Cardiomyocyte Differentiation Of Cord Lining Membrane Stem Cells. Phan T.H., Masilamani J. and Ng Y.H.

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1 Cardiomyocyte Differentiation Of Cord Lining Membrane Stem Cells Phan T.H., Masilamani J. and Ng Y.H. Department of Bioengineering, National University of Singapore 21 Lower Kent Ridge Road, Singapore ABSTRACT There is much research recently for alternative sources of stem cells so that the controversy surrounding embryonic stem cells can be side-stepped. Thus, it is of great interest to investigate cord lining membrane stem cells for their cardiogenic and myogenic differentiation ability. The cells are cultured on selected media, and RT-PCR and gel electrophoresis are performed to determine the level of expression on day 1, 7, 14 and 21 respectively so as establish the most suitable medium and cell type for cardiogenic and myogenic differentiation. INTRODUCTION Heart disease occurs when the heart loses its ability to pump sufficient blood throughout the body, and this is due to a loss of functional cardiomyocytes. Due to the inability of adult cardiomyocytes to regenerate, transplantation of a donor heart or isolated cardiomyocytes are the only solutions. However, the differentiation of cardiomyocytes from embryonic stem cells causes ethical issues to arise. Hence, it is crucial to obtain stem cells from other sources. Mesenchymal and epithelial cells can be obtained from the lining of umbilical cords. Umbilical cords are collected from hospitals thus ensuring that there is no controversy over the use of stem cells from foetuses. Furthermore, there is a plentiful source of umbilical cord as they are routinely discarded from hospitals. The umbilical cord also contains a significantly larger number of stem cells (several hundred million stem cells per cord) as compared to other sources of stem cells. Hence, it is worth exploring how mesenchymal and epithelial cells from the umbilical cord lining can be utilised for both myogenic and cardiogenic differentiation. From previous studies (Wang, 2004) (Coppi, 2007), 5-azacytidine and 5-aza-2 -deoxycytidine have been shown to induce cardiogenic and myogenic differentiation. The purpose of this research is to determine which combination of media (PP1 and PP3) and cells (CLMC and CLEC) are best suited for cardiogenic or myogenic differentiation. MATERIALS AND METHODS Cell Culture Cells were seeded at a density of 3,000 cells/cm 2 on plastic plates precoated with Matrigel (Collaborative Biomedical Products; incubation for 1 h at 37 o C at 1 mg/ml in DMEM) in two

2 complete mediums, PP1 and PP3. PP1 consists of DMEM low-glucose formulation containing 10% horse serum (Gibco/BRL), 0.5% chick embryo extract (Gibco/BRL) and Pen/Strep (5% antibiotics) (Ferrari, 1998). Twelve hours after seeding, 3 µm 5-azacytidine was added to the culture medium for 24 hours. Incubation is then continued in complete medium lacking 5- azacytidine, with medium changes every 3 days (Rosenblatt, 1995). Cells are harvested at day 1, day 7, day 14 and day 21. PP3 consists of DMEM low-glucose formulation and Pen/Strep. Twelve hours after seeding, 3 µm 5-aza-2 -deoxycytidine was added to the culture medium for 24 hours. Incubation then continued in complete medium lacking 5-azaC, with medium changes every 3 days. Cells are harvested at day 1, day 7, day 14 and day 21. The change in medium is to prevent cell death due to overexposure to 5-azacytidine or 5-azaC. RNA Extraction and Reverse Transcriptase Polymerase Chain Reaction Analysis Total RNA was extracted from treated cells using RNeasy Purification Reagent (Qiagen, Valencia, CA), and then a sample (1µg) was reversed transcribed with dntp and Oligo-dT primer at 65 o C for 5 minutes, and then with 5X buffer, DTT and Superscript at 50 o C for 1 hour, and finally 85 o C for 5 minutes. Polymerase chain reaction (PCR) was performed using specific primers as follows: alpha-actin (), beta-actin (), cardiac actin 1 (), natriuretic peptide precursor A (). PCR was performed for 40 cycles, with an initial 10-minute incubation period at 95 o C. Each cycle consists of denaturation at 95 o C for 15 seconds, annealing at 55 o C for 30 seconds, and elongation at 72 o C for 30 seconds. Products are examined on ethidium bromide-stained 1% agarose gel and photographed under UV light. β-actin was used as RNA input control. Primer Assay Gene Description Actin, Alpha 1 Expressed in skeletal muscles. Major constituent of contractile apparatus. Actin, Beta Non-muscle cytoskeletal actin 1 Actin, Alpha, Cardiac Muscle 1 Natriuretic peptide precursor A Found in muscle tissue, major constituent of contractile apparatus. Absence of which causes cardiomyopathy Expression of gene is the first hallmarks of chamber myocardium formation

3 Table 1. A list of primers, the genes tested and the description of their functions. Medium PP1 PP3 Samples CLEC42 P4 CLMC44 P3 CLEC25 P5 CLEC42 P4 CLMC29 P3 CLMC44 P3 Table 2. A list of cell types and medium used. CLEC: Cord Lining Epithelial Cells CLMC: Cord Lining Mesenchymal Cells RESULTS AND DISCUSSION A B Fig. 1. RT-PCR Analysis of cells treated in PP1 medium for the expression of skeletal muscles and cardiomyocytes genes related genes. Beta-Actin () is used as the RNA input control. Alpha-actin () shows the gene expression for skeletal muscles. Cardiac muscle alpha actin (1) and Natriuretic peptide precursor A () shows the cardiac-specific gene expression. A: Gene expression for Cord Lining Epithelial Cells (CLEC) 42P4. B: Gene expresson for Cord Lining Mesenchymal Cells (CLMC) 44P3. For both CLEC42P4 and CLMC44P3, there is equal level of gene expression from the input control from day 1 to day 21. There is equal loading for day 1 to day 21, and thus we shall take into consideration the level of gene expression for day 1 to day 21 for the other primers.

4 For CLEC42P4, and shows an increase in the level of gene expression from day 1 to day 21. For, there is an increase in the level of gene expression from day 1 to day 14, but there is a decrease in gene expression after day 14. For CLMC44P3, shows no change in the level of gene expression from day 1 to day 21. However, for there is a decrease in the level of gene expression after day 14, and for, there is a decrease in gene expression after day 7. For PP1 medium, the increase in level of gene expression from day 1 through day 21 for proves that CLEC is more suitable for myogenic differentiation, while the increase in level of gene expression from day 1 through day 21 for proves that CLEC is more suitable for cardiogenic differentiation too. For PP1 medium, CLMC is not as suitable as there is either no change or a decrease in gene expression for the primers used. C D E F Fig. 2. RT-PCR Analysis of cells treated in PP3 medium for the expression of skeletal muscles and cardiomyocytes genes related genes. Beta-Actin () is used as the RNA input control. Alpha-actin () shows the gene expression for skeletal muscles. Cardiac muscle alpha actin (1) and Natriuretic peptide precursor A () shows the cardiacspecific gene expression. C: Gene expresson for Cord Lining Mesenchymal Cells (CLMC) 29P3. D: Gene

5 expresson for CLMC44P3. E: Gene expression for Cord Lining Epithelial Cells (CLEC) 42P4. F: Gene expression for CLEC 25P5. For CLMC29P3, CLMC44P3, CLEC 42P4, there is equal level of gene expression from the input control from day 1 to day 21. There is equal loading for day 1 to day 21, and thus we shall take into consideration the level of gene expression for day 1 to day 21 for the other primers. For CLEC25P5, there is a decrease in the level of gene expression from the input control on day 21. This decrease is due to cell death after 14 days of incubation. Thus, we shall only take into consideration the level of gene expression for day 1 to day 14 for the rest of primers. For CLMC29P3 and CLMC44P3, and shows a decrease in the level of gene expression from day 1 to day 21, while shows no change in the level of gene expression. For CLEC42P4,, and shows a constant level of gene expression from day 1 to day 21. For CLEC25P5, the result for day 21 is disregarded as there is a decrease in the level of gene expression from the input control on day 21., and show an increase in the level of gene expression from day 1 to day 14. The results demonstrated that for PP3 medium, CLEC is more suitable for the both myogenic and cardiogenic differentiation. This is because there is a higher level of gene expression for all primers from day 1 to day 14 for CLEC25P5, while there is either a constant or decreased level of gene expression for both CLMC samples. Thus, for both mediums, CLEC is a better candidate for both cardiogenic and myogenic differentiation. Comparing PP1 and PP3, the general level of expression for PP1 is higher than the level of expression for PP3 at day 21. Thus, for future experiments, PP1 instead of PP3 should be used as a complete medium. FUTURE WORKS Different primers for RT-PCR, immunohistochemistry staining and western blotting will be used in future tests to better determine the cell type and medium most suitable for cardiogenic and myogenic differentiation.

6 REFERENCES Coppi, P.D.; Siddiqui, M.M.; Jr, G.B. et al. (2007), "Isolation of amniotic stem cell lines with potential for therapy", Nature Biotechnology, Ferrari, G. et al. (1998), Muscle regeneration by bone marrow-derived myogenic progenitors, Science, Rosenblatt, J.D.; Lunt, A.I.; Parry, D.J. and Partridge, T.A. (1995), Culturing satellite cells from living single muscle fiber explants, In Vitro Cell. Dev. Biol. Anim., Wang, H.S.; Peng, S.T.; Hung, S.C. et al. (2004), Mesenchymal Stem Cells in the Wharton s Jelly of the Human Umbilical Cord, Stem Cells,