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ORIGINAL ARTICLE
Year : 2019  |  Volume : 32  |  Issue : 3  |  Page : 978-982

Use of azacytidine in differentiation of mesenchymal stem cells


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shebin El-Kom, Egypt
2 Department of Clinical Pathology, Biala Central Hospital, Biala, Kafr El Sheikh, Egypt

Date of Submission08-Jan-2018
Date of Acceptance10-Feb-2018
Date of Web Publication17-Oct-2019

Correspondence Address:
Rasha A El Morsy
Al Taqua Street, Biala, Kafr El Sheikh
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_893_17

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  Abstract 


Objective
We aimed to study the differentiation of mesenchymal stem cells (MSCs) into cardiomyocytes using azacytidine.
Background
Ischemic injury of cardiomyocytes results in heart failure, as cardiomyocytes do not have the ability to regenerate after death. This has prompted interest in identifying cells capable of replacing the injured myocardium with healthy cells. The ideal candidate for cellular cardiomyoplasty is a less committed cell that can undergo full cardiogenic differentiation which can be found in the adult bone marrow (BM). Now it is believed that the adherent population of cells isolated from BM and expanded in vitro are a potential source of undifferentiated MSCs.
Patients and methods
The study included 30 cases referred to Clinical Pathology Department. Extra BM sample was collected from patients who already had a benign indications for BM examination as hypersplenism and immune thrombocytopenic purpura. MSCs were cultured from BM aspirate and mononuclear cells were separated using ficoll-hypaque solution. Thereafter, MSCs were separated from mononuclear cells fraction using plastic adherence flasks, and MSCs were subcultured in differentiating media containing azacytidine. Differentiation into cardiomyocytes was detected by morphology of cardiomyocytes and immunophenotyping.
Results
Vimentin expression on cardiomyocytes treated with azacytidine (82.46 ± 9.04) was significantly higher than on MSCs not treated with azacytidine (15.18 ± 4.11). Statistically significant difference was found between MSCs and cardiomyocytes for presence of vimentin (P < 0.001).
Conclusion
By using azacytidine, MSCs can be differentiated to cardiomyocytes.

Keywords: azacytidine, bone marrow, cardiomyoplasty, mesenchymal stem cells, vimentin


How to cite this article:
Fathy WM, Ahmedy IA, Motawe SM, El Morsy RA. Use of azacytidine in differentiation of mesenchymal stem cells. Menoufia Med J 2019;32:978-82

How to cite this URL:
Fathy WM, Ahmedy IA, Motawe SM, El Morsy RA. Use of azacytidine in differentiation of mesenchymal stem cells. Menoufia Med J [serial online] 2019 [cited 2019 Nov 12];32:978-82. Available from: http://www.mmj.eg.net/text.asp?2019/32/3/978/268860




  Introduction Top


Myocardial infarction is often followed by progressive deterioration of left ventricular function and eventually leads to overt heart failure [1]. The management of ischemic heart disease includes medications, percutaneous coronary intervention, and surgical operations. These treatments could improve myocardial ischemia and heart failure symptoms. Although the surgical operations make the obstructed artery open again, the damage to myocardial wall is irreversible. The current medical and surgical management options are of limited value. Shortage in donor hearts and high cost are serious problems in heart transplantation [2].

In 2001, Orlic et al. [2] transplanted autologous bone marrow mesenchymal stem cells (BMSCs) into mouse damaged heart and found these stem cells mostly differentiated into cardiomyocytes. This important discovery guided the scientists and clinicians to engage in plenty of researches on stem cells transplantation to treat myocardial infarction. Significant progress has been made in the mesenchymal stem cells (MSCs) research field, such as cell culture condition and technique of inducing differentiation in vitro [3]. The differentiation of myocardial cells from stem cells provides a promising vision for cell treatment of cardiac diseases [4],[5].

MSCs have been described in nearly all postnatal tissues or organs, including umbilical cord blood placenta and bone marrow (BM) [6],[7], among others. Human mesenchymal stem cells (hMSCs) were isolated, and then culture expanded; their multilineage potential was demonstrated by in-vitro methods [3]. Moreover, after treatment with the DNA demethylation agent 5-azacytidine, a murine MSC-like cell line was shown to express cardiac differentiation markers [8]. Owing to the problems of immune rejection and ethical concerns, MSCs have great application in personalized treatment of heart diseases because of the ability of autologous transplantation [9],[10]. Owing to its availability, feasibility, plasticity, and ability to express cardiomyocyte, MSCs have been used in cardiogenic differentiation [11].

Aim

We aimed to study the differentiation of MSCs into cardiomyocytes using azacytidine.


  Patients and Methods Top


The present study was carried out on 30 patients referred to Clinical Pathology Department, El-Menoufia University Hospital, between May 2015 and May 2017. They had other benign indications for BM examination such as hypersplenism and immune thrombocytopenic purpura, and an extra BM sample was aseptically collected. Patients were chosen as follows: age above 18–50 years; good general health condition; BM examination free of infiltration; having negative results for hepatitis C virus, hepatitis B virus, and HIV; patient willing to consent; and not having neoplastic conditions or taking antineoplastic medications. The study was approved by Ethical Committee of El-Menoufia Faculty of Medicine. Informed consent was taken from every patient.

Culture of mesenchymal stem cells

BM sample was diluted (1: 1 ratio) with sterile PBS using kit supplied by Invitrogen (Carlsbad, California, USA) and then layered on top of ficoll-hypaque using kit supplied by Biochrom AG (Berlin, Germany). After collection and seeding of the mononuclear cell fraction (at a concentration of million cells/cm 2), it was allowed to adhere to tissue culture plastic flasks 25 cm 2 (cell star) and was incubated at 37°C and 5% carbon dioxide in 5 ml of the fresh complete nutrient medium (F10), which was composed of the following: low-glucose Dulbecco's modified Eagle's medium with l-glutamine (2 mmol/l), using kit supplied by Euroclone Via Lombardia (Siziano, Italy); 10% fetal bovine serum, using kit supplied by Euroclone Via Lombardia; penicillin–streptomycin, using kit supplied by Sigma-Aldrich Co. (Saint Louis, Missouri, USA) (100 U/ml penicillin and 100 μg/ml streptomycin); fungizone (0.25 μg/ml), using kit supplied by Bioscience (Redhill Surrey, UK); and basic fibroblast growth factor and insulin-like growth factor I (10 ng/ml), using kit supplied by R and D Systems Inc. (Minneapolis, Minnesota, USA). Nonadherent cells were removed by changing half medium every 4 days.

At ninth day, when fibroblast-like cells reached 80–90% confluence [Figure 1] they were harvested by trypsinization and then examined morphologically, and the surface markers CD44 and CD34 were detected by flow cytometric analysis.
Figure 1: Mesenchymal stem cells before treatment with 5-azacytidine (up) and after treatment with 5-azacytidine (down).

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Cellular characterization

The harvested MSCs at day 9 were identified by detecting surface markers CD44 and CD34 using flow cytometric analysis. Cell resuspension was made in 1-ml PBS after washing with PBS and centrifugation at 3200 rpm for 5 min. Overall, 100 μl of cell suspension was added to 10-μl fluorochrome conjugated reagents (phycoerythrin anti-human CD44) using kit supplied by R and D Systems Inc. In another tube, 100 μl of cell suspension was added to 10-μl fluorochrome conjugated reagents (fluorescein isothiocyanate-conjugated anti-CD34) using kit supplied by R and D Systems Inc. After light vortex, cells were incubated in the dark for 30 min. Finally, cells were analyzed by BD FACS Calibur Flow Cytometer (BD Biosciences, San Jose, California, USA). The MSCs were gated out on CD44 and CD34 expression. Proper isotype control was run in parallel with each test sample.

Cardiomyogenic differentiation of mesenchymal stem cells

Around 1 × 104 of MSCs were seeded in 35-mm culture dishes. On the second day after seeding, cells were treated with 10 μmol 5-azacytidine using kit supplied by Sigma-Aldrich Co. Every 3 days, the media was changed. The cells were allowed to differentiate over a period of 12 days. By using inverted and phase contrast microscope, the cells were observed every day for any morphological changes.

Flow cytometric detection of vimentin in cardiomyocytes

Samples were prepared for intracytoplasmic and surface flow cytometric assessment. The cardiomyocytes and MSCs were gated out for expression of vimentin using kit supplied by R and D Systems Inc. Proper isotype control was run in parallel with each test sample.

Statistical analysis

The collected data were analyzed using IBM personal computer and statistical package for the social sciences, SPSS version 11 (SPSS Inc., Chicago, Illinois, USA). Two types of statistics were done: descriptive statistics, for example percentage, mean, and SD, and analytic statistics, for example, Mann–Whitney test (nonparametric test), which is a test of significance used for comparison between two groups not normally distributed having quantitative variables and Student's t-test.

For normally distributed quantitative variables to compare between two studied groups, Pearson's correlation (r) is a test used to measure the association between two quantitative variables. P value of less than 0.05 was considered statistically significant.


  Results Top


At first 2 days after the initial plating, cultured cells showed cytoplasmic projections and formed small clusters. At third and fourth days, the onset of fibroblast could be observed. At seventh day, cells tended to have a multipolar fibroblastoid appearance and a 60% confluence. By ninth day, it increased to reach 80–90% confluence gradually.

Flow cytometric analysis of MSCs showed positive expression for CD44 [Figure 2] (ranging between 67.10 and 97.50 with a mean ± SD of 83.01 ± 9.55) and negative expression for CD34 [Figure 2] (ranging between 0.10 and 2.0, with a mean ± SD of 0.92 ± 0.55) [Table 1].
Figure 2: Flow cytometric pictures representing positive expression of CD44 on mesenchymal stem cells, negative expression of CD34 on mesenchymal stem cells, and vimentin-positive expression on cardiomyocytes.

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Table 1: Descriptive statistics of flow cytometric analysis results of CD34 and CD44 on mesenchymal stem cells

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On the fourth day, ∼20–30% of cells became thinner and aligned parallel to each other, and on the sixth day, they were seen in groups of 6–7 cells. They showed central broadening of cytoplasm on the seventh day, whereas on the eighth day, some of them showed multinucleation extending their cytoplasmic processes with adjacent cells, and on the ninth day, they showed ball stick-like morphology [Figure 1]. The cells were observed under ×10, ×20, and ×40 magnification. Cells were regularly monitored using phase contrast microscope and inverted microscope, and images were captured for analysis.

Results of flow cytometric analysis of preinduction and postinduction BMA-MSCs:

MSCs showed low-positive expression of vimentin, with a mean ± SD of 15.18 ± 4.11, whereas cardiomyocytes showed high-positive expression of vimentin [Figure 2], with a mean ± SD of 82.46 ± 9.04. Vimentin expression on cardiomyocytes was significantly higher than on MSCs (P < 0.001) [Table 2].
Table 2: Descriptive statistics of flow cytometric results of vimentin

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  Discussion Top


Over the past decades, stem cells have gained great interest in clinical research, tissue engineering, and regenerative medicine owing to their ability of self-renewal and potential to differentiate into the various cell types of the organism [12].

Cell therapy has the potential to improve healing of ischemic heart, repopulate injured myocardium, and restore cardiac function [13]. Various stem cell-based approaches for the production of cardiomyocytes have been shown to improve left ventricular function in preclinical animal models. Promising results rapidly led to clinical trials, initially using BM-derived mononuclear cells, then MSC populations, and more recently, cardiac progenitor cells [14],[15].

MSCs are progenitors of connective tissues, which have emerged as important tools for tissue engineering owing to their differentiation potential along various cell types [16]. Accumulating evidence has demonstrated that BMMSCs may trans differentiate into cardiomyocytes, making BMMSCs a promising source of cardiomyocytes for transplantation [17].

Our study aimed to study MSCs differentiation into cardiomyocytes using azacytidine. BMMSCs were obtained from adequate number of patients undergoing BM aspiration for nonmalignant reasons. The obtained cells were isolated and subcultured in differentiating media containing 5-azacytidine. The produced cardiomyocytes were identified by their characteristic morphology and by using immunophenotyping.

In accordance with our study, it was found in a study by Ling et al. that cardiomyogenic differentiation of adult BMMSCs with 5-azacytidine is feasible and appears to improve their in-vivo cardiac differentiation as well as the functional recovery in a rat model of the infarcted myocardium [18].

Results of our study were also in agreement of the study of Haghani et al. [19] who aimed to differentiate the BMMSCs into cardiomyocyte. In their study, BMSCs were exposed to 5-azacytidine for 24 h. Within 2 weeks after the induction of cell differentiation by 5-azacytidine, the cardiomyogenic cells were stained by Fuchsin, and then binucleated cells were counted and compared with the neonate cardiomyocyte as positive control. Results showed that there was no significant difference between the number of binucleated cells within the cardiomyogenic cell group and positive control group; however, a statistically significant difference was observed between both of these groups and undifferentiated cell group.

In another study, Yang et al. [20] analyzed the changes of gene expression profiles during the process that human bone marrow mesenchymal stem cells (hBMMSCs) are induced to differentiate into cardiomyogenic cells with 5-azacytidine, and after 3 weeks of induction, immunocytochemical staining for α-actin, cardiac troponin, and connexin 43 showed all positive results. During this process, multiple genes related with signal transduction, transcription, and growth factors are involved.

In addition, in our study, flow cytometry analysis revealed that MSCs showed positive expression for CD44 whereas they showed negative expression for CD34. This is in accordance with the study of Zhang et al. [21], who investigated the differences between adipose-derived stem cells and MSCs in in-vitro culture and differentiation into cardiomyocytes. In their study, both adipose-derived stem cells and MSCs before treatment with 5-azacytidine were stained positively for CD29, CD44, and CD105, but negatively for CD34 and CD45, α-sarcromeric actin, cardiac troponin T, and von Willebrand factor.

Moreover, in the study of Zhu et al. [22], human umbilical cord mesenchymal stem cells (hUCMSCs) were isolated and purified from the umbilical cords of normal or cesarean term deliveries under sterile conditions. Flow cytometry analysis revealed that CD13, CD29, CD44, CD90, and CD105 were highly expressed on the surface of passage-3 hUCMSCs, but negative for CD31, CD34, CD45, and Human Leukocyte Antigen – DR isotype (HLA-DR).

Likewise, the study by Yu et al. [23], in their study aiming to investigate the involvement of Notch signalling during the course of hBMMSC differentiation into cardiomyocytes using hBMSCs, using flow cytometric analysis revealed that CD29, CD44, and CD90 were highly expressed on the surface of cells in their fifth passage, whereas detection of CD34, CD45, CD54, and HLA-DR was negative.

Comparison between the cardiac marker vimentin secretion by BMMSCs and cardiomyocytes produced in our study revealed statistically significant higher expression of vimentin with cardiomyocytes. These data find support in the study of Antonitsis et al. [24], who investigate the ability of adult hBMMSCs to differentiate toward a cardiomyogenic phenotype in vitro. In their study, BM samples were aspirated from 30 patients undergoing open heart surgery from the anterior iliac crest. Second passaged cells were treated with 10 μmol 5-azacytidine. As control groups, they used cells not expanded in culture and cells untreated with 5-azacytidine. Morphologic characteristics were analyzed by confocal and electron microscopy. The expression of the cytoskeletal protein vimentin and muscle-specific myocin heavy chain was analyzed by immunohistochemistry. Results showed that numerous myofilaments were detected in induced cells which were immunohistochemically positive for myosin heavy chain and vimentin [24].


  Conclusion Top


By using azacytidin, MSCs can be differentiated to cardiomyocytes.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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