Overexpression of CDKN2B (p15INK4B) and altered global DNA methylation status in mesenchymal stem cells of high-risk myelodysplastic syndromes

Abstract Current clinical management approaches for COVID-19 patients are generally based on supportive treatment, which mainly includes respiratory support and restricted fluid input, which is currently a subject of much debate. Systemic Inflammation caused by SARS-CoV-2 may be related to various extrapulmonary comorbidities such as cytokine-mediated neuroinflammation leading to both non-neuronal and neurological consequences in COVID-19. Mesenchymal stem cells (MSCs) are adult stem cells with multipotent properties suitable for medical applications that have been reported as potential therapies in the setting of lung diseases. The immunosuppressive properties of MSCs provide a strong rationale to explore their potential beneficial effects on immune events in COVID-19. Multiple in vivo studies have demonstrated the capability of MSCs to prevent inflammatory responses and reduce lung damage. Recently, the use of MSCs in treating COVID-19 disease has improved long-term pulmonary function, but the specific mechanisms by which MSCs inhibit the severe inflammatory response induced by SARS-CoV-2 have not been elucidated. To the best of our knowledge, this is the first work describing the regulatory effects of MSCs on peripheral blood mononuclear cells (PBMCs) derived from patients with COVID-19 by measuring the pro-inflammatory and anti-inflammatory cytokines expression and secretion. We also examined the effects on the methylation of genes normally suppressed by DNA methylation in PBMCs. Our result showed that MSCs exerted an immune regulatory function on PBMCs in culture, skewing toward a type-2 response. This occurs by a mechanism consistent with a reduction in inflammatory factors (TNF-α, IL-1β, IL-6, IL-18, and IFNγ) protein and mRNA expression levels. In contrast, the anti-inflammatory cytokines (IL-4 and IL-10) increased following co-culture with MSCs. Consistent with these findings, the DNA methylation status of these immune genes seemed relevant to their expression pattern, except for GATA3, IL-1β, and IFNγ genes which showed no significant differences in methylation level between PBMCs with and without MSC exposure. Moreover, in co-culture interaction, MSCs modulated the Th1/Th2 cells in PBMCs compared to unstimulated PBMCs. These data demonstrate that MSCs can exert important immunomodulatory functions that affect virus-associated cytokine storms in pulmonary tissue during the severe respiratory stage.

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The Different Immunoregulatory Functions of Mesenchymal Stem Cells in Patients with Low-Risk or High-Risk Myelodysplastic Syndromes

PLoS ONE ◽

10.1371/journal.pone.0045675 ◽

2012 ◽

Vol 7 (9) ◽

pp. e45675 ◽

Cited By ~ 23

Author(s):

Zhigang Zhao ◽

Zhenling Wang ◽

Qiubai Li ◽

Weiming Li ◽

Yong You ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

High Risk ◽

Myelodysplastic Syndromes ◽

Low Risk

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Azacitidine Treatment in High Risk Myelodysplastic Patients in Complete Haematological Remission Reverts Mesenchymal Stem Cells to a Normal Phenotype

Blood ◽

10.1182/blood.v124.21.1904.1904 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1904-1904 ◽

Cited By ~ 1

Author(s):

Antonella Poloni ◽

Giulia Maurizi ◽

Domenico Mattiucci ◽

Benedetta Costantini ◽

Marianna Mariani ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

High Risk ◽

Myelodysplastic Syndromes ◽

Proliferative Potential ◽

Proliferative Capacity ◽

Normal Phenotype ◽

Haematological Remission

Abstract Introduction. Myelodysplastic syndromes (MDSs) are a heterogeneous group of clonal hematopoietic stem cell (HSC) malignancies that are characterized by ineffective bone marrow hematopoiesis, peripheral blood cytopenias, and a substantial risk for progression to acute myeloid leukemia. Mesenchymal stem cells (MSCs) isolated from bone marrow of patients affected by myelodysplastic syndromes (MDS) play a critical role in myelodysplastic microenvironment showing altered structural epigenetic and functional features. Methods. In this work we evaluated the effect of azacitidine treatment on MSC-MDS. In particular, we analyzed MSC-MDS from 24 high-risk patients at diagnosis and after azacitidine treatment, studying their morphology, proliferative potential, cell cycle activity and their capacity to support haematopoiesis. Results. MDS-MSCs at diagnosis appeared larger and flattened, achieved confluence at a significantly lower rate than donors and displayed reduced proliferative capacity. In particular 40% of samples were unable to expand. This reduced proliferative capacity of MSC-MDS at diagnosis suggested changes in the cell cycle activity. Therefore we studied the gene expression profiles of 37 regulatory genes, observing CDKN2B up-regulation in MDS-MSCs (8 times higher than donors). Notably, after azacitidine treatment MDS-MSCs of patients who reached complete haematological remission (MDS-MSCs-CR) reverted to the typical BM-MSC morphology and recovered a proliferative potential similar to normal BM-MSC achieving confluence at a significantly higher rate. Molecular analysis on MDS-MSC-CR revealed a significant reduction in the expression level of CDKN2B showing correlation between cell cycle progression and expression level of this gene. Moreover, to study the long-term hematopoietic maintaining ability, MDS-MSCs at diagnosis were cultured with CD133+ cells, and they showed a decreased ability to support the growth of myeloid and erythroid progenitors. Conversely, MSC-MDS-CR showed an increased capacity to support haematopoiesis similar to healthy donors. Conclusion. We showed that MDS-MSCs at diagnosis were structurally and functionally altered while MSC-MDS-CR after azacitidine revert to a normal phenotype. It has been supposed that healthy MSCs adopt MDS-MSCs like molecular features when exposed to haematopoietic MDS cells. Our results may confirm these data suggesting that myelodysplastic cells can alter bone marrow microenvironment interacting with MSC and affecting their normal role and functionality. Disclosures No relevant conflicts of interest to declare.

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The Different Immunoregulatory Functions on Dendritic Cells between Mesenchymal Stem Cells Derived from Bone Marrow of Patients with Low-Risk or High-Risk Myelodysplastic Syndromes

PLoS ONE ◽

10.1371/journal.pone.0057470 ◽

2013 ◽

Vol 8 (3) ◽

pp. e57470 ◽

Cited By ~ 20

Author(s):

Zhenling Wang ◽

Xiaoqiong Tang ◽

Wen Xu ◽

Zeng Cao ◽

Li Sun ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Dendritic Cells ◽

High Risk ◽

Myelodysplastic Syndromes ◽

Low Risk

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Faculty Opinions recommendation of Expression of the p16INK4A gene is associated closely with senescence of human mesenchymal stem cells and is potentially silenced by DNA methylation during in vitro expansion.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1088258.541282 ◽

2007 ◽

Author(s):

Helen Gruber

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Human Mesenchymal Stem Cells ◽

P16ink4a Gene ◽

In Vitro Expansion

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Functional characteristics of mesenchymal stem cells derived from bone marrow of patients with myelodysplastic syndromes

Cancer Letters ◽

10.1016/j.canlet.2011.08.030 ◽

2012 ◽

Vol 317 (2) ◽

pp. 136-143 ◽

Cited By ~ 35

Author(s):

Zhi-Gang Zhao ◽

Wen Xu ◽

Hai-Peng Yu ◽

Bing-Ling Fang ◽

Shu-Hong Wu ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Myelodysplastic Syndromes ◽

Functional Characteristics

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Genome-Wide DNA Methylation Pattern of Cancer Stem Cells in Esophageal Cancer

Technology in Cancer Research & Treatment ◽

10.1177/1533033820983793 ◽

2020 ◽

Vol 19 ◽

pp. 153303382098379

Author(s):

Xiying Yu ◽

Ying Teng ◽

Xingran Jiang ◽

Hui Yuan ◽

Wei Jiang

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Esophageal Cancer ◽

Cancer Stem Cells ◽

Side Population ◽

Methylation Status ◽

Methylation Profile ◽

Cpg Dinucleotides ◽

Genome Wide ◽

Differentially Methylated Genes

Background: Cancer stem cells (CSCs) are considered the main cause of cancer recurrence and metastasis, and DNA methylation is involved in the maintenance of CSCs. However, the methylation profile of esophageal CSCs remains unknown. Methods: Side population (SP) cells were isolated from esophageal squamous cell carcinoma (ESCC) cell lines KYSE150 and EC109. Sphere-forming cells were collected from human primary esophageal cancer cells. SP cells and sphere-forming cells were used as substitutes for cancer stem-like cells. We investigated the genome-wide DNA methylation profile in esophageal cancer stem-like cells using reduced representation bisulfite sequencing (RRBS). Results: Methylated cytosine (mC) was found mostly in CpG dinucleotides, located mostly in the intronic, intergenic, and exonic regions. Forty intersected differentially methylated regions (DMRs) were identified in these 3 groups of samples. Thirteen differentially methylated genes with the same alteration trend were detected; these included OTX1, SPACA1, CD163L1, ST8SIA2, TECR, CADM3, GRM1, LRRK1, CHSY1, PROKR2, LINC00658, LOC100506688, and NKD2. DMRs covering ST8SIA2 and GRM1 were located in exons. These differentially methylated genes were involved in 10 categories of biological processes and 3 cell signaling pathways. Conclusions: When compared to non-CSCs, cancer stem-like cells have a differential methylation status, which provides an important biological base for understanding esophageal CSCs and developing therapeutic targets for esophageal cancer.

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Potential effect of tobacco cigarettes smoking on global DNA methylation status and protamines transcripts in human spermatozoa

Middle East Fertility Society Journal ◽

10.1186/s43043-021-00066-3 ◽

2021 ◽

Vol 26 (1) ◽

Author(s):

Mohammed M. Laqqan ◽

Maged M. Yassin

Keyword(s):

Dna Methylation ◽

Cigarette Smoking ◽

Dna Fragmentation ◽

Methylation Status ◽

Human Spermatozoa ◽

Global Dna Methylation ◽

Transcription Level ◽

Heavy Smokers ◽

Protamine 1 ◽

Protamine 2

Abstract Background Epigenetics refers to an alteration in gene expression without alteration in the sequence of DNA and this process may be affected by environmental factors and lifestyle like cigarette smoking. This study was designed to evaluate the potential effect of cigarette smoking on the global DNA methylation status and the transcription level of protamine 1 and protamine 2 in human spermatozoa. A total of 188 semen samples were collected from men with a mean age of 34.9 ± 5.8 years old (98 heavy smokers and 90 non-smokers). The DNA and RNA were isolated from purified spermatozoa, then the status of global DNA methylation and the transcription level of protamine 1 and protamine 2 were evaluated using ELISA and qPCR, respectively. The chromatin non-condensation and DNA fragmentation in human spermatozoa were evaluated using chromomycin A3 staining and TUNEL assay, respectively. Results A significant increase has been found in the status of global DNA methylation in spermatozoa of heavy smokers compared to non-smokers (7.69 ± 0.69 ng/μl vs. 4.90 ± 0.40 ng/μl, P < 0.001). Additionally, a significant reduction has been found in transcription level of protamine 1 (25.49 ± 0.31 vs. 23.94 ± 0.40, P < 0.001) and protamine 2 (28.27 ± 0.39 vs. 23.45 ± 0.30, P < 0.001) in heavy smokers. A downregulation has been found in the transcription level of protamine 1 and protamine 2 with a fold change of 0.497 and 0.047, respectively. A significant increase has been shown in the level of DNA fragmentation and chromatin non-condensation in heavy smokers compared to non-smokers (P < 0.001). On the other hand, a significant positive correlation has been found between sperm chromatin non-condensation, sperm DNA fragmentation, transcription level of protamine 1, transcription level of protamine 2, and global DNA methylation status (r = 0.304, P < 0.001; r = 0.399, P < 0.001; r = 0.216, P = 0.003; r = 0.494, P < 0.001, respectively). Conclusion Tobacco cigarette smoking has a potential influence on the global DNA methylation and the transcription level of protamine genes in human spermatozoa, and consequently, affect negatively on the semen parameters.

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