Identification of Plateled-derived Growth Factor-BB as Cardiogenesis-Inducing Factor in Mouse Embryonic stem cells under Serum-free Conditions

Abstract

Background/Aims: Embryonic stem (ES) cells may represent an alternative source of functionally mature cardiomyocytes for the treatment of heart diseases. ES cells spontaneously differentiate into spheroidal aggregates, also referred to as embryoid bodies (EBs). The identification of growth factors playing a decisive role in cardiogenesis is a crucial issue for the generation of mature cardiomyocytes. Methods: In order to identify growth factors promoting cardiac development, we established a new differentiation protocol using a defined serum-replacement medium (SRM) containing 5µg/ml insulin and 5µg/ml transferrin in combination with Dulbecco’s Modified Eagle Medium (DMEM). Furthermore, we added platelet-derived growth factor-BB (PDGF-BB) or sphingosine-1-phosphate (SPP) to promote cardiac differentiation. Results: Using SRM/DMEM, we obtained a 6-fold increase of cardiac specific myosin heavy chain α and β (cMHCα/β) in relation to 0,2% foetal calf serum (FCS)/DMEM (= 100%). Stimulation of EBs with PDGF-BB in the presence of SRM/DMEM resulted in a further 2,6-fold enhancement in comparison with the SRM/DMEM-induced increase of cMHCα/β (= 100%). A parallel increase in the number of beating EBs was observed. Similar results were obtained after stimulation of EBs with 5µg/ml SPP. Conclusion: We established a serum-free protocol and identify PDGF-BB and SPP as potent factors promoting cardiogenesis in ES cells.

Embryonic stem cells and cardiomyocyte differentiation: phenotypic and molecular analyses

“…This has led to the use of serum-free media conditions to control ES cell fate. For instance, during mES cells differentiation the expression of cardiac specific α and β MHC genes increased 6-fold in serum replacement medium supplemented with insulin and transferrin (SRIT) as compared to serum-containing medium [41]. Other examples involve the substitution of serum by platelet derived growth factor-BB (PDGF BB) or sphingosine 1 phosphate (SPP), both of which increase the number of beating EBs.…”

Cardiac Repair by Embryonic Stem-Derived Cells

“…For example, a number of studies have implicated signaling of BMP and Wnt family members in cardiomyogenic differentiation of ES cells (Winnier et al 1995;Czyz and Wobus 2001;Behfar et al 2002;Kawai et al 2004;Terami et al 2004). In addition, signaling through the FGF (Dell'Era et al 2003;Kawai et al 2004), IGF-II (Morali et al 2000), Crypto (Parisi et al 2003), PDGFBB (Sachinidis et al 2003), CT-1 (Sauer et al 2004), TGF-beta (Behfar et al 2002), and dynorphin B (Ventura et al 2003) pathways enhanced cardiomyogenesis during EB differentiation. Perhaps not unexpectedly, cytokine/growth factor treatments were occasionally observed to promote cardiomyogenic differentiation via rather indirect mechanisms.…”

Extrinsic regulation of cardiomyocyte differentiation of embryonic stem cells

“…Antin et al [1996] demonstrated that insulin and IGF-II promote cardiac development in vivo by both autocrine and paracrine mechanisms. Sachinidis et al [2003b] reported that PDGF-BB is a potent factor that promotes cardiogenesis in ES cells under serum-free conditions.…”