scholarly journals Effect of Passage Number on Human Induced Pluripotent Stem Cell Derived Neurons Culture Contaminants with Non-Neuronal Cell Types

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Dominique Ketsoglou ◽  
Erica Cantor ◽  
Guanglong Jiang ◽  
Xi Wu ◽  
Santosh Phillips ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Neuronal Cell ◽  
Cell Types ◽  
Cell Marker ◽  
Passage Number ◽  
Marker Expression ◽  
Induced Pluripotent ◽  
The Impact ◽  
Glial Precursor

Background: The study of live human neurons has been hindered due to the complexity and potential irreversible damage to the patient during biopsy. However, reprogramming of adult human somatic cells into induced pluripotent stem cells (iPSCs) has proved to be a novel method in the study of the pathophysiology of disease and therapeutic targets of the human nervous system. There are several approaches, and the optimum time (i.e., passage number) to generate highly pure cultures is being studied. Therefore, our laboratory has investigated the effect of passage number on culture contaminants with non-neuronal cell types.     Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood from three cell lines and reprogrammed into iPSCs. Each cell line consisted of three samples that were analyzed after low (5-10), middle (20-26), and high (30-38) number of passages. Cells were maintained in an induction medium for eight days. On day nine, cells were dissociated and replated in a maintenance medium. On day 33, total RNA was extracted from cells. Normalized values for non-neuronal cell marker genes were compared using paired Student’s t-tests and two-way ANOVA, with the cell line and passage number as independent variables. P-values less than 0.05 were considered significant.     Results: Our results showed that lower passage number was associated with decreased astrocyte and chondrocyte marker expression. High passage number was associated with decreased oligodendrocyte and glial precursor marker expression. Of the fibroblast markers evaluated, there were similar trends of expression between all three groups. There was no significant difference in microglial cell marker gene expression between all three groups.     Conclusion and Potential Impact: Low gene expression suggests a purer culture. According to these results, as passage number increases, there is more contaminants with oligodendrocytes and glial precursor cells. Conversely, with low passage numbers, there are more contaminants with astrocytes and chondrocytes. Future studies will identify the impact of these non-neuronal contaminants and implications on research.  

Uncovering the Diversification of Tissue Engineering on the Emergent Areas of Stem Cells, Nanotechnology and Biomaterials

2020 ◽  
Vol 15 (3) ◽  
pp. 187-201 ◽  
Author(s):  
Sunil K. Dubey ◽  
Amit Alexander ◽  
Munnangi Sivaram ◽  
Mukta Agrawal ◽  
Gautam Singhvi ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Immune System ◽  
Clinical Application ◽  
Cell Types ◽  
Patient Specific ◽  
Potential Strategy ◽  
Induced Pluripotent ◽  
Treat All ◽  
The Impact

Damaged or disabled tissue is life-threatening due to the lack of proper treatment. Many conventional transplantation methods like autograft, iso-graft and allograft are in existence for ages, but they are not sufficient to treat all types of tissue or organ damages. Stem cells, with their unique capabilities like self-renewal and differentiate into various cell types, can be a potential strategy for tissue regeneration. However, the challenges like reproducibility, uncontrolled propagation and differentiation, isolation of specific kinds of cell and tumorigenic nature made these stem cells away from clinical application. Today, various types of stem cells like embryonic, fetal or gestational tissue, mesenchymal and induced-pluripotent stem cells are under investigation for their clinical application. Tissue engineering helps in configuring the stem cells to develop into a desired viable tissue, to use them clinically as a substitute for the conventional method. The use of stem cell-derived Extracellular Vesicles (EVs) is being studied to replace the stem cells, which decreases the immunological complications associated with the direct administration of stem cells. Tissue engineering also investigates various biomaterials to use clinically, either to replace the bones or as a scaffold to support the growth of stemcells/ tissue. Depending upon the need, there are various biomaterials like bio-ceramics, natural and synthetic biodegradable polymers to support replacement or regeneration of tissue. Like the other fields of science, tissue engineering is also incorporating the nanotechnology to develop nano-scaffolds to provide and support the growth of stem cells with an environment mimicking the Extracellular matrix (ECM) of the desired tissue. Tissue engineering is also used in the modulation of the immune system by using patient-specific Mesenchymal Stem Cells (MSCs) and by modifying the physical features of scaffolds that may provoke the immune system. This review describes the use of various stem cells, biomaterials and the impact of nanotechnology in regenerative medicine.

scholarly journals Optimizing expression quantitative trait locus mapping workflows for single-cell studies

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Anna S. E. Cuomo ◽  
Giordano Alvari ◽  
Christina B. Azodi ◽  
Davis J. McCarthy ◽  
Marc Jan Bonder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quantitative Trait Locus ◽  
Single Cell ◽  
Quantitative Trait ◽  
Cell Types ◽  
Expression Quantitative Trait Locus ◽  
Eqtl Mapping ◽  
Trait Locus ◽  
The Impact

Abstract Background Single-cell RNA sequencing (scRNA-seq) has enabled the unbiased, high-throughput quantification of gene expression specific to cell types and states. With the cost of scRNA-seq decreasing and techniques for sample multiplexing improving, population-scale scRNA-seq, and thus single-cell expression quantitative trait locus (sc-eQTL) mapping, is increasingly feasible. Mapping of sc-eQTL provides additional resolution to study the regulatory role of common genetic variants on gene expression across a plethora of cell types and states and promises to improve our understanding of genetic regulation across tissues in both health and disease. Results While previously established methods for bulk eQTL mapping can, in principle, be applied to sc-eQTL mapping, there are a number of open questions about how best to process scRNA-seq data and adapt bulk methods to optimize sc-eQTL mapping. Here, we evaluate the role of different normalization and aggregation strategies, covariate adjustment techniques, and multiple testing correction methods to establish best practice guidelines. We use both real and simulated datasets across single-cell technologies to systematically assess the impact of these different statistical approaches. Conclusion We provide recommendations for future single-cell eQTL studies that can yield up to twice as many eQTL discoveries as default approaches ported from bulk studies.

scholarly journals Effects of taurolithocholate, a Ca2+-mobilizing agent, on cell Ca2+ in rat hepatocytes, human platelets and neuroblastoma NG108-15 cell line

1991 ◽  
Vol 273 (1) ◽  
pp. 153-160 ◽  
Author(s):  
J F Coquil ◽  
B Berthon ◽  
N Chomiki ◽  
L Combettes ◽  
P Jourdon ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Bile Acid ◽  
Cell Line ◽  
Rat Liver ◽  
Neuronal Cell ◽  
Rat Hepatocytes ◽  
Cell Types ◽  
Liver Cells ◽  
Human Platelets ◽  
Rat Liver Cells

The monohydroxy bile acid taurolithocholate permeabilizes the endoplasmic reticulum to Ca2+ in rat liver cells. To assess whether this action on the endoplasmic reticulum was restricted to this tissue, the effects of bile acid were investigated in two cell types quite unrelated to rat hepatocyte, namely human platelets and neuronal NG108-15 cell line. The results showed that taurolithocholate (3-100 microM) had no effect on free cytosolic [Ca2+] in human platelets and NG108-15 cells. whereas it increased it from 180 to 520 nM in rat hepatocytes. In contrast, in cells permeabilized by saponin, taurolithocholate initiated a profound release of the stored Ca2+ from the internal Ca2+ pools in the three cell types. The bile acid released 90% of the Ca2+ pools, with rate constants of about 5 min-1 and half-maximal effects at 15-30 microM. The results also showed that, in contrast with liver cells, which displayed an influx of [14C]taurolithocholate of 2 nmol/min per mg, human platelets and the neuronal cell line appeared to be resistant to [14C]taurolithocholate uptake. The influx measured in these latter cells was about 100-fold lower than in rat liver cells. Taken together, these data suggest that human platelets and NG108-15 cells do not possess the transport system for concentrating monohydroxy bile acids into cells. However, they show that human platelets and neuronal NG108-15 possess, in common with liver cells, the intracellular system responsible for taurolithocholate-mediated Ca2+ release from internal stores.

scholarly journals Differential SOD2 and GSTZ1 profiles contribute to contrasting dental pulp stem cell susceptibilities to oxidative damage and premature senescence

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nadia Y. A. Alaidaroos ◽  
Amr Alraies ◽  
Rachel J. Waddington ◽  
Alastair J. Sloan ◽  
Ryan Moseley
Keyword(s):  
Stem Cell ◽  
Regenerative Medicine ◽  
Oxidative Damage ◽  
Dental Pulp ◽  
Stem Cell Marker ◽  
Significant Heterogeneity ◽  
Premature Senescence ◽  
Cell Marker ◽  
Marker Expression ◽  
The Impact

Abstract Background Dental pulp stem cells (DPSCs) are increasingly being advocated as viable cell sources for regenerative medicine-based therapies. However, significant heterogeneity in DPSC expansion and multi-potency capabilities are well-established, attributed to contrasting telomere profiles and susceptibilities to replicative senescence. As DPSCs possess negligible human telomerase (hTERT) expression, we examined whether intrinsic differences in the susceptibilities of DPSC sub-populations to oxidative stress-induced biomolecular damage and premature senescence further contributed to this heterogeneity, via differential enzymic antioxidant capabilities between DPSCs. Methods DPSCs were isolated from human third molars by differential fibronectin adhesion, and positive mesenchymal (CD73/CD90/CD105) and negative hematopoietic (CD45) stem cell marker expression confirmed. Isolated sub-populations were expanded in H2O2 (0–200 μM) and established as high or low proliferative DPSCs, based on population doublings (PDs) and senescence (telomere lengths, SA-β-galactosidase, p53/p16INK4a/p21waf1/hTERT) marker detection. The impact of DPSC expansion on mesenchymal, embryonic, and neural crest marker expression was assessed, as were the susceptibilities of high and low proliferative DPSCs to oxidative DNA and protein damage by immunocytochemistry. Expression profiles for superoxide dismutases (SODs), catalase, and glutathione-related antioxidants were further compared between DPSC sub-populations by qRT-PCR, Western blotting and activity assays. Results High proliferative DPSCs underwent > 80PDs in culture and resisted H2O2−induced senescence (50–76PDs). In contrast, low proliferative sub-populations exhibited accelerated senescence (4–32PDs), even in untreated controls (11-34PDs). While telomere lengths were largely unaffected, certain stem cell marker expression declined with H2O2 treatment and expansion. Elevated senescence susceptibilities in low proliferative DPSC (2–10PDs) were accompanied by increased oxidative damage, absent in high proliferative DPSCs until 45–60PDs. Increased SOD2/glutathione S-transferase ζ1 (GSTZ1) expression and SOD activities were identified in high proliferative DPSCs (10–25PDs), which declined during expansion. Low proliferative DPSCs (2–10PDs) exhibited inferior SOD, catalase and glutathione-related antioxidant expression/activities. Conclusions Significant variations exist in the susceptibilities of DPSC sub-populations to oxidative damage and premature senescence, contributed to by differential SOD2 and GSTZ1 profiles which maintain senescence-resistance/stemness properties in high proliferative DPSCs. Identification of superior antioxidant properties in high proliferative DPSCs enhances our understanding of DPSC biology and senescence, which may be exploited for selective sub-population screening/isolation from dental pulp tissues for regenerative medicine-based applications.

scholarly journals Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells Provide Insights on Parenteral Nutrition Associated Cholestasis in the Immature Liver

2021 ◽  
Author(s):  
T. Hang Nghiem-Rao ◽  
Courtney Pfeifer ◽  
Michelle Asuncion ◽  
Joshua Nord ◽  
Daniel Schill ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Bile Acid ◽  
Parenteral Nutrition ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Computational Techniques ◽  
Adult Liver ◽  
Induced Pluripotent ◽  
The Impact

Abstract Parenteral nutrition-associated cholestasis (PNAC) significantly limits the safety of intravenous parenteral nutrition (PN). Critically ill infants are highly vulnerable to PNAC-related morbidity and mortality, however the impact of hepatic immaturity on PNAC is poorly understood. We examined developmental differences between fetal/infant and adult livers, and used human induced pluripotent stem cell-derived hepatocyte-like cells (iHLC) to gain insights into the contribution of development to altered sterol metabolism and PNAC. We used RNA-sequencing and computational techniques to compare gene expression patterns in human fetal/infant livers, adult liver, and iHLC. We identified distinct gene expression profiles between the human feta/infant livers compared to adult liver, and close resemblance of iHLC to human developing livers. Compared to adult, both developing livers and iHLC had significant downregulation of xenobiotic, bile acid, and fatty acid metabolism; and lower expression of the sterol metabolizing gene ABCG8. When challenged with stigmasterol, a plant sterol found in intravenous soy lipids, lipid accumulation was significantly higher in iHLC compared to adult-derived HepG2 cells. Our findings provide insights into altered bile acid and lipid metabolizing processes in the immature human liver, and support the use of iHLC as a relevant model system of developing liver to study lipid metabolism and PNAC.

scholarly journals Timing and Duration of Gbx2 Expression Delineates Thalamocortical and Dopaminergic Medial Forebrain Bundle Circuitry

2019 ◽  
Author(s):  
Elizabeth Normand ◽  
Catherine Browning ◽  
Mark Zervas
Keyword(s):  
Gene Expression ◽  
Medial Forebrain Bundle ◽  
Neural Circuit ◽  
Temporal Dynamics ◽  
Neuronal Cell ◽  
Cell Types ◽  
Dopamine Neurons ◽  
Genetic Lineage ◽  
Thalamocortical Axons ◽  
Circuit Development

SUMMARYGene expression is a dynamic process, which is highly coordinated during development to ensure the proper allocation and identity of neuronal cell types within the brain. Equally important during neurodevelopment is how cohorts of neurons establish axonal projections that innervate terminal target sites. We sought to bridge the temporal dynamics of gene expression, within a specific genetic lineage, to the establishment of neuronal circuits derived from cohorts of the lineage-specific progenitors. A central goal was to be able to accomplish genetic inducible circuit mapping non-invasively and with commonly available CreER/loxP technology. Specifically, we genetically marked thalamic neuron progenitors that expressed the transcription factor Gbx2 at an early embryonic stage and tracked the formation of lineage-derived thalamocortical axons during embryogenesis. We then assessed the neural circuitry at an early postnatal stage. We show that the temporal specificity of lineage marking provides a high degree of clarity for following neural circuit development. We also determined that the onset and duration of gene expression can delineate subsets of neural circuits derived from a common lineage. For example, we uncovered a novel contribution of Gbx2-expressing progenitors to midbrain dopamine neurons and dopaminergic axons of the medial forebrain bundle. We anticipate that this system can be instructive in elucidating changes in neural circuit development in both normal development and in mutant mice in which neural circuit formation is altered.

scholarly journals Optimising expression quantitative trait locus mapping workflows for single-cell studies

2021 ◽  
Author(s):  
Anna S.E. Cuomo ◽  
Giordano Alvari ◽  
Christina B. Azodi ◽  
Davis J. McCarthy ◽  
Marc Jan Bonder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quantitative Trait Locus ◽  
Single Cell ◽  
Quantitative Trait ◽  
Cell Types ◽  
Expression Quantitative Trait Locus ◽  
Eqtl Mapping ◽  
Trait Locus ◽  
The Impact

AbstractSingle-cell RNA-sequencing (scRNA-seq) has enabled the unbiased, high-throughput quantification of gene expression specific to cell types and states. With the cost of scRNA-seq decreasing and techniques for sample multiplexing improving, population-scale scRNA-seq, and thus single-cell expression quantitative trait locus (sc-eQTL) mapping, is increasingly feasible. Mapping of sc-eQTL provides additional resolution to study the regulatory role of common genetic variants on gene expression across a plethora of cell types and states, and promises to improve our understanding of genetic regulation across tissues in both health and disease. While previously established methods for bulk eQTL mapping can, in principle, be applied to sc-eQTL mapping, there are a number of open questions about how best to process scRNA-seq data and adapt bulk methods to optimise sc-eQTL mapping. Here, we evaluate the role of different normalisation and aggregation strategies, covariate adjustment techniques, and multiple testing correction methods to establish best practice guidelines. We use both real and simulated datasets across single-cell technologies to systematically assess the impact of these different statistical approaches and provide recommendations for future single-cell eQTL studies that can yield up to twice as many eQTL discoveries as default approaches ported from bulk studies.

scholarly journals Maternal Overnutrition Programs Central Inflammation and Addiction-Like Behavior in Offspring

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Larisa Montalvo-Martínez ◽  
Roger Maldonado-Ruiz ◽  
Marcela Cárdenas-Tueme ◽  
Diana Reséndez-Pérez ◽  
Alberto Camacho
Keyword(s):  
Gene Expression ◽  
Fetal Programming ◽  
Gene Expression Pattern ◽  
Cell Types ◽  
Addictive Behavior ◽  
Adaptive Responses ◽  
Inflammatory Phenotype ◽  
Pregnancy And Lactation ◽  
Maternal Overnutrition ◽  
The Impact

Obesity or maternal overnutrition during pregnancy and lactation might have long-term consequences in offspring health. Fetal programming is characterized by adaptive responses to specific environmental conditions during early life stages. Programming alters gene expression through epigenetic modifications leading to a transgenerational effect of behavioral phenotypes in the offspring. Maternal intake of hypercaloric diets during fetal development programs aberrant behaviors resembling addiction in offspring. Programming by hypercaloric surplus sets a gene expression pattern modulating axonal pruning, synaptic signaling, and synaptic plasticity in selective regions of the reward system. Likewise, fetal programming can promote an inflammatory phenotype in peripheral and central sites through different cell types such as microglia and T and B cells, which contribute to disrupted energy sensing and behavioral pathways. The molecular mechanism that regulates the central and peripheral immune cross-talk during fetal programming and its relevance on offspring’s addictive behavior susceptibility is still unclear. Here, we review the most relevant scientific reports about the impact of hypercaloric nutritional fetal programming on central and peripheral inflammation and its effects on addictive behavior of the offspring.

Inducible cAMP early repressor suppresses gastrin-mediated activation of cyclin D1 and c-fos gene expression

2007 ◽  
Vol 292 (4) ◽  
pp. G1062-G1069 ◽  
Author(s):  
Tonje S. Steigedal ◽  
Torunn Bruland ◽  
Kristine Misund ◽  
Liv Thommesen ◽  
Astrid Lægreid
Keyword(s):  
Gene Expression ◽  
Cyclin D1 ◽  
Cell Line ◽  
Transcriptional Activation ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Neuroendocrine Cells ◽  
Cell Cycle Gene ◽  
Pancreatic Cell ◽  
Inducible Camp Early Repressor

The gastric hormone gastrin and its precursors promote proliferation in several gastrointestinal cell types. Here we show that gastrin induces transcription of cell cycle gene cyclin D1 and protooncogene c- fos in the neuroendocrine pancreatic cell line AR42J and that this gastrin response is inhibited by endogenous inducible cAMP early repressor (ICER). The transcriptional repressor ICER is known to downregulate both its own expression and the expression of other genes containing cAMP-responsive elements (CREs). Using siRNA, we also show that CRE promoter elements are the targets of endogenous ICER in AR42J cells as well as in the neuroendocrine cell line RIN5F. Our results suggest that ICER plays an important role in molecular mechanisms governing gastrin-mediated growth by modulating gastrin's transcriptional activation of growth-related genes. Our finding that ICER modulates pituitary adenylate cyclase-activating polypeptide-activated gene expression also indicates a regulatory effect of ICER in the responses of neuroendocrine cells to peptides other than gastrin.

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