Rat hair-follicle-associated pluripotent (HAP) stem cells can differentiate into atrial or ventricular cardiomyocytes in culture controlled by specific supplementation

by Nanako Takaoka, Michiko Yamane, Ayami Hasegawa, Koya Obara, Kyoumi Shirai, Ryoichi Aki, Hiroyasu Hatakeyama, Yuko Hamada, Nobuko Arakawa, Manabu Tanaka, Robert M. Hoffman, Yasuyuki Amoh

There has been only limited success to differentiate adult stem cells into cardiomyocyte subtypes. In the present study, we have successfully induced beating atrial and ventricular cardiomyocytes from rat hair-follicle-associated pluripotent (HAP) stem cells, which are adult stem cells located in the bulge area. HAP stem cells differentiated into atrial cardiomyocytes in culture with the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and cyclosporine A (CSA). HAP stem cells differentiated into ventricular cardiomyocytes in culture with the combination of activin A, BMP4, bFGF, inhibitor of Wnt production-4 (IWP4), and vascular endothelial growth factor (VEGF). Differentiated atrial cardiomyocytes were specifically stained for anti-myosin light chain 2a (MLC2a) antibody. Ventricular cardiomyocytes were specially stained for anti-myosin light chain 2v (MLC2v) antibody. Quantitative Polymerase Chain Reaction (qPCR) showed significant expression of MLC2a in atrial cardiomyocytes and MLC2v in ventricular cardiomyocytes. Both differentiated atrial and ventricular cardiomyocytes showed characteristic waveforms in Ca²⁺ imaging. Differentiated atrial and ventricular cardiomyocytes formed long myocardial fibers and beat as a functional syncytium, having a structure similar to adult cardiomyocytes. The present results demonstrated that it is possible to induce cardiomyocyte subtypes, atrial and ventricular cardiomyocytes, from HAP stem cells.

Asymmetric and symmetric protein arginine methylation in methionine-addicted human cancer cells

by Ashley G. Holtz, Troy L. Lowe, Yusuke Aoki, Yutaro Kubota, Robert M. Hoffman, Steven G. Clarke

The methionine addiction of cancer cells is known as the Hoffman effect. While non-cancer cells in culture can utilize homocysteine in place of methionine for cellular growth, most cancer cells require exogenous methionine for proliferation. It has been suggested that a biochemical basis of this effect is the increased utilization of methionine for S-adenosylmethionine, the major methyl donor for a variety of cellular methyltransferases. Recent studies have pointed to the role of S-adenosylmethionine-dependent protein arginine methyltransferases (PRMTs) in cell proliferation and cancer. To further understand the biochemical basis of the methionine addiction of cancer cells, we compared protein arginine methylation in two previously described isogenic cell lines, a methionine-addicted 143B human osteosarcoma cell line and its less methionine-dependent revertant. Previous work showed that the revertant cells were significantly less malignant than the parental cells. In the present study, we utilized antibodies to detect the asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) products of PRMTs in polypeptides from cellular extracts and purified histone preparations of these cell lines fractionated by SDS-PAGE. Importantly, we observed little to no differences in the banding patterns of ADMA- and SDMA-containing species between the osteosarcoma parental and revertant cell lines. Furthermore, enzymatic activity assays using S-adenosyl-ʟ-[methyl-³H] methionine, recombinantly purified PRMT enzymes, cell lysates, and specific PRMT inhibitors revealed no major differences in radiolabeled polypeptides on SDS-PAGE gels. Taken together, these results suggest that changes in protein arginine methylation may not be major contributors to the Hoffman effect and that other consequences of methionine addiction may be more important in the metastasis and malignancy of osteosarcoma and potentially other cancers.