An In Vivo Analysis of Hematopoietic Stem Cell Potential
http://www.100md.com
《循环研究杂志》
the Department of Cell Biology and Anatomy (R.P.V., P.A.F., T.C.M., R.R.M., C.J.D.), Department of Medicine (Y.E., A.C.L., M.M., H.M., M.O.)
the Cardiovascular Developmental Biology Center (R.P.V., P.A.F., T.C.M., R.R.M., C.J.D.), Medical University of South Carolina
the Department of Veterans Affairs Medical Center (Y.E., A.C.L., M.M., H.M., M.O.), Charleston, SC.
Abstract
Recent studies evaluating hematopoietic stem cell (HSC) potential raise the possibility that, in addition to embryonic sources, adult valve fibroblasts may be derived from HSCs. To test this hypothesis, we used methods that allow the potential of a single HSC to be evaluated in vivo. This was achieved by isolation and clonal expansion of single lineage-negative (LineC), c-kit+, Sca-1+, CD34eC cells from the bone marrow of mice that ubiquitously express enhanced green fluorescent protein (EGFP) combined with transplantation of individual clonal populations derived from these candidate HSCs into a lethally irradiated congenic non-EGFP mouse. Histological analyses of valve tissue from clonally engrafted recipient mice revealed the presence of numerous EGFP+ cells within host valves. A subpopulation of these cells exhibited synthetic properties characteristic of fibroblasts, as evidenced by their expression of mRNA for procollagen 11. Further, we show by Y-chromosomeeCspecific fluorescence in situ hybridization analysis of female-to-male transplanted mice that the EGFP+ valve cells are the result of HSC-derived cell differentiation and not the fusion of EGFP+ donor cells with host somatic cells. Together, these findings demonstrate HSC contribution to the adult valve fibroblast population.
Key Words: adult stem cells bone marrow collagen stem cell plasticity
Introduction
The cardiac valves are composed of individual leaflets, each of which consists of connective tissue surrounded by an outer layer of endocardial endothelial cells. The connective tissue component of valve leaflets is composed of extracellular matrix (ECM) molecules, most prominently interstitial collagen, elastin, and glycosaminoglycans,1 and connective tissue/interstitial cells that include fibroblasts,2eC4 myofibroblasts,5,6 and smooth muscle cells.3,4,7 Given recent findings demonstrating that adult bone marroweCderived hematopoietic stem cells (HSCs) have the potential to give rise to a number of cell types,8eC13 we sought to test the hypothesis that HSCs are a source of valve interstitial cells in the adult.
The ability of HSCs to give rise to cell types not traditionally associated with their lineage is a subject of intense interest. To date, in vivo studies evaluating the potential of stem cells have reported findings that differ with regards to the degree of plasticity that HSCs possess.14eC20 These differing results are likely attributable to the fact that many of the studies evaluating HSC potential were conducted using mixed or enriched populations of stem cells.21eC24 To clearly define the potential of the HSC, we have developed protocols that allow the potential of a single HSC to be evaluated in vivo. This was achieved by combining clonal HSC transplantation25eC28 with the use of transgenic mice that express enhanced green fluorescent protein (EGFP) on a universal promoter.29 When EGFP+ donor HSCs are transplanted into congenic recipient mice, the progeny of donor adult bone marrow stem cells (EGFP+ cells) are readily identifiable in the recipient. Using this strategy, we have previously reported the ability of adult bone marrow HSCs to give rise to kidney mesangial cells,30 brain microglial cells, and perivascular pericyte-like cells.9 These findings, in conjunction with the fact that both kidney mesangial cells and brain microglial cells exhibit fibroblastic/myofibroblastic properties, have led us to speculate that HSCs may be a systemic source of such cells.
Findings reported herein establish that cells derived from adult bone marrow HSCs (1) reproducibly engraft into the cardiac valves of adult recipients, (2) are long-term residents of the valve leaflets, (3) are morphologically indistinguishable from host valve interstitial cells, and (4) express mRNA for procollagen 11.
Materials and Methods
Donor Cell Preparation
Ten- to 12-week-old female pCAGGS::EGFP (EGFP+) mice (C57BL/6-Ly5.2 background),29 provided by M. Okabe (Osaka University, Japan), were used as bone marrow donors. Ten- to 14-week-old male C57BL/6-Ly5.1 mice (Jackson Laboratories, Bar Harbor, Me) were used as irradiated recipients and as the source for radioprotective cells. All animals were treated and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Research Council, Washington, DC, 1996) and with guidelines set by the institutional animal care and use committee, Medical University of South Carolina. Lineage-negative (LineC), c-kit+, Sca-1+, CD34eC cells, which are highly enriched for HSCs, were prepared from the bone marrow total nucleated cells (BM-TNCs) of EGFP mice as described previously.30 For 100-cell transplantation, freshly prepared cells LineC, c-kit+, Sca-1+, CD34eC cells were injected directly into irradiated recipient mice without cell culture. For clonal transplantation, single LineC, c-kit+, Sca-1+, CD34eC cells were deposited into individual wells of Corning 96-well plates, and candidate HSCs were identified during short-term culture in the presence of recombinant rat steel factor (SF) and recombinant human G-CSF (for details, see Masuya et al30). Based on the studies of Weissman and colleagues,31,32 cells with long-term repopulating activity reside in the Sca-1+, c-kit+ fraction of bone marrow mononuclear cells. Therefore, as the 100-cell and clonal cells used in our studies both express Sca-1 and c-kit, their repopulating activity is identical.
Transplantation
Recipient Ly-5.1 mice were prepared with a single 950-cGy dose of total body irradiation using a 4x106 V linear accelerator. To achieve a high efficiency of clonal HSC engraftment, the contents of wells containing 20 or fewer viable clusters of cells were injected into tail veins of the irradiated Ly-5.1 mice.9,30 Five hundred LineC, c-kit+, Sca-1+, CD34eC BM cells from Ly-5.1 mice, which contain only short-term repopulating cells, were also transplanted as radioprotective cells to prevent postirradiation death.25 A separate cohort of mice received 100 noncultured LineC, c-kit+, Sca-1+, CD34eC cells from EGFP+ mice to exclude effects of short-term culture on valve cell potential. In separate experiments to exclude the effects of total body irradiation, busulfan conditioning of recipient mice before donor stem cell transplantation was accomplished by subcutaneous delivery of 22.5 mg/kg busulfan in PBS to pregnant mice on embryonic days E17 and E18 of gestation.33,34 Pups were injected IP with 1.0x106 donor EGFP+ BM-TNC on postnatal day 1 (P1). In all transplantation models, levels of blood chimerism were monitored at 30-day intervals posttransplantation. Flow cytometric analysis of hematopoietic engraftment was performed 2 months after transplantation, as previously described.30 Only mice exhibiting high levels of multilineage hematopoietic engraftment were used for analysis of stem cell contribution to the valve interstitial cell population in the clonally transplanted and 100-cell transplanted, and busulfan-conditioned mice.
Histological Analysis of Engrafted EGFP+ Cells
Hearts from engrafted mice were excised and processed for paraffin sectioning as described previously.30 Histological 3.0-e sections were imaged for EGFP fluorescence using a bandpass filter cube that is optimized for EGFP excitation. Anti-GFP immunolabeling was performed with a polyclonal antibody to GFP (Molecular Probes, Eugene, Ore). Secondary antibodies were purchased from Jackson Immunochemicals (West Grove, Pa). DRAQ5 red fluorescent DNA probe (Biostatus, Shepshed Leicestershire, UK) was used to label nuclei. Epifluorescence/differential interference contrast (DIC) imaging was conducted on a Leica DMRB HC microscope equipped with the narrow bandpass GFP excitation cube. This instrument is supported by a digital imaging workstation that includes a real-time color digital camera (SPOT-RT) and a Gateway Select 1400 personal computer. Images were processed using Adobe Photoshop 7.0.
In Situ Hybridization
A nonradioactive in situ hybridization technique using digoxigenin (DIG)-labeled RNA probes was used as described in Kubo et al,35 to detect expression of procollagen 11 mRNA in valve tissue sections from transplanted mice. A 321-bp fragment of COL1A1 cDNA, in Bluescript SK II phagemid (Stratagene, La Jolla, Calif), kindly provided by Dr Maria Trojanowska (Medical University of South Carolina), was used as template for in vitro transcription. Sense probes and antisense probes for COL1A1 were labeled with DIG-11-UTP using a DIG RNA-labeling kit (Roche, Indianapolis, Ind). The labeled RNA probes were used at a final concentration of 1 ng/e蘈. Hybridization was performed in a humidified chamber for 18 hours at 65°C. After posthybridization washing, DIG-labeled probes were detected using a DIG-nucleotide detection kit according to the protocol of the manufacturer (Roche).
Fluorescence In Situ Hybridization Analysis of Engrafted Valves
To investigate whether stem cell fusion with somatic cells had occurred,36,37 Y-chromosomeeCspecific fluorescence in situ hybridization (FISH) analysis was performed on valve tissue sections from male recipients of female (EGFP+) bone marrow cells. Paraformaldehyde-fixed, paraffin-embedded sections were cleared to PBS, mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, Calif), and imaged using a narrow bandpass GFP excitation cube. The coverslips were subsequently removed and the sections hybridized with a biotinylated Y-chromosome paint probe (Cambio, Cambridge, UK) and visualized with Cy3-conjugated streptavidin (Vector Labs). Nuclei were counterstained with DRAQ5 (Biostatus). Sections were coverslipped with Vectashield (Vector Labs).
Results
Mice Transplanted With a Clonal Population of Cells Derived From a Single EGFP+ HSC Exhibit a High Level of Multilineage Hematopoietic Reconstitution
To evaluate the ability of the HSC to contribute to the adult valve interstitial cell population, single candidate HSCs (LineC, c-kit+, Sca-1+, CD34eC) were isolated from donor (pCAGGS::EGFP) EGFP+ mice29 and clonally expanded in short-term culture. Individual clones were subsequently transplanted into lethally irradiated congenic non-EGFP mice. To abrogate concerns over the effects of culture on HSC potential, parallel experiments were conducted in which 100 noncultured bone marrow LineC, c-kit+, Sca-1+, CD34eC cells were transplanted into lethally irradiated congenic non-EGFP mice.
As the ability to evaluate the in vivo potential of the HSC is dependent on the efficiency of engraftment, transplanted mice were assayed to determine the level of donor HSC engraftment 60 days after transplantation. Figure 1 depicts flow cytometric analysis of the nucleated blood cells of a representative clonally transplanted mouse. The abundance of EGFP+-nucleated cells in the granulocyte/macrophage and B-cell and T-cell lineages indicates that a high level of multilineage hematopoietic reconstitution was achieved. Only those mice exhibiting high levels (>60%) of multilineage hematopoietic reconstitution following transplantation with either clonal cells or 100 LineC, c-kit+, Sca-1+, CD34eC cells were included in this study.
EGFP+ Cells Are Evident in the Valves of Mice Transplanted With a Clonal Population of Cells Derived From a Single EGFP+ HSC
When cardiac valves from mice (n=6) were analyzed at 3 months after transplantation with a clonal population of cells derived from a single EGFP+ HSC, numerous EGFP+ cells were evident within the valve leaflet (Figure 2A through 2C). Analysis of the sectioned valve leaflets at higher magnification revealed that the EGFP+ cells were distributed throughout the leaflets (Figure 2D). As judged by morphology, the engrafted EGFP+ cells were indistinguishable from host (non-EGFP+) valve interstitial cells. Similar to the results obtained when mice were transplanted with a clonal population of HSCs, analysis of mice (n=4) transplanted with 100 noncultured LineC, c-kit+, Sca-1+, CD34eC cells also revealed the presence of a population of EGFP+ cells in the valve leaflets that were morphologically indistinguishable from host (non-EGFP+) valve interstitial cells (Figure 2E). To determine whether the EGFP+ cells in the valves and/or the ability of bone marrow stem cells to generate valve interstitial cells persisted over time, we extended the time between transplantation and analysis to periods up to 12 months. The presence of EGFP+ cells in the valves of mice 1 year posttransplantation clearly demonstrates that engraftment of HSC-derived (EGFP+) cells in the valves and/or the ability of bone marrow stem cells to generate valve interstitial cells persisted over time (Figure 2F).
Engrafted EGFP+ Cells Express mRNA for Procollagen 11
To investigate whether the EGFP+ cells observed in the valves of transplanted mice represented a population of cells that falls outside of those traditionally ascribed to the HSC lineage, we sought to evaluate whether the EGFP+ cells contribute to valve ECM production. To accomplish this, we performed in situ hybridization on sections of valves obtained from mice that had been clonally engrafted with EGFP+ HSCs. As seen in Figure 3A, collagen-synthesizing cells were distributed throughout the valve leaflets of transplanted mice. To determine whether any of the collagen synthesizing cells were of bone marrow origin, the section immediately following the section depicted in Figure 3A was immunolabeled with antibodies to GFP. When the pattern of procollagen 11 expression was compared with the pattern of EGFP immunoreactivity in the serial 3-e sections (compare Figure 3A and 3B), it was evident that a subpopulation of the EGFP+ cells expressed mRNA for procollagen 11. These findings strongly suggest that HSC-derived cells contribute to a valve interstitial cell population that exhibits the synthetic phenotype of fibroblasts.
Engraftment of EGFP+ Cells Into the Valves of Recipient Mice Is Not an Effect of Tissue Injury
In studies evaluating stem cell potential, irradiation is routinely used to establish a bone marrow niche for donor HSC engraftment and expansion. It is generally held that, following irradiation, HSCs engraft into the bone marrow and subsequently give rise to progenitor cells, which circulate in the peripheral blood before engrafting into various tissues. However, as whole body irradiation has the potential to induce injury38eC41 and elicit the homing of hematopoietic-derived cells, we sought to evaluate whether irradiation could also induce the homing and/or engraftment of bone marroweCderived cells to the valves. To evaluate this potential, counts of cells per unit area in histological sections of valves from age-matched control and irradiated mice at 7 days postirradiation were compared. Analysis of cell counts from 3 independent investigators detected no significant difference in cell density in the valves (irradiated 1.056±0.002 cells/unit area versus control=1.075±0.002 cells/unit area, P=0.132).
As an alternative to irradiation and as an additional control experiment, pretransplantation conditioning by whole body irradiation was replaced with intrapartum delivery of the chemotherapeutic agent busulfan. This treatment has been shown to have myelosupressive effects33 and therefore conditions recipient mice for donor bone marrow stem cell engraftment (see Materials and Methods). Mice with high levels of multilineage hematopoietic reconstitution were selected for analysis of stem cell contribution to the valve interstitial cell population. As seen in Figure 4, EGFP+ cells were evident throughout the valve leaflet in busulfan-treated mice. The similarity of results of the 2 different transplantation strategies strongly supports that the tissue engraftment we observe is a physiological event and not an artifact of the experimental systems.
EGFP + Valve Cells Are Derived As a Result of HSC Differentiation and Not From the Fusion of EGFP+ Cell With Host Somatic Cells
Recent reports have called into question the concept of "plasticity" of adult bone marrow stem cells, suggesting that this apparent plasticity is actually the result of fusion of donor stem cells with host somatic cells.36,37 To exclude the possibility that the adult bone marrow HSC-derived fibroblasts are produced by a fusion event, we performed FISH analysis on histological sections of valve leaflets from gender mismatched transplant recipients. Using this strategy, male recipients of (EGFP+) female donor bone marrow cells were hybridized with a Y-chromosomeeCspecific paint probe. Figure 5A is a composite image depicting the superimposition of the EGFP, Y-chromosome paint and the nuclear DRAQ5 fluorescence. EGFP-expressing cells that lack a Y chromosome are clearly evident (asterisks). In the case of other cells, the composite image precludes the clear assignment of fluorescence expression to individual cells (Figure 5A, inset). To clearly identify Y-chromosome probe hybridization in individual cells, the EGFP fluorescence was omitted from the image depicted in Figure 5B. Comparison of the insets in Figure 5A and 5B, which depict the same group of cells, clearly demonstrates that the Y-chromosome+ cell and the EGFP+ cell are indeed separate cells. Similar analysis revealed that in no case could an EGFP+ cell be identified that also hybridized with the Y-chromosome paint probe.
Discussion
It is currently held that adult valve interstitial cells, including fibroblasts,3,4,42 myofibroblasts,5,6 and smooth muscle cells3,4,7 are derived from developmental sources such as the epicardially derived mesenchymal cells that populate primitive embryonic valves/endocardial cushions43 and cells derived from the embryonic epicardium.44eC46 In recent years, evidence has emerged to suggest that in addition to these embryonic sources, adult valve interstitial cell populations may also be derived from sources outside of the valves and, more specifically, from stem cells. That valve interstitial cells may be supplemented by nonvalvular sources in the adult was first indicated by the studies of Koolbergen et al,47 who showed that in patients receiving gender mismatched cryopreserved homograft valve transplants, both donor and recipient cells characterized as fibroblasts were found in the valve leaflets.
Our findings demonstrating that adult HSCs are a source of adult valve interstitial fibroblasts provide the cellular basis for the observations made by Koolbergen et al.47 We base our conclusion that HSCs are a source of valve fibroblasts on the following. First is our finding that HSC-derived EGFP+ cells within the valves express mRNA for procollagen 11, an ECM molecule that is considered to be the signature of the fibroblast. Indeed, collagen synthesis is well described in cardiac fibroblasts outside of the valves.48eC50 Although we are aware that expression of collagen type I is not restricted solely to fibroblasts, its expression in cells that are residents of the valve connective tissue strongly argues that EGFP+/collagen mRNAeCexpressing cells represent HSC-derived fibroblasts. Second, our conclusion that HSCs differentiated into EGFP+ valve fibroblasts is also supported by the fact that cells traditionally associated with hematopoietic lineage do not express collagen type I. Third, the finding that HSCs are a source of fibroblasts is consistent with our overall findings evaluating the differentiation potential of HSCs in other organs. For instance, we have shown that HSCs can give rise to mesangial cells,30 a population of cells that have been described as fibroblastic/myofibroblastic in nature51,52 as well as central nervous system microglia and pericyte-like perivascular cells,9 which also possess both synthetic and contractile properties.
Our demonstration of a HSC origin of valve fibroblasts is compatible with the findings of others evaluating the contributions of cells of bone marrow origin. For example, Wilcox et al53 identified myofibroblast precursors in peripheral blood, and Li et al54 identified vascular smooth muscle cells of recipient origin in the aortic intima of allotransplanted mice. The potential of stem cells to give rise to smooth muscle cells is also indicated by studies demonstrating that smooth muscle cells in the aortic intima following transplant arteriopathy are host bone marrow derived.55 Support for the concept that bone marroweCderived cells have a fibroblastic potential is the presence of circulating fibroblast-like cells, called fibrocytes, in the peripheral blood.56 These circulating cells express both fibroblastic and hematopoietic surface markers.56eC58 As these cells have been shown to enter wounds and are detected in scar tissue, Bucala et al56 have suggested that they represent a systemic source of fibroblasts that may participate with local fibroblasts in wound repair and pathological fibrosis. Collectively, our findings and the work of others strongly support our conclusion that HSCs are indeed a source of new valve fibroblasts in the adult cardiac valves.
The existence of valve interstitial cells derived at different times and from different origins (ie, embryonic epicardium and endocardial cushions44,45 and the adult bone marrow, as reported herein) raises the interesting possibility that these populations of fibroblasts are functionally different and, thus, differ in their susceptibility to and/or participation in valve pathological processes. If correct, this concept may advance our understanding of valvular pathologies that are associated with misregulation of interstitial cellular processes that are inherent to calcification,59,60 fibrosis,61 and cell death and proliferation in response to valve injury2,62 as well as the cellular pathways involved in the progression of these pathologies. In this context, the recent report that adult HSC-derived cells can give rise to osteoblastic precursors13 raises the interesting possibility that calcific valve disease may involve misregulation of this osteogenic potential by HSC-derived valve interstitial cells.
Acknowledgments
This work was supported by NIH grants RO1-HL57375, RO1-HL69123, HL/HD67135, PO1-HL52813, and P20-RR1eC16434 from the National Center for Research Resources, and C06-RR018823 from the Extramural Research Facilities Program of the NIH, NCRR; the Medical University of South Carolina; the Cardiac Developmental Biology Center; and the Office of Research and Development, Medical Research Services, Department of Veterans Affairs. We thank Haiqun Zeng for cell sorting, James D. Duncan and P. Evan Myers for technical assistance, Joyce B. Edmunds for expertise with tissue processing, and the staff of the Department of Radiation Oncology, Medical University of South Carolina, for assistance in the irradiation of mice.
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the Cardiovascular Developmental Biology Center (R.P.V., P.A.F., T.C.M., R.R.M., C.J.D.), Medical University of South Carolina
the Department of Veterans Affairs Medical Center (Y.E., A.C.L., M.M., H.M., M.O.), Charleston, SC.
Abstract
Recent studies evaluating hematopoietic stem cell (HSC) potential raise the possibility that, in addition to embryonic sources, adult valve fibroblasts may be derived from HSCs. To test this hypothesis, we used methods that allow the potential of a single HSC to be evaluated in vivo. This was achieved by isolation and clonal expansion of single lineage-negative (LineC), c-kit+, Sca-1+, CD34eC cells from the bone marrow of mice that ubiquitously express enhanced green fluorescent protein (EGFP) combined with transplantation of individual clonal populations derived from these candidate HSCs into a lethally irradiated congenic non-EGFP mouse. Histological analyses of valve tissue from clonally engrafted recipient mice revealed the presence of numerous EGFP+ cells within host valves. A subpopulation of these cells exhibited synthetic properties characteristic of fibroblasts, as evidenced by their expression of mRNA for procollagen 11. Further, we show by Y-chromosomeeCspecific fluorescence in situ hybridization analysis of female-to-male transplanted mice that the EGFP+ valve cells are the result of HSC-derived cell differentiation and not the fusion of EGFP+ donor cells with host somatic cells. Together, these findings demonstrate HSC contribution to the adult valve fibroblast population.
Key Words: adult stem cells bone marrow collagen stem cell plasticity
Introduction
The cardiac valves are composed of individual leaflets, each of which consists of connective tissue surrounded by an outer layer of endocardial endothelial cells. The connective tissue component of valve leaflets is composed of extracellular matrix (ECM) molecules, most prominently interstitial collagen, elastin, and glycosaminoglycans,1 and connective tissue/interstitial cells that include fibroblasts,2eC4 myofibroblasts,5,6 and smooth muscle cells.3,4,7 Given recent findings demonstrating that adult bone marroweCderived hematopoietic stem cells (HSCs) have the potential to give rise to a number of cell types,8eC13 we sought to test the hypothesis that HSCs are a source of valve interstitial cells in the adult.
The ability of HSCs to give rise to cell types not traditionally associated with their lineage is a subject of intense interest. To date, in vivo studies evaluating the potential of stem cells have reported findings that differ with regards to the degree of plasticity that HSCs possess.14eC20 These differing results are likely attributable to the fact that many of the studies evaluating HSC potential were conducted using mixed or enriched populations of stem cells.21eC24 To clearly define the potential of the HSC, we have developed protocols that allow the potential of a single HSC to be evaluated in vivo. This was achieved by combining clonal HSC transplantation25eC28 with the use of transgenic mice that express enhanced green fluorescent protein (EGFP) on a universal promoter.29 When EGFP+ donor HSCs are transplanted into congenic recipient mice, the progeny of donor adult bone marrow stem cells (EGFP+ cells) are readily identifiable in the recipient. Using this strategy, we have previously reported the ability of adult bone marrow HSCs to give rise to kidney mesangial cells,30 brain microglial cells, and perivascular pericyte-like cells.9 These findings, in conjunction with the fact that both kidney mesangial cells and brain microglial cells exhibit fibroblastic/myofibroblastic properties, have led us to speculate that HSCs may be a systemic source of such cells.
Findings reported herein establish that cells derived from adult bone marrow HSCs (1) reproducibly engraft into the cardiac valves of adult recipients, (2) are long-term residents of the valve leaflets, (3) are morphologically indistinguishable from host valve interstitial cells, and (4) express mRNA for procollagen 11.
Materials and Methods
Donor Cell Preparation
Ten- to 12-week-old female pCAGGS::EGFP (EGFP+) mice (C57BL/6-Ly5.2 background),29 provided by M. Okabe (Osaka University, Japan), were used as bone marrow donors. Ten- to 14-week-old male C57BL/6-Ly5.1 mice (Jackson Laboratories, Bar Harbor, Me) were used as irradiated recipients and as the source for radioprotective cells. All animals were treated and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Research Council, Washington, DC, 1996) and with guidelines set by the institutional animal care and use committee, Medical University of South Carolina. Lineage-negative (LineC), c-kit+, Sca-1+, CD34eC cells, which are highly enriched for HSCs, were prepared from the bone marrow total nucleated cells (BM-TNCs) of EGFP mice as described previously.30 For 100-cell transplantation, freshly prepared cells LineC, c-kit+, Sca-1+, CD34eC cells were injected directly into irradiated recipient mice without cell culture. For clonal transplantation, single LineC, c-kit+, Sca-1+, CD34eC cells were deposited into individual wells of Corning 96-well plates, and candidate HSCs were identified during short-term culture in the presence of recombinant rat steel factor (SF) and recombinant human G-CSF (for details, see Masuya et al30). Based on the studies of Weissman and colleagues,31,32 cells with long-term repopulating activity reside in the Sca-1+, c-kit+ fraction of bone marrow mononuclear cells. Therefore, as the 100-cell and clonal cells used in our studies both express Sca-1 and c-kit, their repopulating activity is identical.
Transplantation
Recipient Ly-5.1 mice were prepared with a single 950-cGy dose of total body irradiation using a 4x106 V linear accelerator. To achieve a high efficiency of clonal HSC engraftment, the contents of wells containing 20 or fewer viable clusters of cells were injected into tail veins of the irradiated Ly-5.1 mice.9,30 Five hundred LineC, c-kit+, Sca-1+, CD34eC BM cells from Ly-5.1 mice, which contain only short-term repopulating cells, were also transplanted as radioprotective cells to prevent postirradiation death.25 A separate cohort of mice received 100 noncultured LineC, c-kit+, Sca-1+, CD34eC cells from EGFP+ mice to exclude effects of short-term culture on valve cell potential. In separate experiments to exclude the effects of total body irradiation, busulfan conditioning of recipient mice before donor stem cell transplantation was accomplished by subcutaneous delivery of 22.5 mg/kg busulfan in PBS to pregnant mice on embryonic days E17 and E18 of gestation.33,34 Pups were injected IP with 1.0x106 donor EGFP+ BM-TNC on postnatal day 1 (P1). In all transplantation models, levels of blood chimerism were monitored at 30-day intervals posttransplantation. Flow cytometric analysis of hematopoietic engraftment was performed 2 months after transplantation, as previously described.30 Only mice exhibiting high levels of multilineage hematopoietic engraftment were used for analysis of stem cell contribution to the valve interstitial cell population in the clonally transplanted and 100-cell transplanted, and busulfan-conditioned mice.
Histological Analysis of Engrafted EGFP+ Cells
Hearts from engrafted mice were excised and processed for paraffin sectioning as described previously.30 Histological 3.0-e sections were imaged for EGFP fluorescence using a bandpass filter cube that is optimized for EGFP excitation. Anti-GFP immunolabeling was performed with a polyclonal antibody to GFP (Molecular Probes, Eugene, Ore). Secondary antibodies were purchased from Jackson Immunochemicals (West Grove, Pa). DRAQ5 red fluorescent DNA probe (Biostatus, Shepshed Leicestershire, UK) was used to label nuclei. Epifluorescence/differential interference contrast (DIC) imaging was conducted on a Leica DMRB HC microscope equipped with the narrow bandpass GFP excitation cube. This instrument is supported by a digital imaging workstation that includes a real-time color digital camera (SPOT-RT) and a Gateway Select 1400 personal computer. Images were processed using Adobe Photoshop 7.0.
In Situ Hybridization
A nonradioactive in situ hybridization technique using digoxigenin (DIG)-labeled RNA probes was used as described in Kubo et al,35 to detect expression of procollagen 11 mRNA in valve tissue sections from transplanted mice. A 321-bp fragment of COL1A1 cDNA, in Bluescript SK II phagemid (Stratagene, La Jolla, Calif), kindly provided by Dr Maria Trojanowska (Medical University of South Carolina), was used as template for in vitro transcription. Sense probes and antisense probes for COL1A1 were labeled with DIG-11-UTP using a DIG RNA-labeling kit (Roche, Indianapolis, Ind). The labeled RNA probes were used at a final concentration of 1 ng/e蘈. Hybridization was performed in a humidified chamber for 18 hours at 65°C. After posthybridization washing, DIG-labeled probes were detected using a DIG-nucleotide detection kit according to the protocol of the manufacturer (Roche).
Fluorescence In Situ Hybridization Analysis of Engrafted Valves
To investigate whether stem cell fusion with somatic cells had occurred,36,37 Y-chromosomeeCspecific fluorescence in situ hybridization (FISH) analysis was performed on valve tissue sections from male recipients of female (EGFP+) bone marrow cells. Paraformaldehyde-fixed, paraffin-embedded sections were cleared to PBS, mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, Calif), and imaged using a narrow bandpass GFP excitation cube. The coverslips were subsequently removed and the sections hybridized with a biotinylated Y-chromosome paint probe (Cambio, Cambridge, UK) and visualized with Cy3-conjugated streptavidin (Vector Labs). Nuclei were counterstained with DRAQ5 (Biostatus). Sections were coverslipped with Vectashield (Vector Labs).
Results
Mice Transplanted With a Clonal Population of Cells Derived From a Single EGFP+ HSC Exhibit a High Level of Multilineage Hematopoietic Reconstitution
To evaluate the ability of the HSC to contribute to the adult valve interstitial cell population, single candidate HSCs (LineC, c-kit+, Sca-1+, CD34eC) were isolated from donor (pCAGGS::EGFP) EGFP+ mice29 and clonally expanded in short-term culture. Individual clones were subsequently transplanted into lethally irradiated congenic non-EGFP mice. To abrogate concerns over the effects of culture on HSC potential, parallel experiments were conducted in which 100 noncultured bone marrow LineC, c-kit+, Sca-1+, CD34eC cells were transplanted into lethally irradiated congenic non-EGFP mice.
As the ability to evaluate the in vivo potential of the HSC is dependent on the efficiency of engraftment, transplanted mice were assayed to determine the level of donor HSC engraftment 60 days after transplantation. Figure 1 depicts flow cytometric analysis of the nucleated blood cells of a representative clonally transplanted mouse. The abundance of EGFP+-nucleated cells in the granulocyte/macrophage and B-cell and T-cell lineages indicates that a high level of multilineage hematopoietic reconstitution was achieved. Only those mice exhibiting high levels (>60%) of multilineage hematopoietic reconstitution following transplantation with either clonal cells or 100 LineC, c-kit+, Sca-1+, CD34eC cells were included in this study.
EGFP+ Cells Are Evident in the Valves of Mice Transplanted With a Clonal Population of Cells Derived From a Single EGFP+ HSC
When cardiac valves from mice (n=6) were analyzed at 3 months after transplantation with a clonal population of cells derived from a single EGFP+ HSC, numerous EGFP+ cells were evident within the valve leaflet (Figure 2A through 2C). Analysis of the sectioned valve leaflets at higher magnification revealed that the EGFP+ cells were distributed throughout the leaflets (Figure 2D). As judged by morphology, the engrafted EGFP+ cells were indistinguishable from host (non-EGFP+) valve interstitial cells. Similar to the results obtained when mice were transplanted with a clonal population of HSCs, analysis of mice (n=4) transplanted with 100 noncultured LineC, c-kit+, Sca-1+, CD34eC cells also revealed the presence of a population of EGFP+ cells in the valve leaflets that were morphologically indistinguishable from host (non-EGFP+) valve interstitial cells (Figure 2E). To determine whether the EGFP+ cells in the valves and/or the ability of bone marrow stem cells to generate valve interstitial cells persisted over time, we extended the time between transplantation and analysis to periods up to 12 months. The presence of EGFP+ cells in the valves of mice 1 year posttransplantation clearly demonstrates that engraftment of HSC-derived (EGFP+) cells in the valves and/or the ability of bone marrow stem cells to generate valve interstitial cells persisted over time (Figure 2F).
Engrafted EGFP+ Cells Express mRNA for Procollagen 11
To investigate whether the EGFP+ cells observed in the valves of transplanted mice represented a population of cells that falls outside of those traditionally ascribed to the HSC lineage, we sought to evaluate whether the EGFP+ cells contribute to valve ECM production. To accomplish this, we performed in situ hybridization on sections of valves obtained from mice that had been clonally engrafted with EGFP+ HSCs. As seen in Figure 3A, collagen-synthesizing cells were distributed throughout the valve leaflets of transplanted mice. To determine whether any of the collagen synthesizing cells were of bone marrow origin, the section immediately following the section depicted in Figure 3A was immunolabeled with antibodies to GFP. When the pattern of procollagen 11 expression was compared with the pattern of EGFP immunoreactivity in the serial 3-e sections (compare Figure 3A and 3B), it was evident that a subpopulation of the EGFP+ cells expressed mRNA for procollagen 11. These findings strongly suggest that HSC-derived cells contribute to a valve interstitial cell population that exhibits the synthetic phenotype of fibroblasts.
Engraftment of EGFP+ Cells Into the Valves of Recipient Mice Is Not an Effect of Tissue Injury
In studies evaluating stem cell potential, irradiation is routinely used to establish a bone marrow niche for donor HSC engraftment and expansion. It is generally held that, following irradiation, HSCs engraft into the bone marrow and subsequently give rise to progenitor cells, which circulate in the peripheral blood before engrafting into various tissues. However, as whole body irradiation has the potential to induce injury38eC41 and elicit the homing of hematopoietic-derived cells, we sought to evaluate whether irradiation could also induce the homing and/or engraftment of bone marroweCderived cells to the valves. To evaluate this potential, counts of cells per unit area in histological sections of valves from age-matched control and irradiated mice at 7 days postirradiation were compared. Analysis of cell counts from 3 independent investigators detected no significant difference in cell density in the valves (irradiated 1.056±0.002 cells/unit area versus control=1.075±0.002 cells/unit area, P=0.132).
As an alternative to irradiation and as an additional control experiment, pretransplantation conditioning by whole body irradiation was replaced with intrapartum delivery of the chemotherapeutic agent busulfan. This treatment has been shown to have myelosupressive effects33 and therefore conditions recipient mice for donor bone marrow stem cell engraftment (see Materials and Methods). Mice with high levels of multilineage hematopoietic reconstitution were selected for analysis of stem cell contribution to the valve interstitial cell population. As seen in Figure 4, EGFP+ cells were evident throughout the valve leaflet in busulfan-treated mice. The similarity of results of the 2 different transplantation strategies strongly supports that the tissue engraftment we observe is a physiological event and not an artifact of the experimental systems.
EGFP + Valve Cells Are Derived As a Result of HSC Differentiation and Not From the Fusion of EGFP+ Cell With Host Somatic Cells
Recent reports have called into question the concept of "plasticity" of adult bone marrow stem cells, suggesting that this apparent plasticity is actually the result of fusion of donor stem cells with host somatic cells.36,37 To exclude the possibility that the adult bone marrow HSC-derived fibroblasts are produced by a fusion event, we performed FISH analysis on histological sections of valve leaflets from gender mismatched transplant recipients. Using this strategy, male recipients of (EGFP+) female donor bone marrow cells were hybridized with a Y-chromosomeeCspecific paint probe. Figure 5A is a composite image depicting the superimposition of the EGFP, Y-chromosome paint and the nuclear DRAQ5 fluorescence. EGFP-expressing cells that lack a Y chromosome are clearly evident (asterisks). In the case of other cells, the composite image precludes the clear assignment of fluorescence expression to individual cells (Figure 5A, inset). To clearly identify Y-chromosome probe hybridization in individual cells, the EGFP fluorescence was omitted from the image depicted in Figure 5B. Comparison of the insets in Figure 5A and 5B, which depict the same group of cells, clearly demonstrates that the Y-chromosome+ cell and the EGFP+ cell are indeed separate cells. Similar analysis revealed that in no case could an EGFP+ cell be identified that also hybridized with the Y-chromosome paint probe.
Discussion
It is currently held that adult valve interstitial cells, including fibroblasts,3,4,42 myofibroblasts,5,6 and smooth muscle cells3,4,7 are derived from developmental sources such as the epicardially derived mesenchymal cells that populate primitive embryonic valves/endocardial cushions43 and cells derived from the embryonic epicardium.44eC46 In recent years, evidence has emerged to suggest that in addition to these embryonic sources, adult valve interstitial cell populations may also be derived from sources outside of the valves and, more specifically, from stem cells. That valve interstitial cells may be supplemented by nonvalvular sources in the adult was first indicated by the studies of Koolbergen et al,47 who showed that in patients receiving gender mismatched cryopreserved homograft valve transplants, both donor and recipient cells characterized as fibroblasts were found in the valve leaflets.
Our findings demonstrating that adult HSCs are a source of adult valve interstitial fibroblasts provide the cellular basis for the observations made by Koolbergen et al.47 We base our conclusion that HSCs are a source of valve fibroblasts on the following. First is our finding that HSC-derived EGFP+ cells within the valves express mRNA for procollagen 11, an ECM molecule that is considered to be the signature of the fibroblast. Indeed, collagen synthesis is well described in cardiac fibroblasts outside of the valves.48eC50 Although we are aware that expression of collagen type I is not restricted solely to fibroblasts, its expression in cells that are residents of the valve connective tissue strongly argues that EGFP+/collagen mRNAeCexpressing cells represent HSC-derived fibroblasts. Second, our conclusion that HSCs differentiated into EGFP+ valve fibroblasts is also supported by the fact that cells traditionally associated with hematopoietic lineage do not express collagen type I. Third, the finding that HSCs are a source of fibroblasts is consistent with our overall findings evaluating the differentiation potential of HSCs in other organs. For instance, we have shown that HSCs can give rise to mesangial cells,30 a population of cells that have been described as fibroblastic/myofibroblastic in nature51,52 as well as central nervous system microglia and pericyte-like perivascular cells,9 which also possess both synthetic and contractile properties.
Our demonstration of a HSC origin of valve fibroblasts is compatible with the findings of others evaluating the contributions of cells of bone marrow origin. For example, Wilcox et al53 identified myofibroblast precursors in peripheral blood, and Li et al54 identified vascular smooth muscle cells of recipient origin in the aortic intima of allotransplanted mice. The potential of stem cells to give rise to smooth muscle cells is also indicated by studies demonstrating that smooth muscle cells in the aortic intima following transplant arteriopathy are host bone marrow derived.55 Support for the concept that bone marroweCderived cells have a fibroblastic potential is the presence of circulating fibroblast-like cells, called fibrocytes, in the peripheral blood.56 These circulating cells express both fibroblastic and hematopoietic surface markers.56eC58 As these cells have been shown to enter wounds and are detected in scar tissue, Bucala et al56 have suggested that they represent a systemic source of fibroblasts that may participate with local fibroblasts in wound repair and pathological fibrosis. Collectively, our findings and the work of others strongly support our conclusion that HSCs are indeed a source of new valve fibroblasts in the adult cardiac valves.
The existence of valve interstitial cells derived at different times and from different origins (ie, embryonic epicardium and endocardial cushions44,45 and the adult bone marrow, as reported herein) raises the interesting possibility that these populations of fibroblasts are functionally different and, thus, differ in their susceptibility to and/or participation in valve pathological processes. If correct, this concept may advance our understanding of valvular pathologies that are associated with misregulation of interstitial cellular processes that are inherent to calcification,59,60 fibrosis,61 and cell death and proliferation in response to valve injury2,62 as well as the cellular pathways involved in the progression of these pathologies. In this context, the recent report that adult HSC-derived cells can give rise to osteoblastic precursors13 raises the interesting possibility that calcific valve disease may involve misregulation of this osteogenic potential by HSC-derived valve interstitial cells.
Acknowledgments
This work was supported by NIH grants RO1-HL57375, RO1-HL69123, HL/HD67135, PO1-HL52813, and P20-RR1eC16434 from the National Center for Research Resources, and C06-RR018823 from the Extramural Research Facilities Program of the NIH, NCRR; the Medical University of South Carolina; the Cardiac Developmental Biology Center; and the Office of Research and Development, Medical Research Services, Department of Veterans Affairs. We thank Haiqun Zeng for cell sorting, James D. Duncan and P. Evan Myers for technical assistance, Joyce B. Edmunds for expertise with tissue processing, and the staff of the Department of Radiation Oncology, Medical University of South Carolina, for assistance in the irradiation of mice.
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