FGF-2 controls the differentiation of resident cardiac precursors into functional cardiomyocytes

Abstract

Recent evidence suggests that the heart possesses a greater regeneration capacity than previously thought. In the present study, we isolated undifferentiated precursors from the cardiac nonmyocyte cell population of neonatal hearts, expanded them in culture, and induced them to differentiate into functional cardiomyocytes. These cardiac precursors appear to express stem cell antigen-1 and demonstrate characteristics of multipotent precursors of mesodermal origin. Following infusion into normal recipients, these cells home to the heart and participate in physiological and pathophysiological cardiac remodeling. Cardiogenic differentiation in vitro and in vivo depends on FGF-2. Interestingly, this factor does not control the number of precursors but regulates the differentiation process. These findings suggest that, besides its angiogenic actions, FGF-2 could be used in vivo to facilitate the mobilization and differentiation of resident cardiac precursors in the treatment of cardiac diseases.

Figure 1

Sca-1 expression in cardiac tissues from WT (FGF-2^+/+) or FGF-2–deficient (FGF-2^–/–) mice. (A) Sca-1 immunostaining in heart sections from FGF-2^+/+ and FGF-2^–/– neonatal and adult mice with or without renovascular hypertension and cardiac hypertrophy (2-kidney 1-clip model [2K1C]). Original magnification, ×20 and ×63 (insets). (B) Immunodetection of Sca-1 and troponin I in the hearts of adult FGF-2^+/+ mice. Original magnification, ×40 (left panels), ×63 (right panels). (C) Percentage of Sca-1⁺ and c-kit⁺ cells as determined by FACS analysis in total heart cells (Total) and in the cardiomyocyte (CM) and nonmyocyte cell (NMC) fractions from FGF-2^+/+ and FGF-2^–/– mice (n = 5). (D) Percentage of Sca-1⁺ cells as determined by FACS analysis in neonatal or adult NMCs isolated from FGF-2^+/+ (white bars) and FGF-2^–/– mice (black bars). NMCs were either depleted or not depleted of cells expressing CD45, CD31, CD34, Flk-1, CD3, CD4, CD8, B220, TER119, Mac-1, and Gr-1. *P < 0.05; NS, not significant as compared with the corresponding nondepleted group; n = 3–6.

Figure 2

Characterization of the NMC population before differentiation. (A) Quantification by FACS analysis of neonatal and adult NMCs from FGF-2^+/+ (white bars) and FGF-2^–/– mice (black bars) expressing CD31, Flk-1, CD45, CD34, TER119, Mac-1, Gr-1, CD3, Sca-1, and c-kit after expansion in vitro (n = 5–12). (B) RT-PCR analysis of cardiac-specific gene expression as well as Sca-1, c-kit, and islet-1 in NMCs after expansion and before differentiation (BD), in neonatal cardiomyocytes (Neo CM) and in the adult heart (Ad heart) from either FGF-2^+/+ or FGF-2^–/– mice. C-actin, cardiac actin. (C) RT-PCR analysis of Snail and cadherin expression in expanded NMCs (BD), as well as in the neonatal (Neo heart) and adult hearts (Ad heart) from FGF-2^+/+ or FGF-2^–/– mice.

Figure 3

Cardiogenic differentiation of Sca-1⁺ cells in vitro. (A–L) Immunodetection of Sca-1 and troponin I in differentiating NMCs isolated from FGF-2^+/+ mice in the absence (None) or presence of FGF-2 and maintained for 1 (A–C), 2 (D–F), or 3 weeks (G–I) in differentiation medium. (M–R) The same experiment using NMCs isolated from FGF-2^–/– mice maintained for 3 weeks in differentiation medium. Original magnification, ×40.

Figure 4

Differentiation of cardiac precursors into functional cardiomyocytes. (A) RT-PCR analysis of cardiac-specific gene expression in NMCs from FGF-2^+/+ or FGF-2^–/– mice maintained in control (C) or differentiation medium (D) in the absence of FGF-2 (None), after 1 week or 3 weeks in culture. (B) Quantification of troponin I⁺ cells and spontaneously contracting cells in culture of differentiated cardiac precursor from FGF-2^+/+ and FGF-2^–/– mice after 3 weeks in control medium (white bars) or differentiation medium (black bars); n = 6–10. *P < 0.05. (C) RT-PCR analysis of cardiac-specific gene expression in NMCs from FGF-2^+/+ or FGF-2^–/– mice maintained in control or differentiation medium in the presence of FGF-2 or FGF-1 after 1 week or 3 weeks in culture.

Figure 5

Multipotent capacity of cardiac precursors. (A) Oil red staining and RT-PCR analysis of PPARγ and lipoprotein lipase (LPL) expression in cultures of NMCs induced to differentiate into adipocytes. (B) Immunodetection of MyoD1 and RT-PCR analysis of MyoD1 expression in cultures of NMCs induced to differentiate into skeletal myoblasts. (C) Immunodetection of osteocalcin and RT-PCR analysis of osteocalcin expression in cultures of NMCs induced to differentiate into osteoblasts. Original magnification, ×20 (A and C), ×30 (C, inset), ×40 (B), ×63 (B, inset).

Figure 6

Factor expression in NMCs. RT-PCR analysis of factor expression in NMCs from FGF-2^+/+ and FGF-2^–/– mice after expansion in vitro (before differentiation) as well as after 3 weeks in control and differentiation medium.

Figure 7

Cell transfer into neonatal mouse recipients. (A) PKH2-GL–labeled cells or cells constitutively expressing the GFP gene under the chicken β-actin promoter (GFP) were injected i.v. into neonatal mice. Ten days thereafter, heart sections were stained for α-actinin. (B) PKH2-GL–labeled FGF-2^+/+ or FGF-2^–/– NMCs were injected into neonatal FGF-2^+/+ or FGF-2^–/– mice. Ten days after injection, heart sections were stained for α-actinin. (C) WT PKH2-GL–labeled NMCs were injected into neonatal ROSA26 transgenic mice, and heart sections were stained for β-gal. Original magnification, ×63 (A and C), ×20 (B).

Figure 8

Cell transfer into adult mouse recipients. (A) PKH2-GL–labeled cells isolated from WT hearts were injected into adult FGF-2^+/+ mice. Original magnification. (B) Troponin I expression of differentiated precursors in WT recipient hearts. Two weeks after cell transfer, heart sections were stained for α-actinin. (C) NMCs isolated from ROSA26 transgenic mice were injected into adult FGF-2^+/+ mice. Sections were immunostained for β-gal 4 weeks after transfer. (D) RT-PCR analysis of LacZ gene expression in hearts from FGF-2^+/+ and FGF-2^–/– mice after transfer of NMCs from ROSA26 transgenic mice (Transferred). None, no cells transferred. Magnification, ×20 (A), ×100 (B), ×40 (C).