A 3-D cardiac muscle construct for exploring adult marrow stem cell based myocardial regeneration

Abstract

Adult bone marrow stromal cells (BMSCs) are capable of differentiating into cardiomyocyte-like cells in vitro and contribute to myocardial regeneration in vivo. Consequently, BMSCs may potentially play a vital role in cardiac repair and regeneration. However, this concept has been limited by inadequate and inconsistent differentiation of BMSCs into cardiomyocytes along with poor survival and integration of neo-cardiomyocytes after implantation into ischemic myocardium. In order to overcome these barriers and to explore adult stem cell based myocardial regeneration, we have developed an in vitro model of three-dimensional (3-D) cardiac muscle using rat ventricular embryonic cardiomyocytes (ECMs) and BMSCs. When ECMs and BMSCs were seeded sequentially onto a 3-D tubular scaffold engineered from topographically aligned type I collagen-fibers and cultured in basal medium for 7, 14, 21, or 28 days, the maturation and co-differentiation into a cardiomyocyte lineage was observed. Phenotypic induction was characterized at morphological, immunological, biochemical and molecular levels. The observed expression of transcripts coding for cardiomyocyte phenotypic markers and the immunolocalization of cardiomyogenic lineage-associated proteins revealed typical expression patterns of neo-cardiomyogenesis. At the biochemical level differentiating cells exhibited appropriate metabolic activity and at the ultrastructural level myofibrillar and sarcomeric organization were indicative of an immature phenotype. Our 3-D co-culture system sustains the ECMs in vitro continuum of differentiation process and simultaneously induces the maturation and differentiation of BMSCs into cardiomyocyte-like cells. Thus, this novel 3-D co-culture system provides a useful in vitro model to investigate the functional role and interplay of developing ECMs and BMSCs during cardiomyogenic differentiation.

Figure 1

Immunophenotyping of undifferentiated rat BMSCs by flow cytometry. Single parameter histograms showing the relative fluorescence intensity of staining (abscissa) and the number of cells analyzed, events (ordinate). Isotype controls were included in each experiment to identify the level of background fluorescence. The intensity and distribution of cells stained for hematopoietic and endothelial markers; CD11b, CD31, CD34, CD44, CD45, CD106 and OX43 (black, shaded peaks) were not significantly different from those of isotype control (grey, shaded peaks) (Panels A-E, H-I), indicating that these cultures were devoid of any potential hematopoietic and/or endothelial cells of bone marrow origin. The fluorescent intensity was greater (shifted to right) when BMSCs were stained with CD73 and CD90 (black) compared to isotype control (grey) (Panels F, G). The predominant population of BMSCs consistently expressed CD90 surface molecule, a property of rat bone marrow-derived mesenchymal/stromal stem cells.

Figure 2

Real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of various key cardiogenic markers, α-MHC (myosin, heavy chain6, cardiac muscle, alpha - Myh6), β-MHC (myosin, heavy chain 7, cardiac muscle, beta - Myh7), α-actin cardiac (actin, alpha, cardiac muscle 1 -Actc1), cTnI (troponin I type 3, cardiac - Tnni3), Gata4 (GATA binding protein 4 - Gata4), ANP (natriuretic peptide precursor A – Nppa), BNP (natriuretic peptide precursor B – Nppb) and Cx43 (gap junction protein, alpha 1 – Gja1) as a function of time (abscissa). ECMs cultured in collagen-gel tubular scaffolds (3-D culture) in basal medium (A, B). ECMs and BMSCs co-cultured in collagen-gel tubular scaffolds (3-D culture) in basal medium (C, D). The calibrator control included BMSCs day 0 sample and; the target gene expression was normalized by a non-regulated reference gene expression, Arbp. The expression ratio (ordinate) was calculated using the REST-XL version 2 software. The values are means ± standard errors for three independent cultures (n=3), *p < 0.05; **p < 0.001.

Figure 3

Expression pattern of various cardiomyogenic markers in ECMs containing myotube constructs by confocal microscopy. Localization of key cardiac myocyte phenotypic markers of day 21 embryonic cardiac myocyte (ECM) tube cultures in basal medium demonstrated the expression of structural and contractile proteins, sMHC (A, C), cTnI (D, F), α-actinin (G, I), microfilament, actin (B-C, E-F, H-I, K-L) and the adherent junction protein, N cadherin (J, L). Dual immunostaining of ECMs tube cultures in basal medium revealed areas of elongated and/or round to polyhedral type of cells arranged in multilayers and organized into cord-like or trabecular type of cellular arrangement (A-L). Nuclei of these cells were large and either oval or elongated and fusiform in appearance and were centrally positioned. Cells were also stained for nuclei (blue, DAPI) and fibrillar actin (green, Alexa 488 phalloidin). Merged images (A-L). (A-C, scale bar 100 μm; D-L, scale bar 50 μm).

Figure 4

Expression pattern of various cardiogenic markers in ECMs containing myotube constructs by confocal microscopy. Localization of key cardiac myocyte phenotypic markers of day 21 embryonic cardiac myocyte (ECM) tube cultures in basal medium demonstrated the expression of transcription factor, Gata4 (A, C), peptide hormone, ANP (D, F), structural intermediate filament, desmin (G, I), gap junction protein, Cx43 (J, L) and the microfilament, actin (B-C, E-F, H-I, K-L). Dual immunostainings of ECMs in the tubular construct showed areas of cells on the exterior surface that were aligned and overlapping in an orderly manner (A-C, G-I), whereas the cells on the luminal surface were elongated and/or round to polygonal in nature and were arranged in multilayers (D-F). Nuclei of these cells were large and either oval or elongated and fusiform in appearance and were centrally positioned. Cells were also stained for nuclei (blue, DAPI) and fibrillar actin (green, Alexa 488 phalloidin). Merged images (A-L). (A-C, G-L scale bar 50 μm; D-F scale bar 100 μm).

Figure 5

Expression pattern of various cardiogenic markers in BMSCs/ECMs containing myotube constructs by confocal microscopy. Localization of key cardiac myocyte phenotypic markers of day 21 embryonic cardiac myocytes (ECMs) and bone marrow stromal cells (BMSCs) tube co-cultures in basal medium demonstrated the expression of structural and contractile proteins, sMHC (A, C), cTnI (D, F), α-actinin (G, I), microfilament, actin (B-C, E-F, H-I, K-L) and the adherent junction protein, N cadherin (J, L). The myofibers or trabeculae of cells were arranged in a pseudosyncytial pattern (A-C, E-F, J-L). Specialized cell junctions such as intercalated disk were noticed along with characteristic branching and cross bridging of these trabeulated cells (white arrows, J-L). Nuclei of these cells were large and either oval or elongated and fusiform in appearance and were centrally located. Cells were also stained for nuclei (blue, DAPI) and fibrillar actin (green, Alexa 488 phalloidin). Images (A-C) show a projection representing 9 sections collected at 2 μm intervals (16 μm). Merged images (A-L). (A-L scale bar 50 μm).

Figure 6

Expression pattern of various cardiogenic markers in BMSCs/ECMs containing myotube constructs by confocal microscopy. Localization of key cardiac myocyte phenotypic markers of day 21 embryonic cardiac myocytes (ECMs) and bone marrow stromal cells (BMSCs) tube co-cultures in basal medium demonstrated the expression of transcription factor, Gata4 (A, C), peptide hormone, ANP (D, F), structural intermediate filament, desmin (G, I), gap junction protein, Cx43 (J, L) and the microfilament, actin (B-C, E-F, H-I, K-L). The co-differentiating cells appeared as parallely arranged trabeculae and displayed the characteristic branching and cross bridges to give a pseudosyncytial arrangement (G-L). These trabeculae displayed specialized cell junctions, the intercalated disks (white arrows, G-I). Nuclei of these cells were large and either oval or elongated and fusiform in appearance and were centrally located. Cells were also stained for nuclei (blue, DAPI) and fibrillar actin (green, Alexa 488 phalloidin). Images (J-L) show a projection representing 9 sections collected at 3 μm intervals (24 μm). Merged images (A-L). (A-C, G-L scale bar 50 μm; D-F scale bar 100 μm).

Figure 7

Expression pattern of various cardiogenic markers in GFP-BMSCs/ECMs containing myotube constructs by confocal microscopy. Immunostaining of key cardiac myocyte phenotypic markers of day 21 embryonic cardiac myocytes (ECMs) and green fluorescent protein labeled bone marrow stromal cells (GFP-BMSCs) tube co-cultures in basal medium demonstrated the expression of contractile proteins, α/β-MHC (A-C), cTnI (G-I), the adherent junction protein, N cadherin (D-F) and GFP (A, C, D, F, G, I). The transdifferentiated GFP-BMSCs were delineated by their co-localization signals (yellow). Nuclei of these cells were large and either oval or elongated and fusiform in appearance and were centrally placed. Cells were also stained for nuclei (blue, DAPI) and GFP-BMSCs (green, GFP). Images (G-I) show a projection representing 16 sections collected at 10 μm intervals (150 μm). Merged images (A-I). (A-I scale bar 50 μm).

Figure 8

Biochemical analysis of cardiomyogenic differentiation of BMSCs/ECMs co-culture in 3-D collagen-gel tubular scaffold. Quantitative analysis of ANP and BNP hormones in myotube culture supernatants over a period of 14 days measured by enzyme linked immunosorbent assay, ELISA. (A) Quantification of ANP (Nppa protein) levels was compared between ECMs only cultures and BMSCs/ECMs co-cultures. *p < 0.05. The data represent the mean ± SEM of triplicate cultures (n = 3). (B) Quantification of BNP (Nppb protein) levels was compared between ECMs only cultures and BMSCs/ECMs co-cultures. *p < 0.05. The data represent the mean ± SEM of triplicate cultures (n = 3).

Figure 9

Nuclear morphometric analysis of BMSCs, ECMs and ECMs/BMSCs cultured in the tubular scaffold under basal conditions. The descriptors of nuclear morphology, viz., area - A (A), perimeter - P (B), the equivalent oblate volume – EOV (C) and the elliptical form factor – EFF (D) were measured by the imaging software for each thresholded and image isolated nucleus. Values of A, P, EFF and EOV were calculated directly from the integrated morphometry subroutine of Meta-Morph image analysis software. The nuclear parameters (A, P, EOV and EFF) of BMSCs nuclei were significantly greater than the nuclear parameters of ECMs and ECMs/BMSCs nuclei (p < 0.001). There were no differences between the nuclear parameters (P, EOV and EFF) for the ECMs and ECMs/BMSCs nuclei (p = 0.101, p = 0.426, p = 0.657), except the nuclear area (ECMs > ECMs/BMSCs, p = 0.008). The data represent the means ± SEM. *p < 0.05; **p < 0.001.

Figure 10

Transmission electron microscopic (TEM) analysis of day 21 tubular constructs. (A) BMSCs cultured in tubular constructs in basal medium revealed euchromatic nuclei (N), cytoplasmic pinocytotic vesicles (p) and junctional complexes (gj, fa), the ultrastructural characteristics of active, immature cells. (B) ECMs under the same culture conditions showed active euchromatic nuclei (N) with nucleolus (n). The cytoplasm revealed myofibrils (mf) myofilaments (f), early sarcomeric units, Z-bodies (Zb) and Z-disks (Z) and numerous mitochondria (mi). (C-D) BMSCs/ECMs co-culture under similar culture conditions demonstrated cytoplasmic myofilaments (f), Z-bodies (Zb), pinocytotic vesicles (p), mitochondria (mi) and numerous dense bodies (Db).