Cell Cycle Characteristics of Haemopoietic Stem Cells

Background: Oxidative stress and chronic hyperglycemia are two major side effects of type 2 diabetes affecting all cell types including mesenchymal stem cells (MSCs). As a cell therapy choice, understanding the behavior of MSCs will provide crucial information for efficient treatment. Methods: Placental mesenchymal stem cells were treated with various concentrations of glucose, metformin, rapamycin, and hydrogen peroxide to monitor their viability and cell cycle distribution. Cellular viability was examined via the MTT assay. Cell cycle distribution was studied by propidium iodide staining and apoptosis was determined using Annexin Vpropidium iodide staining and flow cytometry. Involvement of potential signaling pathways was evaluated by Western blotting for activation of Akt, P70S6K, and AMPK. Results: The results indicated that high glucose augmented cell viability and reduced metformin toxic potential. However, the hydrogen peroxide and rapamycin toxicities were exacerbated. Conclusion: Our findings suggest that high glucose concentration has a major effect on placental mesenchymal stem cell viability in the presence of rapamycin, metformin and hydrogen peroxide in culture.

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Synchronize Human Embryonic Stem Cells at Different Cell Cycle Stage

BIO-PROTOCOL ◽

10.21769/bioprotoc.193 ◽

2012 ◽

Vol 2 (11) ◽

Author(s):

Hui Zhu

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Cell Cycle Stage

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Targeting survivin induced cell cycle arrest and apoptosis in hemangioma stem cells

International Journal of Oral and Maxillofacial Surgery ◽

10.1016/j.ijom.2015.08.411 ◽

2015 ◽

Vol 44 ◽

pp. e315

Author(s):

W. Zhang ◽

J.G. Yang ◽

J.Y. Zhu ◽

Y.F. Zhao

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Cell Cycle Arrest ◽

Cycle Arrest ◽

Hemangioma Stem Cells

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Bmi1 Augments Proliferation and Survival of Cortical Bone-Derived Stem Cells after Injury through Novel Epigenetic Signaling via Histone 3 Regulation

International Journal of Molecular Sciences ◽

10.3390/ijms22157813 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7813

Author(s):

Lindsay Kraus ◽

Chris Bryan ◽

Marcus Wagner ◽

Tabito Kino ◽

Melissa Gunchenko ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Dna Damage ◽

Stem Cell ◽

Histone Modification ◽

Epigenetic Regulation ◽

Critical Role ◽

Proliferative Potential ◽

Therapeutic Effects ◽

Histone 3

Ischemic heart disease can lead to myocardial infarction (MI), a major cause of morbidity and mortality worldwide. Multiple stem cell types have been safely transferred into failing human hearts, but the overall clinical cardiovascular benefits have been modest. Therefore, there is a dire need to understand the basic biology of stem cells to enhance therapeutic effects. Bmi1 is part of the polycomb repressive complex 1 (PRC1) that is involved in different processes including proliferation, survival and differentiation of stem cells. We isolated cortical bones stem cells (CBSCs) from bone stroma, and they express significantly high levels of Bmi1 compared to mesenchymal stem cells (MSCs) and cardiac-derived stem cells (CDCs). Using lentiviral transduction, Bmi1 was knocked down in the CBSCs to determine the effect of loss of Bmi1 on proliferation and survival potential with or without Bmi1 in CBSCs. Our data show that with the loss of Bmi1, there is a decrease in CBSC ability to proliferate and survive during stress. This loss of functionality is attributed to changes in histone modification, specifically histone 3 lysine 27 (H3K27). Without the proper epigenetic regulation, due to the loss of the polycomb protein in CBSCs, there is a significant decrease in cell cycle proteins, including Cyclin B, E2F, and WEE as well as an increase in DNA damage genes, including ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related (ATR). In conclusion, in the absence of Bmi1, CBSCs lose their proliferative potential, have increased DNA damage and apoptosis, and more cell cycle arrest due to changes in epigenetic modifications. Consequently, Bmi1 plays a critical role in stem cell proliferation and survival through cell cycle regulation, specifically in the CBSCs. This regulation is associated with the histone modification and regulation of Bmi1, therefore indicating a novel mechanism of Bmi1 and the epigenetic regulation of stem cells.

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THU0495 NOVEL UNDERSTANDING OF THE PATHOGENESIS OF JUVENILE IDIOPATHIC ARTHRITIS: FOCUS ON MESENCHYMAL STEM CELLS IMPAIRMENT, SENESCENCE AND IMMUNOREGULATORY FUNCTION

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.3845 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 483.2-484

Author(s):

L. Zaripova ◽

A. Midgley ◽

S. Christmas ◽

E. Baildam ◽

R. Oldershaw

Keyword(s):

Oxidative Stress ◽

Cell Cycle ◽

Stem Cells ◽

Cell Proliferation ◽

Mesenchymal Stem Cells ◽

Juvenile Idiopathic Arthritis ◽

Metabolic Activity ◽

Healthy Controls ◽

High Level ◽

Pro Inflammatory Cytokines

Background:Juvenile idiopathic arthritis (JIA) is a well-known chronic rheumatic disease of childhood characterised by progressive joint destruction and severe systemic complications.Immune cells are known to trigger the pathophysiological cascade in JIA, but there is little information regarding the contribution made by Mesenchymal stem cells (MSCs). These cells are able to modulate the immune response and decrease the level of pro-inflammatory cytokines. With addition of regenerative property it makes MSCs potential candidates for clinical application as immunosuppressants in treatment of autoimmune diseases.Objectives:To investigate MSCs proliferation, viability and immunomodulatory function in JIA and healthy children.Methods:MSCs were separated from peripheral blood (PB) and synovial fluid (SF) of JIA patients and healthy controls. Cell proliferation rate was counted by Population doublings per day (PDD) during 9 days, in the last of which alamarBlue™ assays were performed to assess cell viability. Due to measure senescence MSCs were stained with SA-β-galactosidase. Immunofluorescence was used to examine the expression of p16, p21, p53. Oxidative stress was measured with DCFH-DA. Cell cycle analysis was evaluated with Propidium Iodide and analysed by Accuri® C6 Flow Cytometer.Commercially-available bone marrow mesenchymal stem cells (BM-MSCs) were treated with graded concentrations of pro-inflammatory cytokines (0.1-100 ng/ml) with following examination of cell viability. Mixed lymphocyte reactions (MLR) were performed to measure MSC immunomodulatory abilityin vitro.Results:The growth kinetics of JIA-MSCs were different from healthy controls. JIA-MSCs divided slowly and appeared disorganised with large cytoplasm and loads of outgrowth. They demonstrated a decrease in cell proliferation (negative PDD) and metabolic activity. Difference in growth kinetics and metabolic activity were found inside the JIA PB group with some evidence of response following biological treatment. Thus, PB-MSCs from patients treated with TNFi and anti-IL6 medications had notably higher cell proliferation and metabolic activity against JIA patients received other therapy. Considering this difference, it was hypothesised that cytokines obtained in a high amount in PB and SF of JIA patients may influence MSCs viability. To prove this BM-MSCs were treated with cytokines and demonstrated a dose-dependent decrease in metabolic activity significantly after TNFα and IL1, no significantly after treatment with IL6. Both BM-MSCs treated with cytokines and JIA-MSCs displayed high level of reactive oxygen species.Cell cycle analysis revealed that JIA-MSCs were arrested in G0/G1 phase with low number of mitotic cells. In addition, the number of senescence-associated SA-β-gal-positive cells was notably higher in JIA-MSCs. Furthermore, JIA-MSCs expressed high level of immunofluorescence for p16, p21 and p53 which played an important role in regulating the senescence progress of MSCs.Results of MLR showed the ability of BM-MSCs to decrease the percentage of activated T-helpers, T-suppressors, B-cells and natural killers proliferation, while JIA-MSCs lost this property.Conclusion:Taken together current research has demonstrated that under the influence of proinflammatory cytokines JIA-MSCs suffered from oxidative stress and disruption of metabolic activity acquire senescent morphology, shorten of telomere length, arrest in G0 phase of cell cycle and finally loss of immune regulation. We are continuing our research to determine the mechanisms that are responsible for the impaired phenotype with the aim of identifying new therapeutic strategies for the treatment of JIA.Disclosure of Interests: :None declared

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PAWI-2 overcomes tumor stemness and drug resistance via cell cycle arrest in integrin β3-KRAS-dependent pancreatic cancer stem cells

Scientific Reports ◽

10.1038/s41598-020-65804-5 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 3

Author(s):

Jiongjia Cheng ◽

John R. Cashman

Keyword(s):

Cell Cycle ◽

Pancreatic Cancer ◽

Stem Cells ◽

Cancer Stem Cells ◽

Cell Cycle Arrest ◽

Specific Inhibitor ◽

Feedback Mechanism ◽

Major Health Problem ◽

Cycle Arrest ◽

Drug Inhibition

Abstract Today, pancreatic cancer (PC) remains a major health problem in the US. The fact that cancer stem cells (CSCs) become enriched in humans following anti-cancer therapy implicates CSCs as key contributors to tumor dormancy, metastasis, and relapse in PC. A highly validated CSC model (FGβ3 cells) was used to test a novel compound (PAWI-2) to eradicate CSCs. Compared to parental bulk FG cells, PAWI-2 showed greater potency to inhibit cell viability and self-renewal capacity of FGβ3 cells. For FGβ3 cells, dysregulated integrin β3-KRAS signaling drives tumor progression. PAWI-2 inhibited β3-KRAS signaling independent of KRAS. This is clinically relevant. PAWI-2 targeted the downstream TBK1 phosphorylation cascade that was negatively regulated by optineurin phosphorylation via a feedback mechanism. This was confirmed by TBK1 genetic knockdown or co-treatment with TBK1-specific inhibitor (MRT67307). PAWI-2 also overcame erlotinib (an EGFR inhibitor) resistance in FGβ3 cells more potently than bortezomib. In the proposed working model, optineurin acts as a key regulator to link inhibition of KRAS signaling and cell cycle arrest (G2/M). The findings show PAWI-2 is a new approach to reverse tumor stemness that resensitizes CSC tumors to drug inhibition.

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