In Vivo Characterization of Bone Marrow–Derived Fibroblasts Recruited into Fibrotic Lesions
http://www.100md.com
《干细胞学杂志》
a Pathology Division, and
b Investigative Treatment Division, National Cancer Center Research Institute East, Kashiwa, Chiba, Japan;
c Laboratory of Cancer Biology, Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan;
d Department of Biotechnology, Institute of Research and Innovation, Kashiwa, Chiba, Japan
Key Words. Bone marrow–derived fibroblasts ? Green fluorescent protein
Correspondence: Atsushi Ochiai, M.D., Ph.D., Pathology Division, National Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa-City, Chiba 277-8577, Japan. Telephone: 81-4-7134-6855; Fax: 81-4-7134-6865; e-mail: aochiai@east.ncc.go.jp
ABSTRACT
Injury evokes a sequence of events in tissue. Once injury occurs, the host initiates a coordinated repair response. However, if the injury is prolonged, a repeated process of repair and destruction occurs, which subsequently leads to tissue remodeling. During the process, tissue fibroblasts migrate into the injured site and produce collagens and extracellular matrix proteins in response to several extracellular stimuli. Their functions include important roles in growth and differentiation of adjacent epithelia and healing and inflammatory response. Fibroblasts represent the key source of interstitial collagens, but these cells are known to be heterogeneous with respect to a number of phenotypic and functional features . This heterogeneity may arise not only from the activation and differentiation processes that take place in the cells but from their different cellular origins.
Stem cells in the adult have traditionally been thought to be restricted in their potential to differentiate and regenerate tissues in which they reside. Recent advances have revealed that after transplantation of bone marrow (BM), hematopoietic stem cells or nonhematopoietic mesenchymal stem cells, muscle , heart , liver , vascular cells , and other mesenchymal cells of donor origin have been detected. Investigators revealed that BM-derived cells can be progenitors for tissue fibroblasts that are recruited through the circulation to populate peripheral organs . During renal fibrosis, a small number of fibroblasts were BM origin using BM chimera and transgenic reporter mice . We previously reported that cancer-induced stroma generated by the human pancreatic cancer cell line consist of BM-derived fibroblasts and that BM-derived fibroblasts become a major component of cancer-induced stromal cells in the later stage of tumor development . Furthermore, BM-derived fibroblasts were engrafted into multiple organs, and it was found that these cells are recruited into injured tissue . However, the phenotype and functional roles of BM-derived fibroblasts have not been fully understood.
In the study reported here, we investigated the possible relationship between BM-derived fibroblasts and various fibroblast phenotypes. Cancer implantation (skin), excisional wounding (skin), and bleomycin administration (lung) were used to assess whether fibroblast engraftment was modulated by tissue damage and to analyze the phenotypes of BM-derived fibroblasts. Using BM chimera mice expressing enhanced green fluorescent protein (GFP) only in BM-derived cells, we found that excisional wounding is a stimulus for the recruitment of BM-derived fibroblasts within the skin but not in the lung. Furthermore, we found that BM-derived fibroblasts expressed type I collagen, CD45, Thy-1, and - smooth muscle actin (-SMA).
MATERIALS AND METHODS
Recruitment of BM-Derived Fibroblasts into Noninjured Skin and Lung
Sublethally irradiated mice were injected with 1 x 107 GFP-labeled BM cells. GFP phenotyping of BM cells from the recipient mice demonstrated that their marrows had been reconstituted by high levels (>80%) of donor cells 4 weeks after BMT (data not shown). Since sublethal irradiation may induce sequential events of tissue damage, we first evaluated the effects of irradiation on the skin and lung of the BM chimera mice. Histological examination revealed essentially normal skin and lung architecture without fibrosis, although mild perivascular inflammatory cell infiltration was pointed out. Since we focused on fibroblasts as cells with pivotal roles in the fibrosis, BMT in the process of creating BM chimera mice had no significant effects on the morphological analysis of the fibrotic process of the skin and lung. An antibody specific for GFP was used to investigate whether fibroblasts were of BM origin. In the skin of BM chimera mice, GFP-Fbs were mainly found adjacent to striated muscle of the dermis (Fig. 2 A–C). The frequency of GFP-Fbs within the total skin fibroblast was 8.7% ± 4.6%. In the lung, GFP-Fbs were found located around the bronchus and the vessels within the bronchovascular bundle (Fig. 2 D–F). The frequency of GFP-Fbs was 8.9% ± 2.5 % (Table 1).
Figure 2. Microscopic appearance of (A–C) skin and (D–F) lung after bone marrow transplantation. Boxes indicate magnified regions of the (A, B) skin and (D, E) lung. C and F are serial sections of B and E, respectively. Arrowhead indicates GFP+ fibroblasts. Abbreviations: GFP, green fluorescent protein; H.E., hematoxylin and eosin.
Table 1. Population (%) of green fluorescent protein–positive (GFP+) fibroblasts
Increased Recruitment of BM-Derived Fibroblasts into Fibrotic Lesions Induced by Cancer Implantation and Excisional Wounding
After implantation of a transplantable human large cell neuro-endocrine carcinoma of the lung into the skin, stromal fibrosis occurred prominently (Fig. 3A–B). Among the fibroblasts around the cancer nests, numerous fibroblasts showed positive reaction for GFP, and the frequency of GFP-Fbs was 59.7% ± 16.3% (Fig. 3C; Table 2). Upon excisional wounding, the injured area involving the deeper structure of the dermis and tissue was replaced by granulation tissue, which is comprised of many inflammatory cells and fibroblasts. Although GFP-Fbs were observed within the whole layer of skin, these cells have a tendency to locate in the deeper layer (Fig. 2 D–F). The frequency of GFP-Fbs per total fibroblasts was 32.2% ± 4.8%. In contrast, within lung fibrotic lesions induced by bleomycin administration, GFP+ cells were mainly mononuclear cells without spindle cytoplasm, as shown in Figure 3I. The frequency of GFP-Fbs was 7.1% ± 2.4%, which was similar to the frequency found in control lung tissue.
Figure 3. Microscopic appearance of skin and lung after (A–C) cancer implantation, (D–F) excisional wounding, and (G–I) bleomycin administration. Boxes indicate magnified regions of the skin after (A, B) cancer implantation, (D, E) excisional wounding, and (G, H) bleomycin administration. C, F, and I are serial sections of B, E, and H, respectively. Note that numerous GFP+ fibroblasts are found in the fibrotic lesions induced by (C) cancer implantation and (F) excisional wounding, whereas few are found in (I) bleomycin administration. Abbreviation: H.E., hematoxylin and eosin.
Table 2. Summary of phenotypes (%) of green fluorescent protein–positive (GFP+) fibroblasts
BM-Derived Fibroblasts Expressed Collagen Type I
Deposition of collagen type I is a key characteristic finding in tissue fibrotic processes, and fibroblasts are a well-known major producer of this molecule. To determine whether GFP-Fbs express collagen type I and contribute to tissue fibrosis, sections from cancer implantation and excisional wounding were analyzed by confocal immunofluorescence microscopy. When germinal center B cells of the spleen in BMT mice were stained with GFP and type I collagen, numerous B cells showed positive for GFP, but type I collagen–positive cells were hardly observed (Fig. 4A). Any GFP+ cells could not be detected in the mice reconstituted with GFP–/– (wild-type) marrow cells (Fig. 4B). As shown in Figure 4C–E, when GFP-Fbs were intermingled with GFP– fibro-blasts within cancer implantation (Fig. 4C), excisional wounding (Fig. 4D), and noninjured skin (Fig. 4E), both GFP positive and negative fibroblasts expressed type I collagen. This phenomenon was further confirmed using microdissection analysis. Microdissected dermal fibroblasts of the mouse without BMT expressed type I collagen mRNA but did not express GFP mRNA (Fig. 4F, left lane). Microdissected GFP-Fbs around the cancer nests of BMT mice expressed both collagen type I and GFP mRNA (Fig. 4F, right lane). These results indicated that BM-derived fibro-blasts produce type I collagen and contribute to tissue fibrosis.
Figure 4. Colocalization of green fluorescent protein (GFP) and type I collagen on fibroblasts in the fibrotic lesions induced by cancer implantation and excisional wounding. (A): Germinal center B cells of the spleen in bone marrow–transplanted (GFP Tg) mice were stained with GFP and type I collagen. (B): Fibroblasts in cancer-induced stroma in the mice reconstituted with GFP–/– (wild-type) marrow cells were stained with GFP and type I collagen. (C–E): Fibroblasts in (C) cancer implantation, (D) excisional wounding, and (E) noninjured skin were stained with GFP and type I collagen. The upper left panel shows GFP fluorescence. The upper right panel shows cells immunostained with anti-type I collagen antibody in the same area. The lower left panel shows cells stained with DRAQ5 for the discrimination of nucleated cells. The lower right panel shows a composite of both fluorophores. (F): Reverse transcription polymerase chain reaction analysis of type I collagen gene in microdissected GFP+ fibroblasts. GFP– fibroblasts also expressed type I collagen transcripts.
Phenotype of BM-Derived Fibroblasts Within Fibrotic Lesions in the Skin
We assessed further phenotypes of GFP-Fbs with respect to their potential identity with previously identified fibroblast phenotypes. Double immunofluorescence staining for GFP and fibroblast markers (including CD34, C-kit, CD45, Thy-1, and -SMA) was performed with the sections from cancer implantation, excisional wounding, and noninjured skin. We could not determine any GFP+/ CD34+ or GFP+/C-kit+ fibroblasts within the fibrotic lesions of the skin. As shown in Figure 5, some GFP-Fbs showed CD45, and these double-positive cells were intermingled with GFP+/CD45– fibroblasts. The ratio of GFP+/CD45+ fibroblasts per GFP-Fb in the cancer-induced stroma and in the granulation tissue produced by excisional wounding was 43.1% ± 5.5% and 38.5% ± 6.3%, respectively (Fig. 5A, D). GFP-Fbs in noninjured skin also expressed CD45 in similar proportion (37.5% ± 3.5%) (Fig. 5G; Table 2). The cancer-induced stroma and granulation tissue induced by excisional wounding contained GFP+/Thy-1+ fibroblasts, and the frequency of double-positive cells per GFP-Fb was 49.1% ± 6.1% and 46.7% ± 2.8%, respectively (Fig. 5B, E). GFP+/Thy-1+ fibroblasts were also found in noninjured skin, and its frequency was 62.6% ± 15.2 % (Fig. 5H). The frequency of GFP+/-SMA+ fibroblasts (BM-derived myofibroblasts) per GFP-Fb in the cancer-induced stroma was 57.4% ± 2.1%, and this frequency was significantly higher than that in noninjured skin (27.2% ± 5.2%; p = .002) (Fig. 5C, I; Table 2). To further confirm whether BM-derived fibroblasts express CD45 and Thy-1, we performed immunohistochemical staining for GFP/-SMA/CD45, and GFP/-SMA/Thy-1 in serial sections of cancer-induced stroma. In these sections, almost all spindle cells showed positive for both GFP and -SMA, indicating that these cells are BM-derived myofibroblasts. Within this area, many CD45+ spindle cells (Fig. 6A) and Thy-1+ spindle cells (Fig. 6B) were observed. These results showed that BM-derived (myo) fibroblasts express CD45 and/or Thy-1.
Figure 5. Colocalization of (A, D, G) green fluorescent protein (GFP)/CD45, (B, E, H) GFP/Thy-1, and (C, F, I) GFP/ -smooth muscle actin (-SMA) on fibroblasts in the fibrotic lesions induced by cancer implantation (A–C) and excisional wounding (D–F), as well as (G–I) fibroblasts in noninjured skin. The upper left panel shows GFP fluorescence. The upper right panel shows cells immunostained with CD45, Thy-1, or -SMA antibody in the same area. The lower left panel shows cells stained with DRAQ5 for the discrimination of nucleated cells. The lower right panel shows a composite of both fluorophores.
Figure 6. Immunohistochemical detection of CD45+ bone marrow (BM)–derived fibroblasts and Thy-1+ BM-derived fibroblasts. (A): Green fluorescent protein (GFP), alpha-smooth muscle actin (-SMA), and CD45 staining of serial sections of cancer-induced stroma. (B): GFP, -SMA, and Thy-1 staining of serial sections of cancer-induced stroma.
DISCUSSION
We are grateful to Yoko Okuhara and Chie Okumura for technical support and Suzaki Motoko for help in preparing the manuscript. This work was supported in part by the Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare; the Grant for Scientific Research Expenses for Health Labour and Welfare Programs; the Foundation for the Promotion of Cancer Research, 2nd-Term Comprehensive 10-Year Strategy for Cancer Control; and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. T.S. is a recipient of Research Resident Fellowships from the Foundation for Promotion of Cancer Research.
REFERENCES
Garrett DM, Conrad GW. Fibroblast-like cells from embryonic chick cornea, heart, and skin are antigenically distinct. Dev Biol 1979;70:50–70.
Schor SL, Schor AM. Clonal heterogeneity in fibroblast phenotype: implications for the control of epithelial-mesenchymal interactions. Bioessays 1987;7:200–204.
Ronnov-Jessen L, Petersen OW, Koteliansky VE et al. The origin of the myofibroblasts in breast cancer: recapitulation of tumor environment in culture unravels diversity and implicates converted fibroblasts and recruited smooth muscle cells. J Clin Invest 1995;95:859–873.
Dugina V, Alexandrova A, Chaponnier C et al. Rat fibroblasts cultured from various organs exhibit differences in alpha-smooth muscle actin expression, cytoskeletal pattern, and adhesive structure organization. Exp Cell Res 1998;238:481–490.
Jelaska A, Strehlow D, Korn JH. Fibroblast heterogeneity in physiological conditions and fibrotic disease. Springer Semin Immunopathol 1999;21:385–395.
Chang HY, Chi JT, Dudoit S et al. Diversity, topographic differentiation, and positional memory in human fibroblasts. Proc Natl Acad Sci U S A 2002;99:12877–12882.
Ferrari G, Cusella-De Angelis G, Coletta M et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998;279:1528–1530.
Shimizu K, Sugiyama S, Aikawa M et al. Host bone-marrow cells are a source of donor intimal smooth- muscle-like cells in murine aortic transplant arteriopathy. Nat Med 2001;7:738–741.
Kuznetsov SA, Mankani MH, Gronthos S et al. Circulating skeletal stem cells. J Cell Biol 2001;153:1133–1140.
LaBarge MA, Blau HM. Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury. Cell 2002;111:589–601.
Corbel SY, Lee A, Yi L et al. Contribution of hematopoietic stem cells to skeletal muscle. Nat Med 2003;9:1528–1532.
Makino S, Fukuda K, Miyoshi S et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 1999;103:697–705.
Jackson KA, Majka SM, Wang H et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001;107:1395–1402.
Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–705.
Petersen BE, Bowen WC, Patrene KD et al. Bone marrow as a potential source of hepatic oval cells. Science 1999;284:1168–1170.
Theise ND, Badve S, Saxena R et al. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 2000;31:235–240.
Alison MR, Poulsom R, Jeffery R et al. Hepatocytes from non-hepatic adult stem cells. Nature 2000;406:257.
Lagasse E, Connors H, Al-Dhalimy M et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000;6:1229–1234.
Gao Z, McAlister VC, Williams GM. Repopulation of liver endothelium by bone-marrow-derived cells. Lancet 2001;357:932–933.
Asahara T, Masuda H, Takahashi T et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999;85:221–228.
Sata M, Saiura A, Kunisato A et al. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med 2002;8:403–409.
Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:71–74.
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.
Iwano M, Plieth D, Danoff TM et al. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest 2002;110:341–350.
Ishii G, Sangai T, Oda T et al. Bone-marrow-derived myofibroblasts contribute to the cancer-induced stromal reaction. Biochem Biophys Res Commun 2003;309:232–240.
Brittan M, Hunt T, Jeffery R et al. Bone marrow derivation of pericryptal myofibroblasts in the mouse and human small intestine and colon. Gut 2002;50:752–757.
Direkze NC, Forbes SJ, Brittan M et al. Multiple organ engraftment by bone-marrow-derived myofibroblasts and fibroblasts in bone-marrow-transplanted mice. STEM CELLS 2003;21:514–520.
Hashimoto N, Jin H, Liu T et al. Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest 2004;113:243–252.
Mombaerts P, Iacomini J, Johnson RS et al. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 1992;68:869–877.
Sasaki T, Sasaki-Irie J, Penninger JM. New insights into the transmembrane protein tyrosine phosphatase CD45. Int J Biochem Cell Biol 2001;33:1041–1046.
Penninger JM, Irie-Sasaki J, Sasaki T et al. CD45: new jobs for an old acquaintance. Nat Immunol 2001;2:389–396.
Deschaseaux F, Gindraux F, Saadi R et al. Direct selection of human bone marrow mesenchymal stem cells using an anti-CD49a antibody reveals their CD45med,low phenotype. Br J Haematol 2003;122:506–517.
Singer JW, Charbord P, Keating A et al. Simian virus 40-transformed adherent cells from human long-term marrow cultures: cloned cell lines produce cells with stromal and hematopoietic characteristics. Blood 1987;70:464–474.
Koumas L, King AE, Critchley HO et al. Fibroblast heterogeneity: existence of functionally distinct Thy 1+ and Thy 1– human female reproductive tract fibroblasts. Am J Pathol 2001;159:925–935.
Koumas L, Phipps RP. Differential COX localization and PG release in Thy-1+ and Thy-1– human female reproductive tract fibroblasts. Am J Physiol Cell Physiol 2002;283:C599–608.
Koumas L, Smith TJ, Feldon S et al. Thy-1 expression in human fibroblast subsets defines myofibroblastic or lipofibroblastic phenotypes. Am J Pathol 2003;163:1291–1300.
Bucala R, Spiegel LA, Chesney J et al. Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med 1994;1:71–81.
Abe R, Donnelly SC, Peng T et al. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol 2001;166:7556–7562.(Genichiro Ishiia, Takafum)
b Investigative Treatment Division, National Cancer Center Research Institute East, Kashiwa, Chiba, Japan;
c Laboratory of Cancer Biology, Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan;
d Department of Biotechnology, Institute of Research and Innovation, Kashiwa, Chiba, Japan
Key Words. Bone marrow–derived fibroblasts ? Green fluorescent protein
Correspondence: Atsushi Ochiai, M.D., Ph.D., Pathology Division, National Cancer Center Research Institute East, 6-5-1 Kashiwanoha, Kashiwa-City, Chiba 277-8577, Japan. Telephone: 81-4-7134-6855; Fax: 81-4-7134-6865; e-mail: aochiai@east.ncc.go.jp
ABSTRACT
Injury evokes a sequence of events in tissue. Once injury occurs, the host initiates a coordinated repair response. However, if the injury is prolonged, a repeated process of repair and destruction occurs, which subsequently leads to tissue remodeling. During the process, tissue fibroblasts migrate into the injured site and produce collagens and extracellular matrix proteins in response to several extracellular stimuli. Their functions include important roles in growth and differentiation of adjacent epithelia and healing and inflammatory response. Fibroblasts represent the key source of interstitial collagens, but these cells are known to be heterogeneous with respect to a number of phenotypic and functional features . This heterogeneity may arise not only from the activation and differentiation processes that take place in the cells but from their different cellular origins.
Stem cells in the adult have traditionally been thought to be restricted in their potential to differentiate and regenerate tissues in which they reside. Recent advances have revealed that after transplantation of bone marrow (BM), hematopoietic stem cells or nonhematopoietic mesenchymal stem cells, muscle , heart , liver , vascular cells , and other mesenchymal cells of donor origin have been detected. Investigators revealed that BM-derived cells can be progenitors for tissue fibroblasts that are recruited through the circulation to populate peripheral organs . During renal fibrosis, a small number of fibroblasts were BM origin using BM chimera and transgenic reporter mice . We previously reported that cancer-induced stroma generated by the human pancreatic cancer cell line consist of BM-derived fibroblasts and that BM-derived fibroblasts become a major component of cancer-induced stromal cells in the later stage of tumor development . Furthermore, BM-derived fibroblasts were engrafted into multiple organs, and it was found that these cells are recruited into injured tissue . However, the phenotype and functional roles of BM-derived fibroblasts have not been fully understood.
In the study reported here, we investigated the possible relationship between BM-derived fibroblasts and various fibroblast phenotypes. Cancer implantation (skin), excisional wounding (skin), and bleomycin administration (lung) were used to assess whether fibroblast engraftment was modulated by tissue damage and to analyze the phenotypes of BM-derived fibroblasts. Using BM chimera mice expressing enhanced green fluorescent protein (GFP) only in BM-derived cells, we found that excisional wounding is a stimulus for the recruitment of BM-derived fibroblasts within the skin but not in the lung. Furthermore, we found that BM-derived fibroblasts expressed type I collagen, CD45, Thy-1, and - smooth muscle actin (-SMA).
MATERIALS AND METHODS
Recruitment of BM-Derived Fibroblasts into Noninjured Skin and Lung
Sublethally irradiated mice were injected with 1 x 107 GFP-labeled BM cells. GFP phenotyping of BM cells from the recipient mice demonstrated that their marrows had been reconstituted by high levels (>80%) of donor cells 4 weeks after BMT (data not shown). Since sublethal irradiation may induce sequential events of tissue damage, we first evaluated the effects of irradiation on the skin and lung of the BM chimera mice. Histological examination revealed essentially normal skin and lung architecture without fibrosis, although mild perivascular inflammatory cell infiltration was pointed out. Since we focused on fibroblasts as cells with pivotal roles in the fibrosis, BMT in the process of creating BM chimera mice had no significant effects on the morphological analysis of the fibrotic process of the skin and lung. An antibody specific for GFP was used to investigate whether fibroblasts were of BM origin. In the skin of BM chimera mice, GFP-Fbs were mainly found adjacent to striated muscle of the dermis (Fig. 2 A–C). The frequency of GFP-Fbs within the total skin fibroblast was 8.7% ± 4.6%. In the lung, GFP-Fbs were found located around the bronchus and the vessels within the bronchovascular bundle (Fig. 2 D–F). The frequency of GFP-Fbs was 8.9% ± 2.5 % (Table 1).
Figure 2. Microscopic appearance of (A–C) skin and (D–F) lung after bone marrow transplantation. Boxes indicate magnified regions of the (A, B) skin and (D, E) lung. C and F are serial sections of B and E, respectively. Arrowhead indicates GFP+ fibroblasts. Abbreviations: GFP, green fluorescent protein; H.E., hematoxylin and eosin.
Table 1. Population (%) of green fluorescent protein–positive (GFP+) fibroblasts
Increased Recruitment of BM-Derived Fibroblasts into Fibrotic Lesions Induced by Cancer Implantation and Excisional Wounding
After implantation of a transplantable human large cell neuro-endocrine carcinoma of the lung into the skin, stromal fibrosis occurred prominently (Fig. 3A–B). Among the fibroblasts around the cancer nests, numerous fibroblasts showed positive reaction for GFP, and the frequency of GFP-Fbs was 59.7% ± 16.3% (Fig. 3C; Table 2). Upon excisional wounding, the injured area involving the deeper structure of the dermis and tissue was replaced by granulation tissue, which is comprised of many inflammatory cells and fibroblasts. Although GFP-Fbs were observed within the whole layer of skin, these cells have a tendency to locate in the deeper layer (Fig. 2 D–F). The frequency of GFP-Fbs per total fibroblasts was 32.2% ± 4.8%. In contrast, within lung fibrotic lesions induced by bleomycin administration, GFP+ cells were mainly mononuclear cells without spindle cytoplasm, as shown in Figure 3I. The frequency of GFP-Fbs was 7.1% ± 2.4%, which was similar to the frequency found in control lung tissue.
Figure 3. Microscopic appearance of skin and lung after (A–C) cancer implantation, (D–F) excisional wounding, and (G–I) bleomycin administration. Boxes indicate magnified regions of the skin after (A, B) cancer implantation, (D, E) excisional wounding, and (G, H) bleomycin administration. C, F, and I are serial sections of B, E, and H, respectively. Note that numerous GFP+ fibroblasts are found in the fibrotic lesions induced by (C) cancer implantation and (F) excisional wounding, whereas few are found in (I) bleomycin administration. Abbreviation: H.E., hematoxylin and eosin.
Table 2. Summary of phenotypes (%) of green fluorescent protein–positive (GFP+) fibroblasts
BM-Derived Fibroblasts Expressed Collagen Type I
Deposition of collagen type I is a key characteristic finding in tissue fibrotic processes, and fibroblasts are a well-known major producer of this molecule. To determine whether GFP-Fbs express collagen type I and contribute to tissue fibrosis, sections from cancer implantation and excisional wounding were analyzed by confocal immunofluorescence microscopy. When germinal center B cells of the spleen in BMT mice were stained with GFP and type I collagen, numerous B cells showed positive for GFP, but type I collagen–positive cells were hardly observed (Fig. 4A). Any GFP+ cells could not be detected in the mice reconstituted with GFP–/– (wild-type) marrow cells (Fig. 4B). As shown in Figure 4C–E, when GFP-Fbs were intermingled with GFP– fibro-blasts within cancer implantation (Fig. 4C), excisional wounding (Fig. 4D), and noninjured skin (Fig. 4E), both GFP positive and negative fibroblasts expressed type I collagen. This phenomenon was further confirmed using microdissection analysis. Microdissected dermal fibroblasts of the mouse without BMT expressed type I collagen mRNA but did not express GFP mRNA (Fig. 4F, left lane). Microdissected GFP-Fbs around the cancer nests of BMT mice expressed both collagen type I and GFP mRNA (Fig. 4F, right lane). These results indicated that BM-derived fibro-blasts produce type I collagen and contribute to tissue fibrosis.
Figure 4. Colocalization of green fluorescent protein (GFP) and type I collagen on fibroblasts in the fibrotic lesions induced by cancer implantation and excisional wounding. (A): Germinal center B cells of the spleen in bone marrow–transplanted (GFP Tg) mice were stained with GFP and type I collagen. (B): Fibroblasts in cancer-induced stroma in the mice reconstituted with GFP–/– (wild-type) marrow cells were stained with GFP and type I collagen. (C–E): Fibroblasts in (C) cancer implantation, (D) excisional wounding, and (E) noninjured skin were stained with GFP and type I collagen. The upper left panel shows GFP fluorescence. The upper right panel shows cells immunostained with anti-type I collagen antibody in the same area. The lower left panel shows cells stained with DRAQ5 for the discrimination of nucleated cells. The lower right panel shows a composite of both fluorophores. (F): Reverse transcription polymerase chain reaction analysis of type I collagen gene in microdissected GFP+ fibroblasts. GFP– fibroblasts also expressed type I collagen transcripts.
Phenotype of BM-Derived Fibroblasts Within Fibrotic Lesions in the Skin
We assessed further phenotypes of GFP-Fbs with respect to their potential identity with previously identified fibroblast phenotypes. Double immunofluorescence staining for GFP and fibroblast markers (including CD34, C-kit, CD45, Thy-1, and -SMA) was performed with the sections from cancer implantation, excisional wounding, and noninjured skin. We could not determine any GFP+/ CD34+ or GFP+/C-kit+ fibroblasts within the fibrotic lesions of the skin. As shown in Figure 5, some GFP-Fbs showed CD45, and these double-positive cells were intermingled with GFP+/CD45– fibroblasts. The ratio of GFP+/CD45+ fibroblasts per GFP-Fb in the cancer-induced stroma and in the granulation tissue produced by excisional wounding was 43.1% ± 5.5% and 38.5% ± 6.3%, respectively (Fig. 5A, D). GFP-Fbs in noninjured skin also expressed CD45 in similar proportion (37.5% ± 3.5%) (Fig. 5G; Table 2). The cancer-induced stroma and granulation tissue induced by excisional wounding contained GFP+/Thy-1+ fibroblasts, and the frequency of double-positive cells per GFP-Fb was 49.1% ± 6.1% and 46.7% ± 2.8%, respectively (Fig. 5B, E). GFP+/Thy-1+ fibroblasts were also found in noninjured skin, and its frequency was 62.6% ± 15.2 % (Fig. 5H). The frequency of GFP+/-SMA+ fibroblasts (BM-derived myofibroblasts) per GFP-Fb in the cancer-induced stroma was 57.4% ± 2.1%, and this frequency was significantly higher than that in noninjured skin (27.2% ± 5.2%; p = .002) (Fig. 5C, I; Table 2). To further confirm whether BM-derived fibroblasts express CD45 and Thy-1, we performed immunohistochemical staining for GFP/-SMA/CD45, and GFP/-SMA/Thy-1 in serial sections of cancer-induced stroma. In these sections, almost all spindle cells showed positive for both GFP and -SMA, indicating that these cells are BM-derived myofibroblasts. Within this area, many CD45+ spindle cells (Fig. 6A) and Thy-1+ spindle cells (Fig. 6B) were observed. These results showed that BM-derived (myo) fibroblasts express CD45 and/or Thy-1.
Figure 5. Colocalization of (A, D, G) green fluorescent protein (GFP)/CD45, (B, E, H) GFP/Thy-1, and (C, F, I) GFP/ -smooth muscle actin (-SMA) on fibroblasts in the fibrotic lesions induced by cancer implantation (A–C) and excisional wounding (D–F), as well as (G–I) fibroblasts in noninjured skin. The upper left panel shows GFP fluorescence. The upper right panel shows cells immunostained with CD45, Thy-1, or -SMA antibody in the same area. The lower left panel shows cells stained with DRAQ5 for the discrimination of nucleated cells. The lower right panel shows a composite of both fluorophores.
Figure 6. Immunohistochemical detection of CD45+ bone marrow (BM)–derived fibroblasts and Thy-1+ BM-derived fibroblasts. (A): Green fluorescent protein (GFP), alpha-smooth muscle actin (-SMA), and CD45 staining of serial sections of cancer-induced stroma. (B): GFP, -SMA, and Thy-1 staining of serial sections of cancer-induced stroma.
DISCUSSION
We are grateful to Yoko Okuhara and Chie Okumura for technical support and Suzaki Motoko for help in preparing the manuscript. This work was supported in part by the Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare; the Grant for Scientific Research Expenses for Health Labour and Welfare Programs; the Foundation for the Promotion of Cancer Research, 2nd-Term Comprehensive 10-Year Strategy for Cancer Control; and Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. T.S. is a recipient of Research Resident Fellowships from the Foundation for Promotion of Cancer Research.
REFERENCES
Garrett DM, Conrad GW. Fibroblast-like cells from embryonic chick cornea, heart, and skin are antigenically distinct. Dev Biol 1979;70:50–70.
Schor SL, Schor AM. Clonal heterogeneity in fibroblast phenotype: implications for the control of epithelial-mesenchymal interactions. Bioessays 1987;7:200–204.
Ronnov-Jessen L, Petersen OW, Koteliansky VE et al. The origin of the myofibroblasts in breast cancer: recapitulation of tumor environment in culture unravels diversity and implicates converted fibroblasts and recruited smooth muscle cells. J Clin Invest 1995;95:859–873.
Dugina V, Alexandrova A, Chaponnier C et al. Rat fibroblasts cultured from various organs exhibit differences in alpha-smooth muscle actin expression, cytoskeletal pattern, and adhesive structure organization. Exp Cell Res 1998;238:481–490.
Jelaska A, Strehlow D, Korn JH. Fibroblast heterogeneity in physiological conditions and fibrotic disease. Springer Semin Immunopathol 1999;21:385–395.
Chang HY, Chi JT, Dudoit S et al. Diversity, topographic differentiation, and positional memory in human fibroblasts. Proc Natl Acad Sci U S A 2002;99:12877–12882.
Ferrari G, Cusella-De Angelis G, Coletta M et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998;279:1528–1530.
Shimizu K, Sugiyama S, Aikawa M et al. Host bone-marrow cells are a source of donor intimal smooth- muscle-like cells in murine aortic transplant arteriopathy. Nat Med 2001;7:738–741.
Kuznetsov SA, Mankani MH, Gronthos S et al. Circulating skeletal stem cells. J Cell Biol 2001;153:1133–1140.
LaBarge MA, Blau HM. Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury. Cell 2002;111:589–601.
Corbel SY, Lee A, Yi L et al. Contribution of hematopoietic stem cells to skeletal muscle. Nat Med 2003;9:1528–1532.
Makino S, Fukuda K, Miyoshi S et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 1999;103:697–705.
Jackson KA, Majka SM, Wang H et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001;107:1395–1402.
Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–705.
Petersen BE, Bowen WC, Patrene KD et al. Bone marrow as a potential source of hepatic oval cells. Science 1999;284:1168–1170.
Theise ND, Badve S, Saxena R et al. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 2000;31:235–240.
Alison MR, Poulsom R, Jeffery R et al. Hepatocytes from non-hepatic adult stem cells. Nature 2000;406:257.
Lagasse E, Connors H, Al-Dhalimy M et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000;6:1229–1234.
Gao Z, McAlister VC, Williams GM. Repopulation of liver endothelium by bone-marrow-derived cells. Lancet 2001;357:932–933.
Asahara T, Masuda H, Takahashi T et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999;85:221–228.
Sata M, Saiura A, Kunisato A et al. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med 2002;8:403–409.
Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:71–74.
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.
Iwano M, Plieth D, Danoff TM et al. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest 2002;110:341–350.
Ishii G, Sangai T, Oda T et al. Bone-marrow-derived myofibroblasts contribute to the cancer-induced stromal reaction. Biochem Biophys Res Commun 2003;309:232–240.
Brittan M, Hunt T, Jeffery R et al. Bone marrow derivation of pericryptal myofibroblasts in the mouse and human small intestine and colon. Gut 2002;50:752–757.
Direkze NC, Forbes SJ, Brittan M et al. Multiple organ engraftment by bone-marrow-derived myofibroblasts and fibroblasts in bone-marrow-transplanted mice. STEM CELLS 2003;21:514–520.
Hashimoto N, Jin H, Liu T et al. Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest 2004;113:243–252.
Mombaerts P, Iacomini J, Johnson RS et al. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 1992;68:869–877.
Sasaki T, Sasaki-Irie J, Penninger JM. New insights into the transmembrane protein tyrosine phosphatase CD45. Int J Biochem Cell Biol 2001;33:1041–1046.
Penninger JM, Irie-Sasaki J, Sasaki T et al. CD45: new jobs for an old acquaintance. Nat Immunol 2001;2:389–396.
Deschaseaux F, Gindraux F, Saadi R et al. Direct selection of human bone marrow mesenchymal stem cells using an anti-CD49a antibody reveals their CD45med,low phenotype. Br J Haematol 2003;122:506–517.
Singer JW, Charbord P, Keating A et al. Simian virus 40-transformed adherent cells from human long-term marrow cultures: cloned cell lines produce cells with stromal and hematopoietic characteristics. Blood 1987;70:464–474.
Koumas L, King AE, Critchley HO et al. Fibroblast heterogeneity: existence of functionally distinct Thy 1+ and Thy 1– human female reproductive tract fibroblasts. Am J Pathol 2001;159:925–935.
Koumas L, Phipps RP. Differential COX localization and PG release in Thy-1+ and Thy-1– human female reproductive tract fibroblasts. Am J Physiol Cell Physiol 2002;283:C599–608.
Koumas L, Smith TJ, Feldon S et al. Thy-1 expression in human fibroblast subsets defines myofibroblastic or lipofibroblastic phenotypes. Am J Pathol 2003;163:1291–1300.
Bucala R, Spiegel LA, Chesney J et al. Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med 1994;1:71–81.
Abe R, Donnelly SC, Peng T et al. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol 2001;166:7556–7562.(Genichiro Ishiia, Takafum)