BMP-2 and FGF-2 synergistically facilitate adoption of a cardiac phenotype in somatic bone marrow c-kit+/Sca-1+ stem cells

Abstract

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.

Keywords: BMP-2; FGF-2; mBMSCs; paracrine signalling.

Figure 1

Isolation and purification of somatic Lin– c‐kit+/Sca‐1+ mBMSC for use in co‐culture onto cardiac myocytes. (A–L) Representative FACS analysis: A–E depict the characterization of (A) total (unsorted) bone marrow for the presence of (C) c‐kit and (D) Sca‐1 using PE‐conjugated rat anti‐mouse c‐kit and FITC‐conjugated rat anti‐mouse Sca‐1, respectively. F–J show lineage marker depleted (Lin) cell populations. (K) The Lin– fraction was further enriched by FACS and re‐sorted (L) for the presence of both c‐kit and Sca‐1. The Lin– c‐kit+/Sca‐1+ mBMSC population was used for the co‐culture experiments. Autofluorescence served to select the sorting thresholds.

Figure 2

Effect of myocardial infarction on mRNA levels of TGF‐β, BMP‐2, and FGF‐2 in adult murine hearts. (A) Representative pictures of a sham (top) and an infarcted (bottom) dissected 2,3,5‐triphenyl tetrazolium chloride (TTC)‐stained heart. (B–E) Semiquantitative mRNA expression of (B) BNP, (C) TGF‐β, (D) BMP‐2, and (E) FGF‐2, respectively, in the ischemic myocardium (MI) and residual myocardium (MR) compared to sham (S). n= 55 hearts in each group. *p < 0.05 versus sham, **p < 0.05 versus MI or MR.

Figure 3

Effect of mBMSCs/NVCMs co‐culture on mRNA levels of distinct BMP and FGF isoforms. (A–C) Representative fluorescent microscopy pictures of mBMSCs co‐cultured onto 10‐fold excess of NVCMs for 48 hours demonstrate a rare integration of GFP+ mBMSCs into the contracting monolayer of NVCMs (A transmission, B 510‐nm emission, C overlay) (magnification 10‐fold). (D–F) Semiquantitative analysis of BMP‐2, BMP‐4, and FGF‐2 mRNA levels from monocultured mBMSCs and NVCMs (control) and enzymatically dissociated co‐cultered mBMSCs and NVCMs populations. n= 4 independent experiments. *p < 0.05 versus control NVCMs, **p < 0.05 versus control BMSCs.

Figure 4

Effect of BMP‐2 and FGF‐2 on mBMSC incorporation following co‐culture with NVCMs. (A) Representative pictures of co‐cultured GFP+ mBMSCs with a 10‐fold excess of NVCMs in the absence (control) and presence of BMP‐2/FGF‐2. (B) Quantitative analysis of recovered GFP+ cells by enzymatic dissociation and FACS. n= 4 independent experiments. *p < 0.05 versus GFP+ cells/BMP‐2/FGF‐2 absence (–).

Figure 5

Effect of combined BMP‐2/FGF‐2 treatment on calcium cycling in mBMSCs during NVCM co‐culture. (Top) Representative tracings of field‐stimulated calcium transients in co‐cultured NVCM and mBMSCs. Note that BMP‐2/FGF‐2 treatment enhances calcium cycling in the co‐cultured mBMSCs. (Bottom) Quantitative analysis of calcium transient amplitudes in co‐cultured NVCMs and mBMSCs in the absence and presence of BMP‐2/FGF‐2. n= 4 independent experiments. *p < 0.05 versus mBMSC.

Figure 6

Analysis of cardiac marker expression in mBMSC in response to NVCM co‐culture and BMP‐2/FGF‐2 treatment. (A–F) Quantitative analysis of Nkx2.5, GATA‐4, MLC‐2v, Cx‐43, c‐kit, and Sca‐1 gene expression in NVCMs, monocultured mBMSC, and co‐cultured BMSC in the absence and presence of BMP‐2/FGF‐2. n= 4 independent experiments. *p < 0.05 versus mBMSC, **p < 0.05 versus co‐cultured BMSC.

Figure 7

Assessment of calcium handling in re‐cultured mBMSC‐derived GFP+ cells. Representative pictures of re‐cultured mBMSC‐derived GFP+ cells co‐cultured in the absence (A/A′) and presence of BMP‐2/FGF‐2 during co‐culture (B/B′). (C) Representative tracings of field‐stimulated calcium transients (1 Hz, 2 mM extracellular calcium) in re‐cultured mBMSC‐derived GFP+ cells from control (top tracing) and of BMP‐2/FGF‐2‐treated co‐cultures (bottom tracing) under the basal conditions (left) and in response to caffeine (10 mM) (right). (D) Statistical analysis of calcium transient amplitudes in re‐cultured mBMSC‐derived GFP+ cell populations under the basal conditions and in response to nifedipine (15 mM). n= 4 independent experiments. *p < 0.05 versus control GFP+ cells.