c-kit expression identifies cardiovascular precursors in the neonatal heart

Abstract

Directed differentiation of embryonic stem cells indicates that mesodermal lineages in the mammalian heart (cardiac, endothelial, and smooth muscle cells) develop from a common, multipotent cardiovascular precursor. To isolate and characterize the lineage potential of a resident pool of cardiovascular progenitor cells (CPcs), we developed BAC transgenic mice in which enhanced green fluorescent protein (EGFP) is placed under control of the c-kit locus (c-kit(BAC)-EGFP mice). Discrete c-kit-EGFP(+) cells were observed at different stages of differentiation in embryonic hearts, increasing in number to a maximum at about postnatal day (PN) 2; thereafter, EGFP(+) cells declined and were rarely observed in the adult heart. EGFP(+) cells purified from PN 0-5 hearts were nestin(+) and expanded in culture; 67% of cells were fluorescent after 9 days. Purified cells differentiated into endothelial, cardiac, and smooth muscle cells, and differentiation could be directed by specific growth factors. CPc-derived cardiac myocytes displayed rhythmic beating and action potentials characteristic of multiple cardiac cell types, similar to ES cell-derived cardiomyocytes. Single-cell dilution studies confirmed the potential of individual CPcs to form all 3 cardiovascular lineages. In adult hearts, cryoablation resulted in c-kit-EGFP(+) expression, peaking 7 days postcryolesion. Expression occurred in endothelial and smooth muscle cells in the revascularizing infarct, and in terminally differentiated cardiomyocytes in the border zone surrounding the infarct. Thus, c-kit expression marks CPc in the neonatal heart that are capable of directed differentiation in vitro; however, c-kit expression in cardiomyocytes in the adult heart after injury does not identify cardiac myogenesis.

Fig. 1.

Generation and evaluation of c-kit^BAC-EGFP mice. (A) BAC-targeting the c-kit initiation codon; Neo cassette removed before DNA injection. Arrows indicate PCR genotyping primers. (B) 1.1-kb PCR product in founder (lane 1); water and WT in lanes 2 and 3; 1-kb DNA ladder in lane 4. (C) Bright-field and fluorescent images of PN0 c-kit^BAC-EGFP pups. (D) EGFP expression in adult tissues shows predicted pattern of expression. (E) Immunolocalization of EGFP in Leydig and outer region of spermatogonium (arrow) in adult testis, stellate and basket cells in the molecular layer of the cerebellum, and selective hippocampal neurons. (Scale bars: C, 1 mm; D, testis, 1 mm, and others, 100 μm; E, testis, 25 μm and brain, 50 μm.)

Fig. 2.

EGFP expression in c-kit^BAC-EGFP mouse hearts. (A) EGFP expression in the heart wall of 14.5-dpc embryo (fluorescence) and PN 3 (immuno) neonates. In the adult heart rare immunostained cells were observed. Control, no 1° antibody. (B) EGFP/c-kit expression concordance. Anti-EGFP (green); anti-CD117 (red). Note lack of coexpression (Upper) and colocalization within a niche (Lower). (C) Native EGFP expression in the atrioventricular region of a PN 2 heart. (D) Some cells coexpressed Flk-1 and EGFP. (E) Some c-kit⁺ cells coexpress PECAM1, indicating endothelial commitment. Very bright EGFP-expressing cells were commonly observed in the epicardium; these cells commonly coexpressed the hematopoietic lineage marker CD45. (F) c-kit-EGFP⁺ cells in the atrioventricular region showing a gradient of EGFP expression, from less differentiated bright cells (arrows), to less fluorescent striated cells. A, atrium; AV, atrioventricular region; and V, ventricle. Hoechst staining (blue) (B, D–F). (Scale bars: A, 14.5 dpc, 50 μm, and others, 25 μm; B, 10 μm; C, 50 μm; D, 25 μm; D Inset, 10 μm; E Left, 25 μm; E Center, 20 μm; E Right, 5 μm; F, 25 μm.)

Fig. 3.

Isolation and expansion of FACS sorted cardiac c-kit-EGFP cells. (A) Representative FACS from PN c-kit^BAC-EGFP and WT littermate hearts. WT and EGFP histograms were normalized to same maximum. tEGFP overlaps with brightly autofluorescent WT cells; sEGFP identifies a pure c-kit-EGFP⁺ population of cells 5 times brighter than background fluorescence. (B) Morphometry and FACS estimate of EGFP⁺ cells in neonatal hearts. (C) EGFP expression and PCNA (red) staining indicates cycling in c-kit sEGFP⁺ cells cultured for 9 days in basic media. Arrow indicates PCNA staining. (D) EGFP expressing cells at 24 h (Left) and 9 days (Center) post-FACS. Note differences in cell size and shape. At right tEGFP cells plated on feeders expand rapidly (3 days) in a network pattern. (E) Merged bright-field and fluorescent image show after 1 day in culture some tEGFP cells form clusters. (F) Images taken from the same field of 9 days (Left) and 16 days (Right) post-sEGFP⁺ plating in basic media shows expansion of cells. Images are merged fluorescent and bright field. Some cells at both 9 and 16 days are no longer fluorescent. (G) Distribution of committed cells 24 h post-FACS (tEGFP cells). Note lack of endothelial cells (PECAM1) and equal distribution of smooth muscle (αSMA) and cardiac cells (troponin T). Nestin staining suggests a population of undifferentiated cells. Number of experiments is indicated in bars. (H) Costaining 9–16 days post-FACS (sEGFP cells). (I) c-kit (green) cell expressing both αSMA (blue) and troponin T (red) markers. Merge of αSMA and troponin T yields purple. (J) Nestin (red) and c-kit (green) costaining in cells cultured for 9 days. (H and I) DAPI staining (blue). (Scale bar: C, 20 μm; D–F, H, and J, 50 μm; I, 10 μm.)

Fig. 4.

Cardiac phenotype of spontaneously beating c-kit⁺-EGFP cells. (A) Single, contractile EGFP expressing cell at left in single frame from a time series. Line through image indicates area used for sequential scans of fluorescence shown at right. Left edge of line scan shows rhythmic contraction of the top left cell extension at ≈2 Hz. (Scale bar: 25 μm.) (B) APs ranged from nodal-like (Top), to more atrial (Middle) or ventricular (Bottom) morphologies. Note prominent diastolic depolarization phases. Networked c-kit-EGFP⁺ cells fired rhythmical burst-like activity, interrupted by periods of quiescence accompanied by prominent membrane hyperpolarization. (C) TTX (10 μM, n = 5) or (D) Nifedipine (1 μM, n = 5), reversibly abolished APs.

Fig. 5.

c-kit^BAC-EGFP neonatal heart cells are cardiovascular precursors. (A) Modification of commitment outcomes by addition of specific growth factors. Number of experiments indicated in bars. (B) Clonal expansion showing a single EGFP⁺ cell at day 3 (Left) and the clone after 20 days (Right). (C) Immunostaining of clones from a single EGFP⁺ cell after 21 days. Individual cells showing commitment to all 3 heart lineages. (D) Coimmunostaining of clonal populations demonstrates commitment to cardiac (Left) or smooth muscle (Right) and endothelium in the same clone. DAPI staining (blue) in C. (Scale bars: B–D, 50 μm.)

Fig. 6.

Reexpression of c-kit-EGFP after injury in the adult heart. (A) (Left) Stefanini-fixed heart 7 days postsurgery. Note the large number of EGFP⁺ cells at the border zone and within the infarcted (darker) area. (Right) Rare fluorescent cells in sham heart. (B) EGFP cells within infarct. (Left) Fluorescence wide-field image of adult c-kit^BAC-EGFP heart 14 days postcryoablation. Note fluorescent cells lining vessel. (Right) c-kit-EGFP⁺ endothelial cells lining an artery 7 days postinjury are identified by PECAM1 staining; surrounding smooth muscle cells also express EGFP. (C) EGFP⁺ cardiomyocytes at border zone. (Left) Fluorescent and nonfluorescent striated cardiomyocytes. (Center and Right) Clusters of EGFP⁺ myocytes at border zone 7 days postinfarct. (D) Lack of pHH3 in EGFP⁺ cardiac myocytes. (Left) EGFP⁺ and EGFP⁻ myocytes at border zone. (Right) Single pHH3⁺ nonmyocyte within infarct. (Scale bars: A Left, 360 μm; A Right, 280 μm; B Left, 250 μm; B Inset, 100 μm; B Right, 10 μm; C, 22 μm; D Left, 14 μm; D Right, 16 μm.)