Title

 

Authors

E. Arrigoni^1,2, E. Conforti^3,4, S. Niada^1,2, D. Stanco^1,5, M. Piccoli^3,4, L. de Girolamo⁵, L. Anastasia^3,4,  A.T. Brini¹.
 
¹ Dept. of Medical Pharmacology, School of Medicine, Università degli Studi di Milano, Italy
² PhD Student of Graduate School in Pharmacological Science, Università degli Studi di Milano,   Italy
³ IRCCS Policlinico San Donato, Milan, Italy
⁴ Dept. of Medical Chem, Biochem and Biotecnology, Università degli Studi di Milano, Italy
⁵ IRCCS Galeazzi Orthopaedic Institute, Italy

 

Abstract

In the recent years, the research on stem cells seems to offer alternative therapies for a variety of diseases such as heart failure, Parkinson's disease, amyotrophic lateral sclerosis, spinal cord injury, diabetes and skeletal-muscle disorders. Mesenchymal stem cells (MSCs) are one of the most studied progenitor cells that possess a good self-renewal capacity and a great ability to in vitro differentiate into a variety of different cell types including bone, cartilage and fat-like cells (de Girolamo et al., 2009; Chagastelles et al., 2010). Since the number of MSCs that actually differentiate into skeletal or cardiac muscles, upon proper chemical and/or physical stimulation, is often low (Miyahara et al.,2006; Mizuno et al., 2009), we have studied the effect of Reversine, a synthetic purine already known to increase plasticity of terminally differentiated cells (Anastasia et al., 2006; Chen et al., 2007; Lee et al., 2009), on human adipose-derived stem cells (ASCs). In this study, hASCs were pre-treated for 72 hours with several concentrations of Reversine and then differentiated towards osteoblast-like cells and smooth and skeletal muscle cells. Cellular growth and viability were monitored, and the effect of Reversine on osteogenic and myogenic differentiation was also analyzed (Conforti et al., submitted). Concentrations of Reversine between 50nM to 1.5μM do not affect hASCs growth, however, the 5µM concentration reduces cells viability of hASCs and it induces drastic changes in cell morphology with an average cell-loss of about 25% in comparison to control cells treated just with vehicle (0.05% DMSO). These results are in contrast with previous published data on fibroblasts (Anastasia et al. 2006), suggesting that hASCs are more sensitive to chemical insults and they may be more easily manipulated in vitro.   
We also show that 50nM Reversine treatment of osteo-differentiated cells upregulates alkaline phosphatase (ALP) gene expression and enzymatic activity of 46% (p<.01) and 44% (p<.05), respectively. Moreover, Reversine seems to improve ASCs myogenic differentiation: indeed, when hASCs are switched into smooth muscle-like cells, we detect an induction of α-actin gene expression (+89%, p<.001), and when we co-culture hASCs with murine C2C12 cells we observed MHC (Myosin Heavy Chain) positive myotubes formation; about 30% of skeletal muscle like-cells are generated by hASCs, detected by specific human-nuclei staining (p<.01).
We conclude that 50nM Reversine treatment seems to improve the hASCs plasticity promoting their differentiation process, and right now it is our interest to investigate its pharmacological target on different cell types.
 
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Mizuno (2009). J Nippon Med Sch.76:56-66.
Chen (2007). Proc Natl Acad Sci U S A104:10482-7.
Lee (2009). J Biol Chem 284:2891-901.
Anastasia (2006). Cell Death Differ 13:2042-51.
Conforti (2011). J Biol Regul Homeost Agents (submitted).