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Stem Cell Research & Therapy May 2024Induced pluripotent stem cells (iPSCs)-derived kidney organoids are a promising model for studying disease mechanisms and renal development. Despite several protocols...
BACKGROUND
Induced pluripotent stem cells (iPSCs)-derived kidney organoids are a promising model for studying disease mechanisms and renal development. Despite several protocols having been developed, further improvements are needed to overcome existing limitations and enable a wider application of this model. One of the approaches to improve the differentiation of renal organoids in vitro is to include in the system cell types important for kidney organogenesis in vivo, such as macrophages. Another approach could be to improve cell survival. Mesodermal lineage differentiation is the common initial step of the reported protocols. The glycogen synthase kinase-3 (GSK-3) activity inhibitor, CHIR99021 (CHIR), is applied to induce mesodermal differentiation. It has been reported that CHIR simultaneously induces iPSCs apoptosis that can compromise cell differentiation. We thought to interfere with CHIR-induced apoptosis of iPSCs using rapamycin.
METHODS
Differentiation of kidney organoids from human iPSCs was performed. Cell survival and autophagy were analyzed using Cell counting kit 8 (CCK8) kit and Autophagy detection kit. Cells were treated with rapamycin or co-cultured with human monocytes isolated from peripheral blood or iPSCs-macrophages using a transwell co-culture system. Monocyte-derived extracellular vesicles (EVs) were isolated using polyethylene glycol precipitation. Expression of apoptotic markers cleaved Caspase 3, Poly [ADP-ribose] polymerase 1 (PARP-1) and markers of differentiation T-Box Transcription Factor 6 (TBX6), odd-skipped related 1 (OSR1), Nephrin, E-Cadherin, Paired box gene 2 (Pax2) and GATA Binding Protein 3 (Gata3) was assessed by RT-PCR and western blotting. Organoids were imaged by 3D-confocal microscopy.
RESULTS
We observed that CHIR induced apoptosis of iPSCs during the initial stage of renal organoid differentiation. Underlying mechanisms implied the accumulation of reactive oxygen species and decreased autophagy. Activation of autophagy by rapamacin and by an indirect co-culture of differentiating iPSCs with iPSCs-macrophages and human peripheral blood monocytes prevented apoptosis induced by CHIR. Furthermore, monocytes (but not rapamycin) strongly promoted expression of renal differentiation markers and organoids development via released extracellular vesicles.
CONCLUSION
Our data suggest that co-culturing of iPSCs with human monocytes strongly improves differentiation of kidney organoids. An underlying mechanism of monocytic action implies, but not limited to, an increased autophagy in CHIR-treated iPSCs. Our findings enhance the utility of kidney organoid models.
Topics: Humans; Induced Pluripotent Stem Cells; Organoids; Apoptosis; Cell Differentiation; Kidney; Monocytes; Pyridines; Pyrimidines; Sirolimus; Autophagy; Coculture Techniques; Macrophages
PubMed: 38702808
DOI: 10.1186/s13287-024-03739-8 -
Journal of Neuroinflammation Sep 2017Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune inflammatory disease of the central nervous system that preferentially affects the optic nerves, spinal...
BACKGROUND
Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune inflammatory disease of the central nervous system that preferentially affects the optic nerves, spinal cord, and area postrema. A series of evidence suggested that B cells play a fundamental role in the pathogenesis of NMOSD. However, there are still gaps left to be answered in NMOSD pathogenesis suggesting the roles of other immune cells. This study aimed to investigate the monocyte inflammatory characteristics, monocyte subset frequency and cytokine production, and cell-surface molecule expression in NMOSD, multiple sclerosis (MS), and healthy controls (HC).
METHODS
Peripheral blood mononuclear cells of 20 aquaporin 4IgG-positive NMOSD patients, 20 MS patients, and 20 healthy controls were collected to analyze the monocyte subsets and to purify monocytes. To mimic the adaptive immunity, we have activated the monocytes using CD40L and IFN-γ to observe the production of cytokines and expression of cell-surface molecules.
RESULTS
NMOSD monocytes showed a remarkable increase in the production of pro-inflammatory cytokines (IL-6, IL-1β) and increased expression of cell-surface molecules (CD80, HLA, ICAM-1, CD16), as well as a decrease in the levels of anti-inflammatory cytokine IL-10, compared to healthy control (HC) monocytes. As expected, MS monocytes also exhibit increased inflammatory cytokine production and increased cell-surface molecule expression compared to HC monocytes. Further analysis of monocyte subsets revealed that NMOSD monocytes have an increased frequency of the non-classical monocyte subset (CD14CD16) and a decreased frequency of the classical monocyte subset (CD14CD16) compared to HC monocytes. This finding was distinctly different from that of MS monocytes, which had an increased intermediate monocyte (CD14CD16) subset. In addition, these NMOSD non-classical monocyte subsets were highly dedicated, IL-6-producing monocytes.
CONCLUSIONS
Increased expression of cell-surface molecules and a reciprocal dysregulation of inflammatory and anti-inflammatory cytokines in NMOSD monocytes suggest an altered monocyte inflammatory response. CD14CD16 non-classical monocyte subset was more abundant in NMOSD monocytes than in HC or MS monocytes, and NMOSD non-classical monocyte subset had dysregulated IL-6 production, a phenotype which has been reported to be highly associated with NMOSD pathogenesis.
Topics: Adult; Female; Humans; Interleukin-6; Male; Middle Aged; Monocytes; Neuromyelitis Optica; Young Adult
PubMed: 28946890
DOI: 10.1186/s12974-017-0961-z -
International Journal of Molecular... Sep 2018Monocytes/macrophages are important in orchestrating inflammatory responses. However, knowledge of the long noncoding RNA (lncRNA) regulation of monocytic cell...
Monocytes/macrophages are important in orchestrating inflammatory responses. However, knowledge of the long noncoding RNA (lncRNA) regulation of monocytic cell differentiation and diseases remains limited. We aimed to elucidate the role of the 17 kb lncRNA noncoding transcript in T cells () in monocyte functions. Knockdown and chromatin immunoprecipitation (ChIP) assays in THP-1 cells (human monocytic leukemia cell line) revealed that is regulated by the monocyte key transcription factor C/EBPβ and that it binds to the promoter of nearby gene via hnRNP-U. Overexpression of in THP-1 cells resulted in cell cycle G1 arrest, differentiation into macrophages, a marked increase in and mRNA levels, and upregulation of the costimulatory molecules. In contrast to the downregulated observed in lipopolysaccharide (LPS)-treated THP-1 cells, the axis was found to be hyperactivated in peripheral blood mononuclear cells (PBMCs) of first-time diagnosed untreated early rheumatoid arthritis (RA) patients, and their gene expression levels decreased markedly after treatment. Higher initial expression levels were associated with a trend of higher disease activity DAS28 scores. In conclusion, our study suggests that the lncRNA is a regulator of inflammation in monocytes, and its activation participates in monocyte/macrophage differentiation and the pathogenesis of RA.
Topics: Adult; Aged; Arthritis, Rheumatoid; Cell Cycle Checkpoints; Cell Differentiation; Cells, Cultured; Down-Regulation; Female; Humans; Inflammation; Macrophages; Male; Middle Aged; Monocytes; Neoplasm Proteins; RNA, Long Noncoding; Up-Regulation
PubMed: 30231487
DOI: 10.3390/ijms19092806 -
Frontiers in Immunology 2021Monocytes are the third most frequent type of leukocytes in humans, linking innate and adaptive immunity and are critical drivers in many inflammatory diseases. Based on...
Monocytes are the third most frequent type of leukocytes in humans, linking innate and adaptive immunity and are critical drivers in many inflammatory diseases. Based on the differential expression of surface antigens, three monocytic subpopulations have been suggested in humans and two in rats with varying inflammatory and phenotype characteristics. Potential intervention strategies that aim to manipulate these cells require an in-depth understanding of monocyte behavior under different conditions. However, monocytes are highly sensitive to their specific activation state and expression of surface markers, which can change during cell isolation and purification. Thus, there is an urgent need for an unbiased functional analysis of activation in monocyte subtypes, which is not affected by the isolation procedure. Here, we present a flow cytometry-based protocol for evaluating subset-specific activation and cytokine expression of circulating blood monocytes both in humans and rats using small whole blood samples (50 - 100 μL). In contrast to previously described monocyte isolation and flow cytometry visualization methods, the presented approach virtually leaves monocyte subsets in a resting state or fixes them in their current state and allows for an unbiased functional endpoint analysis without prior cell isolation. This protocol is a comprehensive tool for studying differential monocyte regulation in the inflammatory and allogeneic immune response and .
Topics: Adult; Animals; Cytokines; Female; Flow Cytometry; Humans; Male; Monocytes; Rats; Rats, Wistar
PubMed: 33981302
DOI: 10.3389/fimmu.2021.641224 -
Journal of Biomechanics Feb 2017The role of cholesterol content on monocyte biomechanics remains understudied despite the well-established link between cholesterol and monocytes/macrophages in...
The role of cholesterol content on monocyte biomechanics remains understudied despite the well-established link between cholesterol and monocytes/macrophages in atherosclerosis, and the effect on other cell types. In this work, we have investigated the effect of cholesterol on monocyte deformability and the underlying molecular mechanisms. We altered the baseline cholesterol in human monocytic cell line THP-1, and investigated the changes in monocyte deformability using a custom microfluidic platform and atomic force microscopy. We observed that the cholesterol depletion lowered deformability while enrichment increased deformability compared to untreated cells. As a consequence of altered deformability, cholesterol depleted cells spread more on collagen-coated surfaces with elongated morphology, whereas cholesterol enriched cells had a more rounded morphology. We observed that the decreased deformability in cholesterol depleted cells, despite an increase in the fluidity of the membrane, is due to an increase in phosphorylation of Protein Kinase C (PKC), which translates to a higher degree of actin polymerization. Together, our results highlight the importance of biophysical regulation of monocyte response to cholesterol levels.
Topics: Biomechanical Phenomena; Cholesterol; Humans; Mechanical Phenomena; Membrane Fluidity; Monocytes; Phosphorylation; Protein Kinase C
PubMed: 28082022
DOI: 10.1016/j.jbiomech.2016.12.033 -
Journal of Thrombosis and Haemostasis :... Nov 2011Microparticles (MPs) are sub-micron vesicles shed by activated or apoptotic cells, including platelets and monocytes. Increased circulating MPs are associated with...
BACKGROUND
Microparticles (MPs) are sub-micron vesicles shed by activated or apoptotic cells, including platelets and monocytes. Increased circulating MPs are associated with thrombosis; however, their role in thrombogenesis is poorly understood.
OBJECTIVE
To determine how MPs promote thrombin generation and modulate fibrin density and stability.
METHODS
Platelets and monocytes were isolated from healthy donors. Platelets were stimulated with calcium ionophore, thrombin receptor agonist peptide (TRAP) or TRAP/convulxin. Monocytes and human monocytic THP-1 cells were stimulated with lipopolysaccharide (LPS). MPs were isolated, washed by high-speed centrifugation and assessed using the following: transmission electron microscopy (TEM), Nanoparticle Tracking Analysis (NTA), flow cytometry, tissue factor (TF) activity, prothrombinase activity, thrombin generation, and clot formation, density and stability.
RESULTS
MPs from monocytes (M-MPs) and platelets (PMPs) had similar shapes and diameters (100-300 nm). M-MPs had TF activity (16.7 ± 2.4 pm TF per 10(6) MP), supported prothrombinase activity and triggered shorter thrombin generation lag times than buffer controls (5.4 ± 0.5 vs. 84.2 ± 4.8 min, respectively). Compared with controls, M-MPs supported faster fibrin formation (0.24 ± 0.24 vs. 76.7 ± 15.1 mOD min(-1) , respectively), 38% higher fibrin network density and higher clot stability (3.8-fold higher turbidity in the presence of tissue plasminogen activator). In contrast, PMPs did not have TF activity and supported 2.8-fold lower prothrombinase activity than M-MPs. PMPs supported contact-dependent thrombin generation, but did not independently increase fibrin network density or stability. Interestingly, PMPs increased rates of thrombin generation and fibrin formation (1.7- and 1.3-fold, respectively) when mixed with THP-1-derived MPs.
CONCLUSION
MPs from platelets and monocytes differentially modulate clot formation, structure and stability, suggesting unique contributions to thrombosis.
Topics: Blood Coagulation; Blood Platelets; Cell-Derived Microparticles; Fibrin; Humans; Monocytes; Thrombin; Thromboplastin; Thrombosis
PubMed: 21883880
DOI: 10.1111/j.1538-7836.2011.04488.x -
Immunology Aug 2014CD13/Aminopeptidase N is a transmembrane metalloproteinase that is expressed in many tissues where it regulates various cellular functions. In inflammation, CD13 is...
CD13/Aminopeptidase N is a transmembrane metalloproteinase that is expressed in many tissues where it regulates various cellular functions. In inflammation, CD13 is expressed on myeloid cells, is up-regulated on endothelial cells at sites of inflammation and mediates monocyte/endothelial adhesion by homotypic interactions. In animal models the lack of CD13 alters the profiles of infiltrating inflammatory cells at sites of ischaemic injury. Here, we found that CD13 expression is enriched specifically on the pro-inflammatory subset of monocytes, suggesting that CD13 may regulate trafficking and function of specific subsets of immune cells. To further dissect the mechanisms regulating CD13-dependent trafficking we used the murine model of thioglycollate-induced sterile peritonitis. Peritoneal monocytes, macrophages and dendritic cells were significantly decreased in inflammatory exudates from global CD13(KO) animals when compared with wild-type controls. Furthermore, adoptive transfer of wild-type and CD13(KO) primary myeloid cells, or wild-type myeloid cells pre-treated with CD13-blocking antibodies into thioglycollate-challenged wild-type recipients demonstrated fewer CD13(KO) or treated cells in the lavage, suggesting that CD13 expression confers a competitive advantage in trafficking. Similarly, both wild-type and CD13(KO) cells were reduced in infiltrates in CD13(KO) recipients, confirming that both monocytic and endothelial CD13 contribute to trafficking. Finally, murine monocyte cell lines expressing mouse/human chimeric CD13 molecules demonstrated that the C-terminal domain of the protein mediates CD13 adhesion. Therefore, this work verifies that the altered inflammatory trafficking in CD13(KO) mice is the result of aberrant myeloid cell subset trafficking and further defines the molecular mechanisms underlying this regulation.
Topics: Animals; CD13 Antigens; Cell Adhesion; Cell Movement; Humans; Macrophages, Peritoneal; Mice; Mice, Knockout; Monocytes; U937 Cells
PubMed: 24627994
DOI: 10.1111/imm.12279 -
International Journal of Molecular... May 2022Cell lines of monocyte/macrophage origin are often used as model systems to study monocyte/macrophage biology. A relevant question is how similar these cell lines are to...
Quantitative Analysis of the Transcriptome of Two Commonly Used Human Monocytic Cell Lines-THP-1 and Mono Mac 6-Reveals Their Arrest during Early Monocyte/Neutrophil Differentiation.
Cell lines of monocyte/macrophage origin are often used as model systems to study monocyte/macrophage biology. A relevant question is how similar these cell lines are to their in vivo counterparts? To address this issue, we performed a detailed analysis of the transcriptome of two commonly used human monocyte/macrophage cell lines, Mono Mac 6 and THP-1. Both of these cell lines originate from leukemic cells with myelo-monocytic characteristics. We found that both Mono Mac 6 and THP-1 represent cells of very immature origin. Their transcriptomes show more similarities to immature neutrophils than cells of the monocyte/macrophage lineage. They express significant levels of N-elastase, proteinase 3, cathepsin G, and azurocidin but very low levels of CD14, ficolin, and complement factor P. All major MHC class II genes are also expressed at low levels. They show high levels of lysozyme and low levels of one of the immunoglobulin Fc receptors, FCGRIIA, which is characteristic of both neutrophils and monocytes. THP-1, but not Mono Mac 6, also expresses the high-affinity receptor for IgG, FCGRIA. Both cell lines lack the expression of the connective tissue components fibronectin, proteoglycan 4, and syndecan 3, which are characteristics of tissue macrophages but are absent in blood monocytes, indicating that they originate from bone marrow precursors and not yolk sac-derived hematopoietic cells. Both of these cell lines seem, therefore, to represent cells arrested during early myelo-monocytic development, at a branch point between neutrophil and monocyte differentiation. Their very immature phenotype indicates that great care should be taken when using these cell lines as models for normal monocyte/macrophage biology.
Topics: Cell Differentiation; Cell Line; Humans; Monocytes; Neutrophils; Transcriptome
PubMed: 35628628
DOI: 10.3390/ijms23105818 -
Scientific Reports Jan 2024Obstructive sleep apnea syndrome (OSAS) and obesity go hand in hand in the majority of patients and both are associated with a systemic inflammation, immune disturbance...
Obstructive sleep apnea syndrome (OSAS) and obesity go hand in hand in the majority of patients and both are associated with a systemic inflammation, immune disturbance and comorbidities such as cardiovascular disease. However, the unambiguous impact of OSAS and obesity on the individual inflammatory microenvironment and the immunological consequences of human monocytes has not been distinguished yet. Therefore, aim of this study was to investigate the impact of OSAS and obesity related factors on the inflammatory microenvironment by performing flow cytometric whole blood measurements of CD14/CD16 monocyte subsets in normal weight OSAS patients, patients with obesity but without OSAS, and patients with OSAS and obesity, compared to healthy donors. Moreover, explicitly OSAS and obesity related plasma levels of inflammatory mediators adiponectin, leptin, lipocalin and metalloproteinase-9 were determined and the influence of different OSAS and obesity related factors on cytokine secretion and expression of different adhesion molecules by THP-1 monocytes was analysed. Our data revealed a significant redistribution of circulating classical and intermediate monocytes in all three patient cohorts, but differential effects in terms of monocytic adhesion molecules CD11a, CD11b, CD11c, CX3CR1, CD29, CD49d, and plasma cytokine levels. These data were reflected by differential effects of OSAS and obesity related factors leptin, TNFα and hypoxia on THP-1 cytokine secretion patterns and expression of adhesion molecules CD11b and CD49d. In summary, our data revealed differential effects of OSAS and obesity, which underlines the need for a customized therapeutic regimen with respect to the individual weighting of these overlapping diseases.
Topics: Humans; Leptin; Monocytes; Obesity; Sleep Apnea, Obstructive; Cytokines
PubMed: 38172514
DOI: 10.1038/s41598-023-49921-5 -
Experimental Cell Research Jan 2018The taxanes Docetaxel and Paclitaxel are two of the standard chemotherapies for patients with metastatic breast cancer. The functional effect of Docetaxel and Paclitaxel...
The taxanes Docetaxel and Paclitaxel are two of the standard chemotherapies for patients with metastatic breast cancer. The functional effect of Docetaxel and Paclitaxel on human innate immune cells of the myeloid lineage is not well established, nor is the effects these agents have on differentiation of monocytes into macrophages and dendritic cells. Therefore, the aim with this project was to determine the effects of Docetaxel and Paclitaxel on primary human monocyte differentiation, activation and function. For this purpose, primary human monocytes were isolated from healthy donors and cultured with or without Docetaxel and Paclitaxel. We found that Docetaxel promoted the differentiation of primary human monocytes into pro-inflammatory macrophages with an M1 phenotype and an ability to present antigens to T cells. Monocytes treated with Docetaxel also displayed an elevated secretion of IL-8 and IL-1β, but did not promote generation of monocytic myeloid-derived suppressor cells. In conclusion, Docetaxel appears to have an immune stimulatory effect that would be beneficial for an anti-tumorigenic type of immune response, whereas Paclitaxel seems to have less effect on myeloid cells.
Topics: Antineoplastic Agents; Carcinogenesis; Cell Differentiation; Dendritic Cells; Docetaxel; Female; Humans; Immunity, Innate; Macrophages; Monocytes; Myeloid Cells; Neoplasms; Paclitaxel; T-Lymphocytes; Taxoids
PubMed: 29269075
DOI: 10.1016/j.yexcr.2017.12.018