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Short Communication |
From the Department of Pathology,*
University of North
Carolina at Chapel Hill, Chapel Hill; and the Department of
Pediatrics,
Duke University Medical Center,
Durham, North Carolina
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We tested the concept that adult-derived stem cells will differentiate into cardiac myocytes in the tissue microenvironment of the heart in vivo using a clonal stem cell line (WB-F344) derived from the adult liver.17-22 The WB-F344 cell line was isolated and cloned from the liver of a young adult male Fischer 344 rat and established as a propagable cell line under conditions that excluded differentiated hepatocytes or biliary epithelial cells as the cells of origin.17-22 WB-F344 cells are diploid, anchorage-dependent, express contact inhibition in vitro, and are not tumorigenic. They exhibit a poorly differentiated nonhepatocytic simplified epithelial cell phenotype in vitro. After exposure to differentiating stimuli in vitro, WB-F344 cells express phenotypes with characteristics of hepatocytes.20 Most important, in the context of the present study, WB-F344 cells transplanted into syngeneic rat liver give rise to differentiated hepatocytes and biliary epithelial cells in vivo.21,22,23 We transplanted WB-F344 cells into normal hearts of adult female nude mice to examine the engraftment and differentiation potential of this male adult-derived stem cell line in the cardiac microenvironment in vivo. We present evidence that the liver-derived WB-F344 stem cells modulate their lineage commitment after transplantation into the cardiac tissue microenvironment in the adult heart and differentiate into cardiac myocytes in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male WB-F344 cells were genetically tagged with the E. coli lac Z gene (ß-galactosidase) by infection with the CRE BAG2 retrovirus.21 WB-F344 cells were cultured in Richter’s minimal essential medium supplemented with 10% fetal calf serum, in a 5% CO2, 95% air environment, at 37°C. Cells intended for transplantation were harvested by trypsinization, washed in cell culture medium, and resuspended in serum-free medium at 2 x 105 cells/ml. Female homozygote nude mice (Tac:Cr:(NCr)-nufBR) were anesthetized with ketamine (50 mg/kg) and xylazine (2.5 mg/kg) injected intraperitoneally. Cell injections were performed with the needle attached directly to a tuberculin syringe or a butterfly infusion system and introduced over the subxyphoid area and directed cephalad toward the left lateral chest. When blood was aspirated from the left ventricle, the cells (50 µl of 2 x 105 cells/ml) were injected, presumably in the wall of the ventricle, as the needle was withdrawn. Control animals were handled identically, and received sham injections of serum-free medium. The mice were euthanized 6 weeks later by inhalation of isofluorane. National Institutes of Health guidelines for the usage of laboratory animals were strictly followed. The experimental protocol was approved by the Duke Institutional Animal Care and Use Committee.
Histochemistry and Immunohistochemistry
The hearts were dissected and sliced coronally into three blocks, which were immediately frozen in liquid nitrogen, and stored at -80°C. Five serial cryostat sections (8 µm) were obtained from each block and used to screen for ß-galactosidase activity, using a ß-galactosidase-staining kit from Boehringer Mannheim (Mannheim, Germany). For the ß-galactosidase reaction, cryosections on glass slides were fixed for 10 minutes in 2% formaldehyde (Mallinckrodt, Paris, Kentucky), 0.2% glutaraldehyde (Fisher, Pittsburgh, PA), in 0.1 mol/L sodium phosphate buffer, pH 7.3. The sections were then extensively washed in this buffer and incubated at 37°C with the ß-galactosidase substrate, 3-indolyl-ß-D-galactopyranoside (X-gal; Boehringer Mannheim, Mannheim, Germany) for 1 to 4 hours. If ß-galactosidase-positive cells were not found on the initial screen, the heart blocks were not sectioned further. Blocks that contained ß-galactosidase-expressing cells were selected and serially sectioned obtaining 70 serial 8-µm sections. These sections were kept at -80°C until needed. The ß-galactosidase expressed in the cytoplasm of WB-F344 cells is bacterial in origin and is optimally activated at pH 7 to 7.5, whereas mammalian (lysosomal) ß-galactosidase is activated at lower pH (pH 3.0 to 6.0). We monitored the pH of all solutions used in the ß-galactosidase reactions and strictly maintained the pH at 7.3. This rigorous control of the pH enhanced the possibility that only bacterial ß-galactosidase activity was detected.
Demonstration of cardiac troponin T was achieved using a monoclonal antibody (mAb) (designated mAb 13-11) that recognizes a cardiac-specific troponin T epitope.24 For this purpose, frozen tissue sections were fixed in 1% formaldehyde in phosphate-buffered saline permeabilized with 0.1% Triton, and incubated with mAb 13-11, as previously described.25 A goat fluorescein-labeled anti-mouse antibody was used as a secondary antibody. Normal mouse serum was substituted for the primary antibody in control immunostaining reactions.
Electron Microscopy
Fresh frozen heart muscle sections were fixed in 1% formaldehyde while still adherent on the glass slides as described above for the ß-galactosidase reaction. After performing the ß-galactosidase reaction, the sections were postfixed in 1% osmium tetraoxide, dehydrated in a graded ethanol series, and infiltrated in L. R. White resin (Ted Pella, Redding, CA). After separating the glass from the resin, ß-galactosidase-positive myocytes were selected and microsectioned at 70 nm. The thin sections were examined with a LEO EM 910 transmission electron microscope at 80 kV. No lead citrate or uranyl acetate was used for postfixation or counterstaining.
Laser Capture Microdissection (LCM)
After performing the ß-galactosidase reaction, tissue sections
were washed in phosphate-buffered saline and dehydrated according to
the National Institutes of Health protocol for LCM (Ref 26
;
http://dir.nichd.nih.gov/1 cm/LCMTAP.htm). Single
ß-galactosidase-expressing myocytes and endogenous (host) myocytes
were independently microdissected from recipient heart sections using
an Arcturus Pixcell II laser capture microdissection instrument and the
instructions supplied with the instrument. Four WB-F344-derived
ß-galactosidase-positive myocytes were captured on one cap membrane
and two cells on another (Figure 1, c and d)
. Twenty myocytes that did not express ß-galactosidase (host
myocytes) were captured on a third membrane. The caps were inserted
into Eppendorf tubes containing 50 µl of a digestion
buffer suggested by the National Institutes of Health protocol
(0.04% proteinase K, 1 mmol/L ethylenediaminetetraacetic
acid, 10 mmol/L Tris-HCl, pH 8.0, and 1% Tween-20). The digestion was
allowed to continue overnight at 37°C. The resulting nucleic acid
fraction served as a template for amplification of a rat
Y-chromosome-specific repetitive DNA sequence using primers previously
described:27
5'-GGTTCTAGACTGTAAAACCCAGAC-3' and
5'-ACTTAAAACTAAGCTTATTGGCCA-3'. A portion (10 µl) of the polymerase
chain reaction (PCR) reactions were analyzed on 1.5% agarose
gels (Figure 1e)
and the remaining PCR product was cloned into a TA
cloning vector (InVitrogen, Carlsbad, CA). The resulting clones
were screened for the Y-chromosome-specific repetitive DNA sequence by
PCR, and subsequently, positive clones were subjected to automated DNA
sequencing.
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The FISH reaction was performed on fresh-frozen 8-µm tissue sections that immediately preceded or followed a section that demonstrated ß-galactosidase-expressing myocytes. A rat Y-probe,27 consisting of a Y-chromosome-specific repetitive DNA sequence in pUC18 plasmid (kindly provided to us by Dr. Barbara Hoebee, Bilthoven, The Netherlands) was used to test whether the ß-galactosidase-expressing cells originated from the transplanted WB-F344 cells. The Y-probe-containing plasmid was labeled with digoxigenin by nick translation (Boehringer Mannheim). FISH was performed on fresh frozen tissue sections fixed in 1:1 (v/v) acetone-methanol solution. DNA was denatured in a solution of 70% formamide (Omnipur, Gibbstown, NJ) in 2x standard saline citrate (0.3 mol/L NaCl, 30 mmol/L sodium citrate) at 70°C for 12 minutes.28 The probe was denatured at 72°C and applied to the tissue section overnight at 37°C in a humidified chamber. The sections were washed three times for 10 minutes each in 50% formamide in 2x standard saline citrate solution at 42°C, followed by two washes in 2x standard saline citrate for 5 minutes each and incubated with a sheep anti-digoxigenin-rhodamine-labeled antibody (Roche, Indianapolis, IN). After this incubation, the slides were washed, covered using a mounting medium containing 4,6-diamidino-2-phenylindole counter stain (Vectashield; Vector Laboratories, Inc., Burlingame, CA), and examined using a Zeiss confocal fluorescence microscope equipped with a triple-band pass filter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. WB-F344-derived myocytes were found on the initial screen in the
left ventricular myocardium of two mice. Hearts from the other eight
mice did not demonstrate ß-galactosidase-expressing cells on the
initial screen and were not sectioned further. It is conceivable that
in these mice the injected cells entered the left ventricle and became
scattered throughout the systemic circulation. Alternately,
ß-galactosidase-positive myocytes might be present within aspects of
the heart tissues that were not sectioned and analyzed in the present
study. Future studies will examine these possibilities.
ß-galactosidase expression was confirmed to originate exclusively
from engrafted WB-F344 cells in the two mice with the following
experiments. We monitored the timing for development of the blue
precipitate during incubation with the X-gal substrate and found the
blue color developed within the first 1 to 2 hours of incubation. We
allowed the incubation to continue for an additional 2 to 3 hours for
easier visualization by light microscopy. Taking advantage of the male
origin of WB-F344 cells engrafted into female recipient hosts, we
confirmed that the ß-galactosidase expressing cells had a rat
Y-chromosome-specific DNA sequence. Additionally, we ruled out the
possibility that ß-galactosidase activity might have originated from
endogenous inflammatory cells, such as macrophages, which could have
invaded the myocardium in response to the injection of WB-F344 cells.
We elicited a cellular inflammatory response in a control mouse by
injecting thioglycollate in the peritoneal cavity and examined the
inflammatory cells for ß-galactosidase activity. Although the
inflammatory response was intense, no cells demonstrated
ß-galactosidase activity. Collectively, these results lead us to
conclude that the ß-galactosidase-positive myocytes in the recipient
hearts originated from the engrafted donor WB-F344 stem cells.
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PCR amplification of their DNA, cloning, and sequencing of the PCR
product confirmed that the ß-galactosidase-positive myocytes
contained the rat Y-chromosome-specific repetitive DNA sequence (Figure 1e)
. As a positive control, we harvested WB-F344 cells from culture and
demonstrated that they contained this sequence (Figure 1e
, lane 2). In
contrast, endogenous (female) myocytes that were microdissected from
tissue sites proximal to the ß-galactosidase-positive myocytes did
not contain the rat Y-chromosome-specific sequence (Figure 1e
, lane 5).
We used FISH to show that ß-galactosidase-positive myocytes contained
the rat Y-chromosome. The FISH reaction was performed on fresh frozen
sections while the ß-galactosidase reaction was performed on fixed
tissue sections. To circumvent these differences in tissue preparation,
we used tissue obtained from immediately adjacent 8-µm-thick serial
sections, causing a shift in register of 8 µm or more. Using this
serial section approach, the cytoplasmic ß-galactosidase reaction
product was demonstrated in one tissue section, and the
rhodamine-stained Y-chromosome in the nucleus of the same myocyte in
the adjacent section (see Figure 3
). A cardiac phenotype was
confirmed in ß-galactosidase-expressing cells demonstrating that they
expressed a cardiac-specific protein, using a monoclonal antibody that
recognizes a cardiac-specific troponin T epitope, which is
conserved across species.24
The
ß-galactosidase-expressing cells demonstrated localization of the
fluorescence to striations consistent with troponin T localization in
the sarcomere (Figure 1, f and g)
.
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; a, b, c, and f). To provide a measure of the size range among the
engrafted WB-F344 cells, we grouped the cells into three sizes, <20
µm, 20 to 50 µm, and 50 to 110 µm in length. We counted 44 cells
that were <20 µm in length, 34 cells that were 20 to 50 µm in
length, and 53 cells that were 50 to 110 µm in length. Some of the
shortest cells may have been captured in cross-section. This
distribution is consistent with the possibility that WB-F344-derived
cells were at different stages of maturation 6 weeks after injection,
as suggested by the electron microscopic findings (Figure 1c)
. The
longest of the ß-galactosidase-positive cells were well
differentiated with clear sarcomere striations (Figure 1b)
identified
by light microscopy, whereas cells that measured 20 to 50 µm in
length were less well differentiated, exhibiting less well-developed
sarcomeres by light microscopy. Striations and sarcomeres in the latter
group of cells were readily demonstrated by electron microscopy.
WB-F344-derived myocytes were identified by the presence of the
electron-dense crystalloid precipitates (ß-galactosidase reaction
product) in their cytoplasm (Figure 2)
.
Nascent striations were present in the small (
20 µm, Figure 2c
)
cells whereas larger cells demonstrated well-organized and
differentiated sarcomeres (Figure 2a)
. In the well-differentiated
myocytes, intercalated disks and apparent gap junctions formed
anatomical couplings between ß-galactosidase-positive WB-F344-derived
myocytes and endogenous (host) myocytes that did not contain any X-gal
reaction precipitate (Figure 2, a and b)
. The presence of anatomical
couplings between stem cell-derived and host myocytes suggests strongly
that these WB-F344-derived myocytes participate in the function of the
cardiac syncytium. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is generally believed that adult mammalian cardiac myocytes have undergone terminal differentiation and can no longer divide to generate new myocytes. Instead, the residual myocytes respond to the cardiac workload after damage by undergoing hypertrophy. Depending on the extent and cause of injury, hypertrophied myocytes may not successfully support the cardiac output, and cardiac decompensation and heart failure result. The great impact of heart disease on mortality and quality of life and the scarcity of donor hearts for organ transplantation have led several investigations to examine transplantation of different types of cells into the heart in an attempt to achieve repair.30-39 Cells used in these studies were precommitted to a muscle lineage before transplantation in vivo. Embryonic cardiomyocytes derived from embryonic stem cells39 and fetal cardiomyocytes survive in the cardiac environment in vivo, and form primitive couplings with host cells.30 Similarly, transplanted skeletal muscle myoblasts31-37 develop into skeletal muscle myotubes and survive uncoupled with native cardiomyocytes for up to 3 months in recipient hearts. Cardiac myocyte cell lines have also been generated for potential therapeutic usage in the failing heart and for understanding myocyte function in vitro.40,41 A cell line derived from mouse atrial myocytes and propagated in vitro maintained an adult cardiac myocyte phenotype in culture.41 Bone marrow stromal cells have been transformed in vitro into a cardiomyogenic cell line with 5-azacytidine40 treatment. This cell line has been suggested,42 but has not been tested in vivo, as a source of cardiomyocytes for cell replacement therapy in cardiomyopathies. Our study goes further and demonstrates that cells from a stem cell line that is neither of embryonic origin nor committed to a muscle lineage will engraft in the adult heart in vivo and differentiate into well-organized mature cardiac myocytes.
Stem cell differentiation has been suggested to be regulated by two independent pathways. One pathway that allows their exit from the stem-cell state requires negatively active events that involve the silencing of regulators that repress the expression of specific gene.43 A second pathway that is positively acting involves regulation by transcription factors that allow stem cells to enter commitment into a specific lineage.43 The response of this WB-F344 liver-derived stem cell line in the heart in vivo as shown here, and a recent preliminary communication that reports that bone marrow stem cells differentiate in the ischemic heart into fetal myocytes (Orlic D, Kajstura J, Chimenti S, Li B, Anderson S, Bodine D, Pickel J, Leri A, Nadal-Ginard B, Anversa P: Exogenous hematopoietic stem cells can regenerate infarcted myocardium. Late Breaking Science Abstracts, American Heart Association, 2000, New Orleans), demonstrate that the adult heart tissue microenvironment expresses the appropriate signals that allow the exit of these extracardiac cells from their stem-cell state and differentiation into myocytes.
This raises the possibility that adult-derived human stem cells can be isolated from a patient, propagated in culture, and used to support the patient’s diseased heart. The potential therapeutic advantages of a clonal stem cell line such as WB-F344 would be the ability to expand the cell number in culture ex vivo and generate a well-characterized population of cells. These could be reintroduced into the patient or stored for future therapeutic interventions, avoiding the complications of allograft transplant rejection.
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Acknowledgements |
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Footnotes |
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Supported in part by National Heart, Lung, and Blood Institute grants R01 HL42250, R01 HL20749, and R01 CA29323 from the National Cancer Institute.
Accepted for publication February 16, 2001.
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