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Circulation Research. 1996;78:205-216

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(Circulation Research. 1996;78:205-216.)
© 1996 American Heart Association, Inc.

Articles

Establishment of the Mesodermal Cell Line QCE-6

A Model System for Cardiac Cell Differentiation

Carol A. Eisenberg, David M. Bader

From the Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY.

Correspondence to Dr Carol A. Eisenberg, Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, SC 29425.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Abstract The QCE-6 cell line was derived from precardiac mesoderm of the Japanese quail. As previously reported, these cells are able to differentiate into two distinct cardiac cell types with myocardial or endocardial endothelial cell properties. This present communication describes in detail the derivation of this cell line and further characterizes the nontreated and induced myocardial and endothelial phenotypes of these cells. The QCE-6 cells exhibit an epithelial morphology, as well as the pattern of protein expression, that is characteristic of precardiac mesoderm. Treatment with retinoic acid, basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-ß2, and TGF-ß3 induces these cells to differentiate and produce mixed cultures of epithelial and mesenchymal cells. The epithelial cells express myosin, desmin, and cardiac troponin I in a punctate pattern throughout the cytoplasm. These sarcomeric proteins become organized in a premyofibrillar pattern when TGF-ß1, platelet-derived growth factor (PDGF)-BB, and insulin-like growth factor (IGF) II are added in combination along with retinoic acid, bFGF, TGF-ß2, and TGF-ß3. Also, these treatments induce Na+,K+-ATPase expression. When the QCE-6 cells are cultured on collagen type I, the mesenchymal cells that are promoted by retinoic acid, bFGF, TGF-ß2, and TGF-ß3 will invade the gel. These mesenchymal cells are positive for QH1 and JB3, which are both markers for presumptive endocardial cells within the early cardiogenic mesoderm. The addition of both PDGF-BB and IGF II to QCE-6 cell cultures will inhibit the ability of retinoic acid, bFGF, TGF-ß2, and TGF-ß3 to induce both the mesenchymal morphology and QH1 and JB3 expression. Collectively, these results suggest that the process of cardiac cell differentiation is regulated by multiple signals and that early cardiogenic mesoderm contains a bipotential stem cell that can give rise to both the myocardial and endocardial lineages. More important, since the QCE-6 cells are representative of early cardiogenic cells, this cell line offers a unique model system to study cardiac cell differentiation.


Key Words: cardiac development • mesoderm • cell line • growth factors


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
The difficulties with isolating and maintaining progenitor cells that give rise to the cardiac lineage have proved to be an obstacle in studying the early development of the vertebrate heart. In the chick, these cardiac progenitors are found within paired fields of mesodermal cells, which are lateral to the anterior portion of the primitive streak.1 At this early stage, the study of these cells is very difficult because of their low number and poor survival in long-term culture. Manipulations involving the early cardiac cells in other species, such as mouse or frog, have not been very successful. Consequently, there is limited information available about the cellular and molecular events that occur during the commitment and differentiation of the precardiac cells. With congenital heart defects affecting 8 of every 1000 babies born,2 it is of medical importance to elucidate events that occur during normal progenitor cell maturation and the mechanisms by which abnormal cells contribute to these deformities. To study cardiac cell development, it is necessary to develop an in vitro culture system.

Presently, there are several cell lines that possess properties that are ascribed to cardiomyocytes. However, none of these appears to be suitable for conveniently modeling progenitor cells of the early precardiac mesoderm. The mouse ES-D3 cell line, derived from totipotential cells of the blastocyst, can form embryoid bodies that contain cardiac myocytes.3 Although these cells recapitulate aspects of heart development,4 they are of limited utility because of the pluripotential nature of the parental cells along with the inability to isolate cell lineages as they develop. Multiple phenotypes are likewise seen with the teratoma-derived P19 and blastocyst-derived hamster ES cell lines.5 6 In addition, the W1, AT-1, H9c2, and MCM1 cell lines are inappropriate in vitro models for studying differentiation, since they already possess a profile of a fully differentiated cardiomyocyte.7 8 9 10 The MEQC1 and MEQC2 cell lines were derived from quail cardiac rhabdomyosarcomas of late-stage embryos11 and thus may not be suitable representatives of early cardiac cells. Moreover, their requirement for coculture with the noncardiac 3T3 cells greatly complicates the analysis of cardiac differentiation. Hence, the generation of a culture system composed of early nondifferentiated mesodermal cells having cardiogenic potential would serve as a useful tool by which to examine further cardiac development.

In birds, cardiac development can be traced to gastrulation, with the mesoderm located on either side of Hensen's node giving rise to heart tissue.1 12 Results from classic descriptive studies suggest that this cardiogenic mesoderm is the source of both the myocardial and endothelial cells found within the heart. Sabin13 first described the delamination of endothelial cells from cardiogenic plates containing the myocardial primordia. Autoradiographic studies by Rosenquist and DeHaan14 and Stalsberg and DeHaan15 support the observation that small clusters of endothelial cells arise from the primordial myocardial areas and migrate to form the endocardium. Manasek16 identified the migratory cells as preendocardium by transmission electron micrography. These observations were confirmed by Virágh et al,17 who noted the detaching of preendocardial cells from the cardiogenic areas of Hamburger and Hamilton18 (HH) stage-9 chicken embryos. Immunochemistry studies using the QH1 monoclonal antibody, which recognizes quail endothelial cells, further verified these cells as endocardial angioblasts.19 20 Additional studies by Wunsch et al21 have suggested that some of these endocardial cells are the precursors to cushion mesenchyme that will later form the valves and septa of the heart.22 These results indicate that endocardium is closely related to the myocardium in origin. What mechanism(s) directs precardiac mesoderm cells to differentiate into myocardial and endocardial cells is presently unknown. However, several studies suggest that soluble factors may be involved in promoting cardiac differentiation of committed cardiac progenitors. Various members of the TGF-ß family have been shown to be important during cardiac myocyte differentiation.23 24 25 PDGF-BB enhances cardiac formation in mesodermal explants taken from axolotl embryos.24 Additional findings have shown that bFGF plays a critical role in the proliferation and contractility of mesodermal explants taken from chicken embryos.26 Furthermore, retinoic acid has been shown to affect early cardiac development.27 28 29

With the goal of preparing a permanent source of cardiac stem cells, a cell line of cardiogenic mesodermal origin was established. This cell line, QCE-6, was obtained from MCA-treated tissue explants taken from HH stage-4 Japanese quail embryos (Coturnix coturnix japonica) and has been stable for 5 years in culture. An initial description of this cell line, which contained a preliminary phenotypic characterization, was recently published.30 That report described the ability of the QCE-6 cells to differentiate into either myocardial or endocardial endothelial cell types. This present communication provides a more extensive characterization of the nontreated (ie, progenitor cell) and induced myocardial and endocardial endothelial phenotypes that can be exhibited by the QCE-6 cells. Moreover, the derivation of the QCE-6 cells is presented here in sufficient detail to assess how representative this cell line is of precardiac stem cells. The present studies demonstrate that multiple signals, provided by exogenous factors, regulate the differentiation and diversification of the QCE-6 cells. The manifestation of both myocardial and endocardial endothelial phenotypes by the QCE-6 cells suggests that these two cell types may share a common lineage in vivo. Since this cell line is representative of early cardiogenic mesoderm, those factors that effect QCE-6 differentiation may have relevance in situ. Hence, the QCE-6 cell line is a unique tool by which to study the mechanism(s) by which the precardiac mesoderm differentiates into the cells of the myocardium and endocardium.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
Derivation of a Clonal Cell Line
Fertile Japanese quail eggs (Coturnix coturnix japonica), obtained from Truslow Farms Inc, Chestertown, Md, were incubated at 37°C with 60% relative humidity. After 20 to 30 hours of incubation, developing quail embryos were removed from the egg using the technique of New31 and placed ventral side up in 35-mm Petri dishes. Using fine glass needles, mesoderm from the presumptive cardiac regions1 32 was isolated from HH stage-4 quail embryos under sterile conditions. Explants were placed directly into transformation medium (see below) in the presence or absence of MCA (1 µg/mL, Sigma Chemical Co). The tissue fragments were cultured in fibronectin-coated (2.0 µg/cm2) eight-well Lab-Tek Chamber Slides (Nunc).

The primary cultures were incubated in a moist 5% CO2 atmosphere at 37°C. Tissue explants were in the transformation medium for 6 days,33 with one change of medium at day 3. Explants cultured in the absence of MCA did not survive beyond 14 days. The MCA-treated cultures had become nearly confluent by day 6 and were transferred into a fibronectin-coated 24-well plate containing medium without MCA. Each well contained {approx}1x103 cells. Subculturing was accomplished using 0.5% trypsin-EDTA (Sigma) in PBS (Ca2+ and Mg2+ free). On day 14, cells were taken only from wells exhibiting substantial cell growth and were plated at a density of 1x103 cells per milliliter in fibronectin-coated 96-well plates (200 µL per well). Partial characterization of these cells was performed at this time by immunofluorescence microscopy with anti-sarcomeric MHC antibody, MF20 (see below). At the third passage, on day 26, cells from confluent wells were subjected to differential adherence34 and then cloned by limiting dilution into two 96-well plates. These culture vessels contained medium without MCA but enriched with 1% chicken embryo extract (GIBCO) and 20% newborn calf serum. At passage 4, clones were sib-selected. The process of sib selection consisted of plating the cells in duplicate cultures; one culture was expanded, while the other was processed for immunohistochemistry with MF20. Only 5 of 16 proliferating clones exhibited MF20 staining. Each of these positive clones was recloned twice and sib-selected again with MF20 and desmin D3 antibodies at passages 5 and 7. Only four clones continued to exhibit positive staining. With time, three of these clones began to exhibit very low growth activity and did not survive continual passage. Because of the decline in cell growth during the early passages, these initial clones were kept in a medium enriched with tryptose phosphate broth, chicken serum, newborn calf serum, and chicken embryo extract. Previous studies have suggested that some component(s) in the chicken embryo extract is essential for successful adaptation of the cells to continuous culture conditions.34 Even under these conditions, only one clone (referred to as clone 4B), continued to proliferate, albeit slowly. By passage 10, this clone no longer manifested reactivity to the MF20 antibody. At this time, the amount of newborn calf serum in the growth medium was reduced to 10%. By passage 15, this clone had begun to demonstrate an increased rate of growth with a change in cell shape. Cloning rings were then used to isolate the subclone referred to as QCE-6.35

Tissue Culture Media
The transformation medium consisted of Eagle's MEM with Earle's salts, nonessential amino acids, and L-glutamine (Sigma), supplemented with 5% newborn calf serum (Hazelton), 2% chicken serum (Sigma), 10% tryptose phosphate broth (GIBCO), 2% sodium bicarbonate, penicillin (100 U/mL, Sigma), and streptomycin (100 µg/mL, Sigma). For the QCE-6 cell line, a variety of media was tested for its optimal growth after establishment. The growth medium that provided maximal viability consisted of MEM with Earle's salts, nonessential amino acids, and L-glutamine, enriched with 10% iron-supplemented newborn calf serum (Sigma), 2% chicken serum, 10% tryptose phosphate broth, 2% sodium bicarbonate, penicillin (100 U/mL), and streptomycin (100 µg/mL). Before media preparation, the calf and chicken sera were inactivated by heating at 56°C for either 30 or 60 minutes, respectively.

Cell Growth Properties
To determine the growth rate of clone 4B at passage 13, the cells were plated at 2x105 cells per well of fibronectin-coated six-well cluster dishes (Corning). Cell counts were performed in triplicate every 4 days, for 20 days, with a hemocytometer using trypan blue exclusion. For the QCE-6 cell line, cells were seeded at lower concentrations of 1x103 and 1x104 cells per well. The cultures were counted every 2 days for a period of 10 days. The medium was changed every 4 days in both the clone 4B and QCE-6 cultures. The data were plotted on semilog paper, with the log phase of growth being denoted by the linear part of the resulting curve. The number of cell generations during an interval of time (g) was calculated using the following formula: g=(log N-log No)/log 2, where N is the number of cells at the end of the time interval, and No is the number of cells at the beginning.36

Cell Culture Substrates
Tissue culture polystyrene dishes (Corning) were coated with plasma fibronectin (from bovine plasma [Sigma] or from human plasma [NY Blood Center]) by incubation at 37°C for at least 2 hours.37 All tissue culture substrates were coated with a 2.0 µg/cm2 concentration of fibronectin unless otherwise stated. The dishes were washed twice with PBS (Ca2+ and Mg2+ free) immediately before plating the cells. Solid gels of polymeric type I collagen (Boehringer Mannheim) were prepared according to manufacturer's instructions.

Immunofluorescence Staining of Cells
Passaged cells were plated at 50% confluence in fibronectin (2.0 µg/cm2)–coated eight-well Lab-Tek Chamber Slides (Nunc) for a minimum of 24 hours. The cells were rinsed twice with PBS, fixed in 100% methanol for 10 minutes at -20°C, and immediately rehydrated with PBS. The slides were blocked with PBS containing 1.0% BSA for 60 minutes at room temperature and incubated with either MF20,38 desmin D3,39 muscle-specific Na+,K+-ATPase,40 cTnI,41 cytokeratin (Biomedical Technologies, Inc), vimentin, N-cadherin (Sigma), cingulin,42 JB3,21 or QH143 antibodies. The antibodies were used as described in the original references or the manufacturer's specifications. F-actin was visualized by fixing the cells in 100% acetone for 30 minutes at -20°C before a 1-hour incubation at room temperature with rhodamine-phalloidin (Molecular Probes, Inc). After three washes with PBS, the slides were incubated with secondary antibodies of fluorescein isothiocyanate–conjugated goat anti-mouse IgG used at a 1:40 dilution or fluorescein isothiocyanate–conjugated goat anti-rabbit IgG (Organon Teknika-Cappel) used at a 1:3000 dilution in PBS/1.0% BSA and applied for 1 hour at room temperature. Control stainings were performed by substituting PBS/1.0% BSA for the primary antibody. Cell nuclei were stained with DAPI (Sigma) according to the manufacturer's instructions. After three PBS rinses, the slides were fixed in 4% formalin for 3 minutes at room temperature. The slides were mounted with 90% glycerol in Tris (pH 8.0) containing 100 mg/mL diazobicyclooctane (Aldrich, an antiquenching agent), viewed under a phase-fluorescence Leitz Dialux microscope, and photographed with either Kodak Ektachrome 400 color slide film or TriXPan 400 black and white print film (Eastman Kodak Co).

Electron Microscopy
Cell plates were washed gently with buffer (0.1 mol/L sodium phosphate, pH 7.3) and fixed for 30 minutes at room temperature with 2.5% glutaraldehyde in buffer. After three 10-minute washes with buffer, cells were postfixed with 1% osmium tetroxide in buffer for 60 minutes at room temperature, washed again in buffer, and dehydrated through a graded ethanol series. Infiltration was completed with LX-112 resin (Ladd Research), and cells were embedded on the culture dish with BEEM capsules. Ultrathin 60-nm sections were cut using a diamond knife (Diatome USA) on an MT-5000 ultramicrotome (RMC, Inc). Sections were stained with 1.5% uranyl acetate and 0.1% lead citrate and examined in a JEOL 100 CX-II electron microscope (JEOL, Inc) at 80 kV.

Retinoic Acid and Growth Factor Treatment
QCE-6 cells were plated at 0.25 times confluence into Lab-Tek chamber slides, which had been precoated with 50 µg/cm2 fibronectin (see above). Cultures were treated with 2x10-8 mol/L all-trans retinoic acid (Sigma) in various combinations with 250 ng/mL human recombinant bFGF, 10 ng/mL human recombinant TGF-ß1, porcine TGF-ß2, chicken recombinant TGF-ß3, human recombinant PDGF-BB from R & D Systems, and 50 ng/mL human recombinant IGF II from Collaborative Research Incorporated. The source of TGF-ß isoforms was dictated by commercial availability. These proteins are extremely well conserved, as each TGF-ß isoform shares >97% sequence identity among mammalian and avian species.44 All manipulations were carried out under subdued light, with the retinoic acid being diluted in medium from a 1x10-2 mol/L stock prepared in dimethyl sulfoxide and kept at -70°C.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
*Results
down arrowDiscussion
down arrowReferences
 
Derivation of the QCE-6 Cell Line
Fig 1Down shows a schematic diagram that illustrates the origins of the QCE-6 cells. Precardiac mesodermal fragments were taken from quail embryos analogous to HH stage 4 and cultured in the presence or absence of the immortalizing agent MCA. By day 2 of culture, beating cells became visible in both control and MCA-treated explants. During the next few days, there was a pronounced depression in contractile activity as the cells adjusted to the in vitro culture surface. After day 5, beating cells were no longer apparent in any of the cultures. Moreover, there was also a noticeable decline in cell growth. By day 14, viable cells were seen only in those cultures derived from MCA-treated mesodermal fragments. At this time, an aliquot of these cells was removed and subjected to immunofluorescence microscopy to identify cardiogenic cells within these cultures. Thus, the majority of the cells from the 2-week-old MCA-treated mesodermal cultures were positive for MF20, an anti–sarcomeric myosin antibody that reacts to early cardiac cells.32 45 However, as these cultures were expanded, there was a decrease in myosin expression and cell viability.



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  		Figure 1. A schematic diagram depicting the establishment of the QCE-6 cell line. Mesoderm from presumptive cardiogenic regions was isolated from quail embryos, analogous to HH stage 4 in chicken. These explants were placed directly into fibronectin (2.0 µg/cm2)–coated chamber slides containing medium with or without MCA. Treatment with MCA was used only during the first 6 days of culture. By day 14 (passage 2), cultures exposed to MCA continued to proliferate, whereas untreated cells did not survive. On day 26 (passage 3), cells were first cloned by limiting dilution. MF20-positive clones, identified by sib selection at passage 4, were recloned twice at passages 5 and 6. At passage 7, parental clone 4B was established after sib selection with the MF20 antibodies. By passage 10, clone 4B lost all MF20 reactivity. At passage 15, clone 4B underwent an adaptation event and was subcloned during the subsequent passage. The QCE-6 cells are a resultant subclone, which has been stable for 5 years.

In an attempt to isolate cells that retained their myosin expression, cells from these cultures were cloned at passage 3. Several of these clones contained myosin-positive cells, but none exhibited myosin expression within every cell. Moreover, there was a reduction in myosin expression among all these clones. The myosin-positive clones were recloned at passages 5 and 6 and reselected for MF20 reactivity. As these clones were passaged, they all demonstrated a decrease and subsequent loss of myosin expression. When clone 4B, the parental clone of the QCE-6 cell line, was selected at passage 7, myosin was expressed by only 10% of the cells within this culture and appeared as faint punctate material in the cytoplasm.30 By passage 10, these cells no longer reacted to the MF20 antibody. At passage 15, clone 4B underwent an adaptation event (described below) and was subcloned to produce the QCE-6 cells. To understand the adaptation event that produced the QCE-6 cells, a morphological analysis of the parental clone 4B is presented.

Morphology of Clone 4B by Light and Electron Microscopy
Light microscopy revealed that clone 4B consisted of cells that were predominantly of fusiform-fibroblastic shape (Fig 2ADown). In addition, this clone exhibited polygonal-shaped cells, elongated spindle-shaped cells, and large asymmetrical cells with multiple cytoplasmic processes. In sparse culture, these long cytoplasmic processes were more abundant and were often found terminating at neighboring cells. When confluent, the cells formed flat monolayers and appeared somewhat epithelioid. Regardless of their morphology, all the cells manifested perinuclear granules, and the majority of the cells were mononucleated, with <5% demonstrating binucleation or multinucleation at any time (Fig 2ADown).



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  		Figure 2. Morphology of clone 4B. Phase (A) and electron (B and C) microscopy of clone 4B cells in culture. Numerous microtubules (arrowheads) can be seen within the cytoplasm and in proximity to the cell membrane (B). Few adherence junctions (arrow) are found between cells (C). Bar=50 µM (A) and 1 µM (B and C).

Electron microscopy of clone 4B revealed that these cells possessed numerous microtubules and rough endoplasmic recticulum. Also, a large number of dense bodies were apparent, some of which appeared morphologically as fat droplets. Many secretory vacuoles and some adherence junctions were present in the cell membrane. The most prominent characteristic of these cells was the extensive cytoskeletal filaments within the cytoplasm and bordering the cell membrane (Fig 2BUp and 2CUp).

Adaptation and Growth of QCE-6 Cells
Clone 4B had a doubling time of 10.9 days on fibronectin-coated tissue culture polystyrene dishes (Fig 3Down). At passage 15, it was observed that the clone 4B culture attained confluence much more rapidly than previously and possessed an altered cell shape. This result indicated that the cells had adapted to culture conditions.34 Morphologically, the culture at confluence consisted of a mixed cell population containing areas of polygonal-shaped cells, epithelial-shaped cells, and fibroblastic-shaped cells. Clone 4B was then recloned, establishing the QCE-6 cell line. The doubling time for the QCE-6 cells was calculated to be {approx}10.3 hours, when the initial input was either 1x103 or 1x104 cells per well of a six-well dish (Fig 3Down). Hence, the proliferation rate of subconfluent cultures was independent of the initial seeding density. These results were obtained when the cells were plated on a fibronectin substrate, which was found to be essential for optimal growth. In addition, these cells were sensitive to postconfluent inhibition of cell division and would begin detaching from the cell culture dish shortly after confluence was reached.



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  		Figure 3. Growth rate of 4B and QCE-6 cells. The growth rate of clone 4B was determined at passage 13 ({square}), with a calculated generation time of 10.9 days. After the establishment of the QCE-6 cell line, the growth rate was determined after plating the cells at both 1x103 ({blacksquare}) and 1x104 ({bullet}) cells per well. The calculated generation time during log phase was 10.3 hours.

Morphology of QCE-6 Cells by Light and Electron Microscopy
QCE-6 propagates as a mononucleated cell line, although binucleation and multinucleation can be seen in <2% of the cells within the population at any one time. The cells exhibit a pleiomorphic shape in nonconfluent or sparse cultures. When confluent, they form flat monolayers and appear epithelioid, possessing a polygonal outline (Fig 4ADown).



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  		Figure 4. Morphology of the QCE-6 cells. Phase (A) and electron (B and C) microscopy of QCE-6 cells in culture. Very few microtubules (arrowheads) are present within the cytoplasm (B). Many adherence junctions (arrows) are found between cells (C). Bar=50 µmol/L (A) and 1 µmol/L (B and C).

When viewed by electron microscopy, these cells exhibited many mitochondria, microtubules, and smooth endoplasmic recticulum. It was difficult to visualize the cytoskeletal filaments because of the large number of ribosomes scattered freely throughout the cytoplasm. Many adherence junctions could be seen at the cell membrane and, occasionally, a protruding microvillus (Fig 4BUp and 4CUp).

Protein Expression by the QCE-6 Cells
The phenotype of the QCE-6 cells was analyzed by staining with antibodies to proteins associated with various cell types of the early embryo. These immunofluorescence studies indicated that the QCE-6 cells have a pattern of protein expression that is characteristic of early cardiogenic mesoderm. The QCE-6 cells demonstrated positive staining with antibodies to the intermediate filament proteins cytokeratin and vimentin (Fig 5ADown and 5BDown), which are epithelial and mesodermal markers, respectively. These cells expressed N-cadherin (Fig 5CDown), a cell-cell adhesion protein associated with early cardiogenic mesoderm. The cells also expressed cingulin (Fig 5DDown), a protein found in the zonula occludens of epithelial cells.42 Staining with rhodamine-phalloidin demonstrated an epithelial-like F-actin pattern with reactivity at the subplasmalemma, albeit very weak in intensity (Fig 5EDown). F-actin expression was augmented significantly when the concentration of fibronectin on which the QCE-6 cells were plated was increased from 2.0 to 50 µg/cm2. Under these conditions, this cytoskeletal protein was displayed at the cell membrane and along stress fibers (Fig 5FDown). Increasing fibronectin concentrations had no effect on the pattern of expression of the other proteins listed above. Thus, the QCE-6 cells express a protein profile consistent with precardiac mesoderm, which exists as an epithelium during early embryonic development.46 Moreover, they do not express markers associated with more differentiated cardiac phenotypes.



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  		Figure 5. Phenotype of the QCE-6 cells. Immunofluorescence microscopy of QCE-6 cells in culture is shown in all panels. Cells were stained with either antibodies to anti-cytokeratin (A), anti-vimentin (B), anti–N-cadherin (C), anti-cingulin (D), or rhodamine-phalloidin (E and F). Cells display an epithelial-like F-actin pattern (E) when plated on a low concentration of fibronectin (2.0 µg/cm2). F-actin expression increased (F) when plated on a higher concentration of fibronectin (50.0 µg/cm2). DAPI was used for staining cell nuclei (A, B, and D). Bar=50 µmol/L.

Differentiation of the QCE-6 Cells Is Promoted by Retinoic Acid and Growth Factors
The phenotype of the QCE-6 cells would suggest that this cell line is representative of the tissue from which they were derived, ie, precardiac mesoderm. However, if these cells are true representatives of this tissue, they should have a cardiac potential. To ascertain whether the QCE-6 cells could express more differentiated cardiac phenotypes, they were exposed to a variety of factors that have been shown to be present in the early embryo. To date, seven factors have been shown to mediate the differentiation of the QCE-6 cells: retinoic acid, bFGF, TGF-ß1, TGF-ß2, TGF-ß3, PDGF-BB, and IGF II.

A protein that is expressed by both myocardial and endocardial cells, but is absent from nontreated QCE-6 cells, is Na+,K+-ATPase (data not shown). However, extensive experimentation with various growth factor treatments indicated that this enzyme will be exhibited by the QCE-6 cells if retinoic acid, bFGF, and TGF-ß1 are present in the cultures (Fig 6Down). The addition of both TGF-ß2 and TGF-ß3 can replace the requirement for TGF-ß1, although significantly lower levels of Na+,K+-ATPase expression will be demonstrated (as judged by the intensity of staining). Interestingly, the QCE-6 cells exhibited the highest levels of Na+,K+-ATPase expression when treated with the combination of all three TGF-ß isoforms, along with retinoic acid and bFGF. The supplementation of these factors with PDGF-BB and IGF II had no stimulatory or inhibitory effect on the production of this protein (Fig 6Down).



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  		Figure 6. Effects of various factor combinations on myocardial and endocardial endothelial phenotypes expressed by the QCE-6 cells. Immunofluorescence microscopy shows QCE-6 cells in culture treated with either retinoic acid, bFGF, and TGF-ß1 (row I); retinoic acid, bFGF, TGF-ß2, and TGF-ß3 (row II); retinoic acid, bFGF, TGF-ß1, TGF-ß2, and TGF-ß3 (row III); or retinoic acid, bFGF, TGF-ß1, TGF-ß2, TGF-ß3, PDGF-BB, and IGF II (row IV). Cells were stained with either antibodies to Na+,K+-ATPase, MHC, cTnI, or QH1. In response to retinoic acid, bFGF, and TGF-ß1, Na+,K+-ATPase becomes expressed, whereas MHC, cTnI, and QH1 staining is not observed. Treatment with retinoic acid, bFGF, TGF-ß2, and TGF-ß3 induces the expression of both MHC and cTnI in a punctate pattern within the cytoplasm of cells that exhibit an epithelial morphology, whereas QH1 staining is displayed by mesenchymal cells that appear to migrate over the epithelial layer. Under these same conditions, the epithelial cells stain faintly for Na+,K+-ATPase. When TGF-ß1 is added to this mix of factors, Na+,K+-ATPase expression by the QCE-6 cells increases significantly, whereas the organization of both MHC and cTnI expression has changed to a more linear array. No migratory cells are present under these conditions, yet QH1 reactivity is still observed among a subpopulation of the epithelial cells. When PDGF-BB and IGF II are added to the factor mixture, Na+,K+-ATPase continues to be displayed, with both MHC and cTnI becoming organized in a fibrillar pattern. Note the increased size of those cells that exhibit the sarcomeric proteins in a filamentous pattern. Under these conditions, positive reactivity toward the QH1 antibody is lost. Bar=50 µmol/L.

In addition to Na+,K+-ATPase expression, these factors also induced the expression of definable cardiac phenotypes. Specifically, cultures exposed to retinoic acid, bFGF, TGF-ß2, and TGF-ß3 would contain a mixed population of cells with either myocardial or endocardial characteristics (see below). Quantitative analysis of the number of cells expressing either phenotype was obtained from cell counts of microscopic fields. It should be noted that the proportion of cells within a culture that exhibited either phenotype varied from 5% to 50%, according to the lot of serum added to the medium. Furthermore, the ratio of cells undergoing myocardial versus endocardial differentiation varied greatly, according to the lot of serum. For example, in one experiment in which 1x106 QCE-6 cells were cultured with a given serum sample, retinoic acid, bFGF, TGF-ß2, and TGF-ß3 induced 20% of the cells to exhibit the myocardial phenotype, 40% to exhibit the endocardial phenotype, and 30% to exhibit the nontreated phenotype. With another serum sample, 50% of the cells expressed the myocardial phenotype, while 40% showed the endothelial phenotype. Importantly, the serum batches only affected the number of cells undergoing differentiation; ie, they did not affect the function of retinoic acid, bFGF, TGF-ß1, TGF-ß2, TGF-ß3, PDGF-BB, and IGF II on the differentiation of the QCE-6 cells. The effects of these seven factors in mediating cardiac cell differentiation will be described in detail below.

Myocardial Differentiation of the QCE-6 Cells
To determine whether the QCE-6 cells were capable of expressing more differentiated cardiac phenotypes, cultures treated under various conditions were immunostained with antibodies specific for sarcomeric MHC and cTnI. These studies demonstrated that four factors were absolutely required for myocardial protein expression: retinoic acid, bFGF, TGF-ß2, and TGF-ß3 (Fig 6Up). If any one of these four factors was not added to the cultures, the QCE-6 cells would not exhibit express expression of these myocardial proteins. Moreover, the requirement for TGF-ß was isoform specific, because substitution of TGF-ß1 for either TGF-ß2 and/or TGF-ß3 was not sufficient for myocardial differentiation (Fig 6Up).

Although treatment of the QCE-6 cells with retinoic acid, bFGF, TGF-ß2, and TGF-ß3 will promote myocardial protein expression, these presumptive myocardial cells are not fully differentiated. As is demonstrated by the pattern of MHC and cTnI expression (Fig 6Up), the sarcomeric proteins are expressed in a punctate fashion within the cytoplasm after treatment with these four factors. The expression pattern of these proteins can be modified when either TGF-ß1, PDGF-BB, and/or IGF II is combined with retinoic acid, bFGF, TGF-ß2, and TGF-ß3. When TGF-ß1 supplements the other four factors, the sarcomeric proteins become expressed in a linear array. A more filamentous arrangement of MHC and cTnI proteins is obtained among the myocardial derivatives of the QCE-6 cells when either PDGF-BB or IGF II is the supplementing factor (data not shown). The greatest enhancement of this filamentous (premyofibrillar) expression of these proteins that has yet been demonstrated is when all seven factors-retinoic acid, bFGF, TGF-ß1, TGF-ß2, TGF-ß3, PDGF-BB, and IGF II-are used to treat the QCE-6 cells (Fig 6Up). This premyofibrillar staining pattern is more noticeable with the MHC protein (Fig 6Up). Moreover, all cells that exhibited a more filamentous MHC expression pattern demonstrated a significant increase in cell size. The enhancement in cell size by the QCE-6 cells correlated absolutely with increased organization of the myofibrillar proteins, regardless of the serum lot that was used. This concurrent cell enlargement with increased sarcomeric protein expression is a process frequently observed with cardiomyocytes in cultures (Y. Sugi, personal communication, 1995).

The expression of a myocardial phenotype by the QCE-6 cells was only apparent in some of the cells in the treated cultures. These myocardial cell derivatives retain the epithelial morphology of the nontreated QCE-6 cells. Most of these epithelial cells will demonstrate proteins characteristic of myocardium. However, treatment of the QCE-6 cells with retinoic acid, bFGF, TGF-ß2, and TGF-ß3 also induced the appearance of a second cell type that possesses a distinct mesenchymal morphology. An analysis of this second induced phenotype is described in the next section.

Endocardial Endothelial Differentiation of the QCE-6 Cells
Nontreated QCE-6 cells have an epithelial morphology. Treatment of the QCE-6 cells with retinoic acid, bFGF, TGF-ß2, and TGF-ß3 will produce cultures containing two distinct cell morphologies: epithelial and mesenchymal. When the QCE-6 cells were plated on fibronectin-coated dishes and exposed to these four factors, the resulting mesenchymal cells appeared to migrate over one another (Fig 7ADown). To analyze more fully the mesenchymal nature of these cells, the cells were plated on top of collagen type I gels, with the factors added to the medium overlying the gels. In the absence of growth factors, QCE-6 cells cultured on collagen gels remained epithelial. However, in accordance with the results obtained on fibronectin-coated dishes, a subpopulation of the cells exposed to retinoic acid, bFGF, TGF-ß2, and TGF-ß3 migrated through the collagen gel (Fig 7BDown). Since it has been postulated that the preendocardial cells arise from an epithelial-to-mesenchymal transformation of precardiac mesoderm, treated cultures were immunostained with the endothelial cell marker QH1. As shown in Fig 7CDown, only the migratory cells reacted positively to this antibody. The phenotype of the mesenchymal derivatives of the QCE-6 cells was analyzed further by staining with the endocardial endothelial marker JB3.21 This antibody recognizes a fibrillin-like extracellular matrix protein that is produced by endocardial precursors of the early heart-forming fields. Also, JB3 is produced by cushion mesenchymal cells as well as their endocardium-derived progenitors of the atrioventricular and outflow tract regions of the heart. JB3 antibody staining of QCE-6 cultures treated with retinoic acid, bFGF, TGF-ß2, and TGF-ß3 demonstrated that this extracellular matrix protein was associated with the migratory cells (Fig 7DDown).



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  		Figure 7. Endocardial endothelial phenotype of the QCE-6 cells. Hoffman optic (A and B) and immunofluorescence (C and D) microscopy of QCE-6 cells grown on fibronectin-coated dishes (A and D) or collagen type-I gels (B and C) is shown. Arrows in panel A indicate mesenchymal cells. Cells were treated with retinoic acid, bFGF, TGF-ß2, and TGF-ß3 (A through D) and stained with either the QH1 (C) or JB3 (D) antibodies. Bar=100 µmol/L.

Since the different TGF-ß isoforms had differential effects on Na+,K+-ATPase and myofibrillar protein expression by the QCE-6 cells, their effects on QH1 expression were analyzed in detail. As demonstrated for the expression of a mesenchymal morphology, the minimal requirements for the expression of QH1 by the QCE-6 cells consisted of retinoic acid, bFGF, TGF-ß2, and TGF-ß3 (Fig 6Up). Treatment of the QCE-6 cells with retinoic acid, bFGF, and either TGF-ß1 (Fig 6Up), TGF-ß2, or TGF-ß3 did not produce QH1 reactivity. Moreover, the coaddition of TGF-ß1 with retinoic acid, bFGF, and either TGF-ß2 or TGF-ß3 failed to promote the endothelial phenotype (as noted by the absence of QH1 antibody reactivity) in the QCE-6 cells (data not shown). Interestingly, the coaddition of TGF-ß1 prevented the QCE-6 cells from expressing a mesenchymal morphology in response to retinoic acid, bFGF, TGF-ß2, and TGF-ß3, yet QH1 expression was still induced in these cultures (Fig 6Up). The further addition of PDGF-BB and IGF II, along with the other factors, prevented QH1 expression by the QCE-6 cells (Fig 6Up).

Diversification of the QCE-6 Cells
A schematic diagram summarizing the results after factor treatments is shown in Fig 8Down. The QCE-6 cells have an epithelial morphology and express proteins found in early cardiogenic mesoderm: cytokeratin, vimentin, N-cadherin, and cingulin. After treatment with retinoic acid, bFGF, TGF-ß2, and TGF-ß3, the cells exhibit two morphologies. The cells showing an epithelial morphology express proteins characteristic of myocardial cells, such as myosin, desmin, cTnI, titin, and {alpha}-actinin. When TGF-ß1, PDGF-BB, and/or IGF II are combined with the other four factors, the sarcomeric proteins become organized into a more filamentous pattern. TGF-ß1 also promotes Na+,K+-ATPase expression by the QCE-6 cells, if added in the presence of retinoic acid and bFGF. The cells demonstrating a mesenchymal morphology are QH1, JB3, and cytotactin positive, indicating that they are of the endothelial lineage. Yet the endocardial endothelial phenotype can be inhibited by the supplementation of two growth factors that provoke further myocardial differentiation (as noted by filamentous MHC staining pattern) of the QCE-6 cells: PDGF-BB and IGF II. Importantly, the QCE-6 cells will remain epithelial and not express any of these markers for either the myocardial or endothelial phenotypes if any one of the four primary inducing factors (retinoic acid, bFGF, TGF-ß2, and TGF-ß3) is absent from the cultures.



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  		Figure 8. A schematic diagram depicting the morphological and phenotypic changes of the QCE-6 cells after treatment with retinoic acid and growth factors. The expression pattern of sarcomeric proteins is indicated within the cell.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
up arrowResults
*Discussion
down arrowReferences
 
The derivation of a clonal cell line that is representative of cardiogenic mesoderm would provide a useful tool for studying early heart development. A cell line would provide easy access to large numbers of cardiogenic mesodermal cells and supply an alternative to the tedious and time-consuming task of microdissecting large numbers of early embryos. Furthermore, such a cell line would provide a pure population of cardiogenic mesodermal cells without contaminating cell types that could complicate analysis. This communication describes the isolation and properties of an avian cardiogenic mesodermal cell line, QCE-6.

This cell line was derived from primary cell explants of cardiogenic mesoderm taken from HH stage-4 Japanese quail embryos. Immortalization of the cells was attributed to the presence of the MCA, since explants grown in the absence of this drug did not survive beyond 2 weeks in culture. To obtain a clonal cell line, the cells were plated at limiting dilution at the third passage and then selected for myosin expression using the MF20 antibody. More than 50% of the cells from a selected colony reacted positively to this antibody. Despite attempts to isolate a pure population of myosin-positive cells by recloning these cells at passages 5 and 6, all the clones showed a decrease in myosin expression over time. When clone 4B, the parental clone of the QCE-6 cell line, was isolated, only 10% of the cells manifested MF20 reactivity. By passage 10, all the cells were negative for this antibody. At passage 15, there was a dramatic change in clone 4B cell morphology, indicating that the cells underwent a culture adaptation event. Subsequently, these cells were subcloned to produce the QCE-6 cell line.

Clone 4B initially appeared as a fibroblast-like cell, possessing secretory vessels, numerous cytoskeletal filaments, and ribosomes that were primarily associated with the endoplasmic recticulum. These cellular characteristics have been observed in other nondifferentiated mesodermal cells.22 After adaptation, the cells displayed an epithelial morphology, with fewer cytoplasmic filaments and ribosomes scattered throughout the cytoplasm. In all, the QCE-6 cells resemble a very early nondifferentiated epithelial cell. Moreover, their coexpression of vimentin and cytokeratin is characteristic of newly gastrulated mesoderm.47 These results suggest that during the adaptation event the cells retained a more primitive48 and, most likely, more stable cell culture phenotype. A change in cell shape is a phenomenon that has occurred during the adaptation of other cell lines, ie, MDCK cells.49 Nevertheless, these changes in cell shape and growth did not alter the capacity of these cells to express many of the same proteins that are characteristic of precardiac mesoderm.

The QCE-6 cells resemble developing cardiogenic mesoderm with regard to their cell morphology and profile of protein expression. When cultivated on a fibronectin substrate, these cells exhibit an epithelial configuration. Analogously, cardiogenic mesoderm continues morphologically as an epithelium throughout development of the heart.46 50 The QCE-6 cells express cytokeratin, N-cadherin, and vimentin, as does the precardiac mesoderm.51 52 53 In addition, the subplasmalemmal staining pattern of F-actin is similar to that described by Tokuyasu and Maher54 in the early-forming myocardium of HH stage-8 to -11 chicken embryos. Moreover, the QCE-6 cells can be induced to differentiate and manifest phenotypes associated with cells of the heart.

To date, seven factors have been shown to regulate QCE-6 cell differentiation: retinoic acid, bFGF, TGF-ß1, TGF-ß2, TGF-ß3, PDGF-BB, and IGF II. No decline in cell proliferation was noticed when these cells were induced to differentiate. Whether these factors also modulate the differentiation of cardiogenic mesoderm in vivo has yet to be determined. However, several studies have demonstrated their presence and/or influence on heart tissue. Retinoic acid is present at Hensen's node early during development.55 bFGF has been detected in the chicken myocardium as early as HH stage 9+56 and throughout the maturation of the heart.57 58 Similarly, IGF II is abundant in the splanchnic mesoderm.59 60 Its expression is later restricted to the heart musculature,61 62 where it is thought to govern cardiomyocyte growth and maturation.63 TGF-ß1 is found within the cardiogenic field,64 and its expression continues through heart development in the ventricular myocardium65 and endocardial cushions.64 66 67 This growth factor was found to enhance heart formation in axolotl mesodermal explants.24 There is evidence suggesting that TGF-ß2 and TGF-ß3 likewise play important roles during cardiac morphogenesis,68 when they are coordinately expressed.69 70 Finally, like TGF-ß1, PDGF-BB promotes cardiac development in axolotl mesodermal explants.24

That other factors may contribute to the differentiation of the QCE-6 cells is indicated by studies showing that the number of cells exhibiting differentiated phenotypes can vary according to serum lot. However, it should be noted that the functional properties of retinoic acid, bFGF, TGF-ß1, TGF-ß2, TGF-ß3, PDGF-BB, and IGF II did not change with different serum batches. Regardless of the serum batch, expression of both the myocardial or endocardial phenotypes required retinoic acid, bFGF, TGF-ß2, and TGF-ß3. Moreover, the coaddition of TGF-ß1, PDGF-BB, and/or IGF II promoted the increased organization of the myofibrillar proteins, while inhibiting the expression of the endocardial phenotype. Thus, the activities demonstrated by these factors were independent of the batch of serum, yet the comparative analysis of multiple serum lots indicates that additional factors modulate the diversification of the QCE-6 cells. That the ratio of cells induced to display a myocardial versus an endocardial endothelial phenotype can vary suggests that the mechanism of differentiation of a bipotential stem cell involves more than simply producing one daughter cell for each cell type. This is not surprising considering that there are far greater numbers of myocardial than endothelial cells in the early heart. Because of the complications using serum to study the diversification of the QCE-6 cells, current studies have used defined media culture conditions to elucidate the mechanisms that regulate their differentiation into cardiac myocytes and endothelial cells (C.A. Eisenberg and R.R. Markwald, unpublished data, 1995).

In avian development, cells that give rise to the heart are located within the lateral plate mesoderm that is established between the ectoderm and endoderm during gastrulation. As this mesoderm spreads cephalad, the precardiac cells cluster within bilateral cardiogenic fields. After the appearance of the amniocardiac coelomic vesicles, these cardiac progenitor cells become restricted to the splanchnic mesoderm closely associated with the endoderm.71 It is known that the myocardium originates from cells within the splanchnic mesoderm.1 71 However, several studies have indicated that endothelial precursor cells also arise from this cell layer. Virágh et al17 observed that cells that give rise to endothelium are present throughout the fusing heart fields. These precursor cells have been observed in the splanchnic mesoderm by QH1 staining19 72 and display a mesenchymal morphology.20 Moreover, experiments have shown that these early endocardial cells are derived from a progenitor that also has myocardial potential and may also coexpress early markers for both lineages.73 Interestingly, there are data to suggest that presumptive endocardial precursor cells may briefly express muscle-specific myosin.45 74

The ability of the QCE-6 cells to express either myocardial or endothelial phenotypes, after treatment with specific growth factors, suggests that this cell line represents the proposed bipotential cardiac progenitor cell.73 75 In view of the events that lead to the establishment of this cell line, this bipotentiality may have been an attribute of clone 4B. Although the original explant of HH stage-4 cardiogenic mesoderm produced many beating cells, it should be noted that it also contained other cell populations. Primarily, there were endocardial endothelial cells that grew outward from the explant and possibly less-differentiated cardiogenic mesoderm cells. It is possible that the early cloned cells (from passages 4 to 10) might have coexpressed the endothelial marker QH1 (for which they were never tested) along with myosin. Hence, it may be hypothesized that the MCA treatment immortalized a bipotential stem cell committed to the cardiac lineage. Accordingly, the adaptation event that gave rise to the QCE-6 cells produced a more primitive and stable phenotype but did not alter the cardiac potential. Moreover, it appears that these cells do not have limits on their differentiation capacity. This is indicated by recent experiments that involve mixing the QCE-6 cells with cells from early-stage embryos, with the former developing into contractile cardiomyocytes (C.A. Eisenberg and R.R. Markwald, unpublished data, 1995).

In summary, the QCE-6 cells were established from a population of early cardiac cells. Because the QCE-6 cells display the phenotype of developing mesoderm, they can be used to study the migration, cell-to-cell interactions, and gene and protein expression of this germ layer. Moreover, since the QCE-6 cells are representative of early cardiogenic cells, they are a unique tool for studying the cellular and molecular processes that occur during cardiac differentiation.


*    Selected Abbreviations and Acronyms
 
bFGF = basic fibroblast growth factor
cTnI = cardiac troponin I
DAPI = 4',6-diamidino-2-phenylindole
HH = Hamburger and Hamilton (stages in embryonic growth)
IGF = insulin-like growth factor
MCA = 20-methylcholanthrene
MHC = myosin heavy chain
PDGF = platelet-derived growth factor
TGF = transforming growth factor


*    Acknowledgments
 
This study was supported by grants from the National Institutes of Health (HL-34318 and HL-37675). Dr Bader is an Established Investigator of the American Heart Association and is a recipient of the Irma T. Hirschl Award. We thank Drs Roger Markwald, Yukiko Sugi, and Leonard Eisenberg for helpful comments and critical reading of this manuscript. We are grateful to Leona Cohen-Gould for her help in the EM work.

Received April 21, 1995; accepted October 10, 1995.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
up arrowResults
up arrowDiscussion
*References
 

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