BMP-2 and FGF-2 synergistically facilitate adoption of a cardiac phenotype in somatic bone marrow c-kit+/Sca-1+ stem cells.
The aim of this study was to explore the effect of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2)- paracrine factors implicated in both cardiac embryogenesis and cardiac repair following myocardial infarction (MI)-on murine bone marrow stem cell (mBMSC) differentiation in an ex vivo cardiac microenvironment. For this purpose, green fluorescent protein (GFP) expressing hematopoietic lineage negative (lin-) c-kit ligand (c-kit) and stem cell antigen-1 (Sca-1) positive (GFP-lin-/c-kit+/sca+) mBMSC were co-cultured with neonatal rat ventricular cardiomyocytes (NVCMs). GFP+ mBMSC significantly induced the expression of BMP-2 and FGF-2 in NVCMs, and approximately 4% GFP+ mBMSCs could be recovered from the co-culture at day 10. The addition of BMP-2 in concert with FGF-2 significantly enhanced the amount of integrated GFP+ mBMSCs by 5-fold ( approximately 20%), whereas the addition of anti-BMP-2 and/or anti-FGF-2 antibodies completely abolished this effect. An analysis of calcium cycling revealed robust calcium transients in GFP+ mBMSCs treated with BMP-2/FGF-2 compared to untreated co-cultures. BMP-2 and FGF-2 addition led to a significant induction of early (NK2 transcription factor related, locus 5; Nkx2.5, GATA binding protein 4; GATA-4) and late (myosin light chain kinase [MLC-2v], connexin 43 [Cx43]) cardiac marker mRNA expression in mBMSCs following co-culture. In addition, re-cultured fluorescence-activated cell sorting (FACS)-purified BMP-2/FGF-2-treated mBMSCs revealed robust calcium transients in response to electrical field stimulation which were inhibited by the L-type calcium channel (LTCC) inhibitor, nifedipine, and displayed caffeine-sensitive intracellular calcium stores. In summary, our results show that mBMSCs can adopt a functional cardiac phenotype through treatment with factors essential to embryonic cardiogenesis that are induced after cardiac ischemia. This study provides the first evidence that mBMSCs with long-term self-renewal potential possess the capability to serve as a functional cardiomyocyte precursor through the appropriate paracrine input and cross-talk within an appropriate cardiac microenvironment.
Degeorge
Brent R
BR
Center for Translational Medicine, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
Rosenberg
Marc
M
Eckstein
Volker
V
Gao
Erhe
E
Herzog
Nicole
N
Katus
Hugo A
HA
Koch
Walter J
WJ
Frey
Norbert
N
Most
Patrick
P
D000818:Animals; D000831:Animals, Newborn; D000067696:Ataxin-1; D000067528:Ataxins; D001854:Bone Marrow Cells / Q000166:cytology*; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D018920:Coculture Techniques; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D009203:Myocardial Infarction / Q000235:genetics / Q000473:pathology; D009206:Myocardium / Q000473:pathology*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D038081:Organogenesis / Q000187:drug effects / Q000235:genetics; D010641:Phenotype; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D019089:Stem Cell Factor / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism
D000067696:Ataxin-1; D000067528:Ataxins; C000593750:Atxn1 protein, mouse; C000593751:Atxn1 protein, rat; D055396:Bone Morphogenetic Protein 2; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D019089:Stem Cell Factor; D016212:Transforming Growth Factor beta; D016222:Fibroblast Growth Factor 2; D049452:Green Fluorescent Proteins; D002118:Calcium