Abstract:
:Many studies demonstrated that human adult cardiac progenitor cells in the form of cardiospheres (CSps) could represent a powerful candidate for cardiac cell therapy. To achieve the clinical translation of this biotechnological product, the development of well-defined culture conditions is required to optimize their proliferation and differentiation. Thrombin, a serine protease acting through the protease-activated receptor 1 (PAR-1) signalling to modulate many cellular functions such as proliferation and differentiation in several cell types, is one of the factors included in the CSps medium. Therefore, the assessment of the effective dependence of the thrombin related cellular effects from PAR-signalling is strategic both for understanding the biological potential of these cells and for the GMP translation of the medium formulation, using synthesised analogs. In this study the effects of thrombin on human CSps and their potential relationship with the specific proteolytic activation of PAR-1 have been investigated in different culture conditions, including thrombin inhibitor hirudin and PAR-1 agonist/ antagonist peptides TFLLR and MUMB2. In this study we show that, in the presence of thrombin and TFLLR, CSps, in which PAR-1 expression was evidenced by immunofluorescence and western blot analysis, increase their proliferation activity (BrdU assay). Such increased proliferative rate was consistently associated with a higher phosphorylation level of the cell cycle inhibitor GSK3. Concerning the assessment of the potential effects of thrombin and its agonist on differentiation, both western blot and real-time PCR analysis for stemness, cardiac and vascular markers (such as cKit, cx43 and KDR) showed that CSps commitment was substantially unaffected, except for GATA4 mRNA, whose transcription was down-regulated in the presence of the natural protease, but not after treatment with TFLLR. In conclusion, activation of PAR-1-dependent signalling is important to support CSps proliferative potential, keeping unaltered or at best stable their differentiation properties. The availability of thrombin agonists, such as TFLLR, able to guarantee the required growth effect without affecting CSps lineage commitment, could represent a technological improvement for cost-effective, easy-to-handle and GMPtranslatable synthetic media.
journal_name
J Biol Regul Homeost Agentsjournal_title
Journal of biological regulators and homeostatic agentsauthors
Fabrizi C,Angelini F,Chimenti I,Pompili E,Somma F,Gaetani R,Messina E,Fumagalli L,Giacomello A,Frati Gsubject
Has Abstractpub_date
2011-04-01 00:00:00pages
S43-51issue
2 Suppleissn
0393-974Xissn
1724-6083pii
6journal_volume
25pub_type
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