Mesenchymal Stern Cells from Adult Human Bone Marrow Differentiate into a Cardiomyocyte Phenotype In Vitro

Abstract

A method for isolating adult human bone marrow mesenchymal stem cells (MSCs) was established, and the ability of human MSCs to differentiate into cells with characteristics of cardiomyocytes in vitro was investigated. Selected MSC surface antigens were analyzed by flow cytometry. The MSCs at Passage 2 were treated with 5-azacytidine to investigate their differentiation into cardiomyocytes. Characteristics of the putative myogenic cells were determined by immunohistochemistry and transmission electron and confocal microscopies. The expression of myogenic specific genes was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time quantitative PCR, and DNA sequencing. The MSCs were spindle-shaped with irregular processes and were respectively positive for CD(13), CD(29), CD(44), CD(71) and negative for CD(3), CD(14), CD(15), CD(33), CD(34), CD(38), CD(45), and HLA-DR. The myogenic cells differentiated from MSCs were positive for beta-myosin heavy chain (beta-MHC), desmin, and alpha-cardiac actin. When the myogenic cells were stimulated with low concentration of K(+) (5.0 mM), an increase in intracellular calcium fluorescence was observed. Myofilament-like structures were observed in electron micrographs of the differentiated myogenic cells. The mRNAs of beta-MHC, desmin, alpha-cardiac actin, and cardiac troponin T were highly expressed in the myogenic cells. These results indicate that 5-azacytidine can induce human MSCs to differentiate in vitro into cells with characteristics commonly attributed to cardiomyocytes. Cardiomyocytes cultured from bone marrow sources are potentially valuable for repairing injured myocardium.

5-Azacytidine induces changes in electrophysiological properties of human mesenchymal stem cells

“…It is noticeable that 5-azacytidine did not exhibit any significant acute toxicity in the micromolar range of concentrations, which were postulated to induce cardiogenesis [9,14,15].…”

“…Some authors reported successful differentiation of hMSC with concentrations between 5 and 10 µM [14,15], suggesting that human cells may require higher concentrations than their murine counterparts, possibly because of higher activity of cytidine deaminase which degrades 5-azacytidine [47]. It must be emphasized, however, that there are also reports of lack of differentiation in rat MSC even with concentrations of 10 µM 5-azacytidine [13].…”

“…However, conflicting results have been published with respect to MSC from other species. In rat MSC, 5-azacytidine was ineffective in promoting cell expansion Cell Research | www.cell-research.com 5-Azacytidine and human mesenchymal stem cells 950 npg or differentiation into cardiomyocytes [13], whereas other studies have claimed to obtain cardiomyocytes from hMSC following induction with the drug [14,15].…”

Progress and prospects: Cell based regenerative therapy for cardiovascular disease

“…Despite these differences in origin, several studies show that bone marrow-derived mesenchymal progenitor cells can acquire a cardiomyocyte phenotype under certain conditions in vitro and in vivo. These cells are capable of differentiating into cells with cardiomyocyte features in vitro, either through coculture with cardiomyocytes 44 or exposure to 5-azacytidine, 45 a cysteine analog capable of altering the expression of certain genes that may regulate differentiation. The cells are positive for b-myosin heavy chain, cardiac troponin T, a-cardiac actin and desmin.…”

“…The cells are positive for b-myosin heavy chain, cardiac troponin T, a-cardiac actin and desmin. 45 Furthermore, these cells exhibit Ca 2+ transients, several types of action potentials and respond to a and b adrenergic stimulation with an increase in contractility, phosphorylation of ERK1/2 and an increase in cAMP. 45 After injection in vivo, mesenchymal progenitor cells tagged with 99m Tc 46 were detected in the heart, and the lac-Z positive cells showed phenotypic features of cardiomyocytes on immunohistochemical examination.…”