ◽  
Pluripotent Stem Cell ◽  
Antiretroviral Treatment ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Similar Response ◽  
Infection Treatment ◽  
Culture Model ◽  
Key Features ◽  
Induced Pluripotent

AbstractHIV-Associated Neurocognitive Disorders (HAND) affect over half of HIV-infected individuals worldwide, despite antiretroviral therapy (ART). Therapeutically targetable mechanisms underlying HAND remain elusive. We developed a human-induced pluripotent stem cell (HiPSC) based model; whereby, we independently differentiate HiPSCs into neurons, astrocytes, and microglia and systematically combine to generate a tri-culture with or without HIV-infection and ART. scRNAseq analysis on tri-cultures including HIV-infected microglia revealed inflammatory signatures in the microglia and EIF2 signaling in all three cell types. Remarkably, EFZ alone induced a similar response to infection. Treatment with the antiretroviral compound Efavirenz (EFZ) mostly resolved these signatures; However, EFZ increased RhoGDI and CD40 signaling in the HIV-infected microglia. This activation was associated with a persistent increase in TNFa expression. This work establishes a tri-culture that recapitulates key features of HIV infection in the CNS and provides a new model to examine the effects of HIV infection and its treatment with antiretrovirals.

scholarly journals Generation of a Panel of Induced Pluripotent Stem Cells From Chimpanzees: a Resource for Comparative Functional Genomics

2014 ◽  
Author(s):  
Irene Gallego Romero ◽  
Bryan J Pavlovic ◽  
Irene Hernando-Herraez ◽  
Nicholas E Banovich ◽  
Courtney L Kagan ◽  
...  
Keyword(s):  
Somatic Cells ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Model Systems ◽  
Sequencing Data ◽  
Differentiation Potential ◽  
Culture Techniques ◽  
Human Ipscs ◽  
Wide Range ◽  
Induced Pluripotent

Comparative genomics studies in primates are extremely restricted because we only have access to a few types of cell lines from non-human apes and to a limited collection of frozen tissues. In order to gain better insight into regulatory processes that underlie variation in complex phenotypes, we must have access to faithful model systems for a wide range of tissues and cell types. To facilitate this, we have generated a panel of 7 fully characterized chimpanzee (Pan troglodytes) induced pluripotent stem cell (iPSC) lines derived from fibroblasts of healthy donors. All lines appear to be free of integration from exogenous reprogramming vectors, can be maintained using standard iPSC culture techniques, and have proliferative and differentiation potential similar to human and mouse lines. To begin demonstrating the utility of comparative iPSC panels, we collected RNA sequencing data and methylation profiles from the chimpanzee iPSCs and their corresponding fibroblast precursors, as well as from 7 human iPSCs and their precursors, which were of multiple cell type and population origins. Overall, we observed much less regulatory variation within species in the iPSCs than in the somatic precursors, indicating that the reprogramming process has erased many of the differences observed between somatic cells of different origins. We identified 4,918 differentially expressed genes and 3,598 differentially methylated regions between iPSCs of the two species, many of which are novel inter-species differences that were not observed between the somatic cells of the two species. Our panel will help realise the potential of iPSCs in primate studies, and in combination with genomic technologies, transform studies of comparative evolution.

scholarly journals In Vitro and In Vivo Models to Study Nephropathic Cystinosis

Cells ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 6
Author(s):  
Pang Yuk Cheung ◽  
Patrick T. Harrison ◽  
Alan J. Davidson ◽  
Jennifer A. Hollywood
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Primary Cultures ◽  
Model Systems ◽  
In Vivo Model ◽  
Nephropathic Cystinosis ◽  
In Vivo Models ◽  
Induced Pluripotent

The development over the past 50 years of a variety of cell lines and animal models has provided valuable tools to understand the pathophysiology of nephropathic cystinosis. Primary cultures from patient biopsies have been instrumental in determining the primary cause of cystine accumulation in the lysosomes. Immortalised cell lines have been established using different gene constructs and have revealed a wealth of knowledge concerning the molecular mechanisms that underlie cystinosis. More recently, the generation of induced pluripotent stem cells, kidney organoids and tubuloids have helped bridge the gap between in vitro and in vivo model systems. The development of genetically modified mice and rats have made it possible to explore the cystinotic phenotype in an in vivo setting. All of these models have helped shape our understanding of cystinosis and have led to the conclusion that cystine accumulation is not the only pathology that needs targeting in this multisystemic disease. This review provides an overview of the in vitro and in vivo models available to study cystinosis, how well they recapitulate the disease phenotype, and their limitations.

scholarly journals The utilisation of organoids and macrophages derived from Human induced pluripotent stem cells as model systems to investigate host-bacterial interactions

2019 ◽  
Vol 1 (1A) ◽  
Author(s):  
Christine Hale ◽  
Leanne Kane ◽  
Matthew Dorman ◽  
Nicholas Thomson
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Genetically Engineered ◽  
Model Systems ◽  
Host Cells ◽  
Bacterial Interactions ◽  
Induced Pluripotent

Using human induced pluripotent stem cell (hiPSC) technology we are developing methods to examine host-bacterial interactions. Due to the fact that undifferentiated human induced pluripotent stem cells are amenable to genetic engineering, can be cultured indefinitely and can further be differentiated into multiple cell types, we are exploiting both organoid and macrophage systems to investigate the interactions between host cells and diarrhoeal pathogens, including enterotoxigenic Escherichia coli and Vibrio cholerae. Utilising both wild type and relevant knockout hiPSC lines we are probing both initial interactions and subsequent utilisation of pathways for the effects of toxins. The further analysis of genetically engineered bacteria extend the usefulness of this model system, and complement the availability of mutant host cells, towards the simultaneous genetic analysis of both pathogen and host.

scholarly journals Standards for Deriving Nonhuman Primate-Induced Pluripotent Stem Cells, Neural Stem Cells and Dopaminergic Lineage

2018 ◽  
Vol 19 (9) ◽  
pp. 2788 ◽  
Author(s):  
Guang Yang ◽  
Hyenjong Hong ◽  
April Torres ◽  
Kristen Malloy ◽  
Gourav Choudhury ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
In Vitro Models ◽  
The Body ◽  
Model Systems ◽  
Induced Pluripotent

Humans and nonhuman primates (NHP) are similar in behavior and in physiology, specifically the structure, function, and complexity of the immune system. Thus, NHP models are desirable for pathophysiology and pharmacology/toxicology studies. Furthermore, NHP-derived induced pluripotent stem cells (iPSCs) may enable transformative developmental, translational, or evolutionary studies in a field of inquiry currently hampered by the limited availability of research specimens. NHP-iPSCs may address specific questions that can be studied back and forth between in vitro cellular assays and in vivo experimentations, an investigational process that in most cases cannot be performed on humans because of safety and ethical issues. The use of NHP model systems and cell specific in vitro models is evolving with iPSC-based three-dimensional (3D) cell culture systems and organoids, which may offer reliable in vitro models and reduce the number of animals used in experimental research. IPSCs have the potential to give rise to defined cell types of any organ of the body. However, standards for deriving defined and validated NHP iPSCs are missing. Standards for deriving high-quality iPSC cell lines promote rigorous and replicable scientific research and likewise, validated cell lines reduce variability and discrepancies in results between laboratories. We have derived and validated NHP iPSC lines by confirming their pluripotency and propensity to differentiate into all three germ layers (ectoderm, mesoderm, and endoderm) according to standards and measurable limits for a set of marker genes. The iPSC lines were characterized for their potential to generate neural stem cells and to differentiate into dopaminergic neurons. These iPSC lines are available to the scientific community. NHP-iPSCs fulfill a unique niche in comparative genomics to understand gene regulatory principles underlying emergence of human traits, in infectious disease pathogenesis, in vaccine development, and in immunological barriers in regenerative medicine.

scholarly journals Establishment and Preliminary Characterization of Three Astrocytic Cells Lines Obtained from Primary Rat Astrocytes by Sub-Cloning

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1502
Author(s):  
Fabio Caradonna ◽  
Gabriella Schiera ◽  
Carlo Maria Di Liegro ◽  
Vincenzo Vitale ◽  
Ilenia Cruciata ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cell Types ◽  
Brain Parenchyma ◽  
Brain Cell ◽  
The Other ◽  
Cellular Events ◽  
Rat Astrocytes ◽  
The Brain ◽  
Brain Cell Types

Gliomas are complex and heterogeneous tumors that originate from the glial cells of the brain. The malignant cells undergo deep modifications of their metabolism, and acquire the capacity to invade the brain parenchyma and to induce epigenetic modifications in the other brain cell types. In spite of the efforts made to define the pathology at the molecular level, and to set novel approaches to reach the infiltrating cells, gliomas are still fatal. In order to gain a better knowledge of the cellular events that accompany astrocyte transformation, we developed three increasingly transformed astrocyte cell lines, starting from primary rat cortical astrocytes, and analyzed them at the cytogenetic and epigenetic level. In parallel, we also studied the expression of the differentiation-related H1.0 linker histone variant to evaluate its possible modification in relation with transformation. We found that the most modified astrocytes (A-FC6) have epigenetic and chromosomal alterations typical of cancer, and that the other two clones (A-GS1 and A-VV5) have intermediate properties. Surprisingly, the differentiation-specific somatic histone H1.0 steadily increases from the normal astrocytes to the most transformed ones. As a whole, our results suggest that these three cell lines, together with the starting primary cells, constitute a potential model for studying glioma development.

scholarly journals Reprogramming of EBV-immortalized B-lymphocyte cell lines into induced pluripotent stem cells

Blood ◽  
2011 ◽  
Vol 118 (7) ◽  
pp. 1801-1805 ◽  
Author(s):  
Su Mi Choi ◽  
Hua Liu ◽  
Pooja Chaudhari ◽  
Yonghak Kim ◽  
Linzhao Cheng ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
B Lymphocyte ◽  
Disease Modeling ◽  
Genetic Disorders ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Inherited Disease ◽  
Induced Pluripotent ◽  
Lymphocyte Cell

AbstractEBV-immortalized B lymphocyte cell lines have been widely banked for studying a variety of diseases, including rare genetic disorders. These cell lines represent an important resource for disease modeling with the induced pluripotent stem cell (iPSC) technology. Here we report the generation of iPSCs from EBV-immortalized B-cell lines derived from multiple inherited disease patients via a nonviral method. The reprogramming method for the EBV cell lines involves a distinct protocol compared with that of patient fibroblasts. The B-cell line–derived iPSCs expressed pluripotency markers, retained the inherited mutation and the parental V(D)J rearrangement profile, and differentiated into all 3 germ layer cell types. There was no integration of the reprogramming-related transgenes or the EBV-associated genes in these iPSCs. The ability to reprogram the widely banked patient B-cell lines will offer an unprecedented opportunity to generate human disease models and provide novel drug therapies.

scholarly journals Glutaminase isoform expression in cell lines derived from human colorectal adenomas and carcinomas

2003 ◽  
Vol 370 (2) ◽  
pp. 403-408 ◽  
Author(s):  
Abigail TURNER ◽  
John D. McGIVAN
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Carcinoma Cell ◽  
Cell Types ◽  
Colorectal Adenomas ◽  
Model Systems ◽  
Mrna Species ◽  
Ht29 Cells ◽  
Isoform Expression ◽  
Glutaminase Activity

This paper describes some properties of glutamine oxidation and glutaminase isoform expression in cell lines derived from human colorectal adenomas and carcinomas. The slow-growing adenoma-derived cell line AA/C1, and the rapidly proliferating carcinoma cell line HT29, both required glutamine for growth. The rate of 14CO2 production from [U-14C]glutamine was faster in AA/C1 cells than in HT29 cells. Conversely HT29 cells showed faster rates of glucose oxidation and lactate production. The activity of glutaminase was 3 times higher in AA/C1 cell extracts than in extracts of HT29 cells. Glutaminase activity in the two cell lines had similar Km values for glutamine, but the activity in AA/C1 cells had a higher K0.5 for activation by phosphate. Glutaminase activity in extracts of both cells was inhibited by glutamate. Western blotting showed the presence, in both cell lines, of isoform(s) of glutaminase with an molecular mass of 63kDa, intermediate between that of kidney glutaminase and liver glutaminase. PCR-based analysis showed that an mRNA species identical to the kidney-type isoform glutaminase C was present in both cell types as was an additional mRNA species identical to the liver-type glutaminase isoform from human breast tumour cells. Northern blotting using isoform-specific cDNA probes demonstrated that mRNA for both glutaminase isoforms was expressed at significant levels in both cell types. Similar results to those in AA/C1 cells and HT29 cells were obtained in two further adenoma and carcinoma cell lines respectively. These results contrast with those reported previously in hepatocyte/hepatoma model systems with respect to fuel selection, glutaminase activity and isoform expression. They also constitute the first demonstration of simultaneous expression of two glutaminase isoforms in a single cell type.

Sign in / Sign up

Export Citation Format

Share Document