Comparison of Various Bone Marrow Fractions in the Ability to Participate in Vascular Remodeling After Mechanical Injury
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《干细胞学杂志》
a Departments of Cardiovascular Medicine and
b Advanced Clinical Science and Therapeutics, University of Tokyo Graduate School of Medicine, Tokyo, Japan;
c Department of Physiology, Keio University School of Medicine, Tokyo, Japan;
d PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan;
e CREST, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan
Key Words. Endothelial cells ? Hematopoietic stem cells ? Progenitor ? Smooth muscle cells ? Transdifferentiation
Correspondence: Masataka Sata, M.D., Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Telephone: 81-3-3815-5411; Fax: 81-3-3814-0021; e-mail: msata-circ@umin.ac.jp
ABSTRACT
Recent evidence suggests that bone marrow–derived cells may participate in regeneration of remote organs . Bone marrow contains both hematopoietic and nonhematopoietic cells. Hematopoietic stem cells (HSCs) are defined as having the capacity for self-renewal and the ability to differentiate into all mature hematopoietic lineages . Although it was assumed that HSCs give rise to hematopoietic cells, recent reports proposed the possibility that HSCs may have the broader potential to differentiate into nonhematopoietic cells, including epithelial cells , hepatocytes , cardiomyocytes , and vascular cells . In contrast, others cast doubt on the pluripotency of adult HSCs under physiological conditions by analyzing uninjured organs of bone marrow chimeric mice .
There might be two possibilities that account for the discrepancy. First, the HSCs used differ in their purity . Most of the studies analyzed the CD34–, c-Kit+, Sca-1+, Lineage– (CD34–KSL) bone marrow cells , which have been assumed as the most primitive HSCs . However, even in the best case series reported , only one in five recipients showed successful engraftment after single-cell transplantation, indicating that the CD34–KSL fraction represents a heterogeneous population containing nonhematopoietic cells. It is possible that nonhematopoietic cells among the CD34–KSL cells might be responsible for the pluripotency. Second, the apparent discrepancy could merely derive from the analysis of noninjured versus injured tissues . We reported that the mode of injury is crucial for the recruitment of bone marrow–derived cells to vascular remodeling . Thus, it remains unclear whether a highly purified single HSC can contribute to vascular remodeling after severe vascular injuries, which are essential for bone marrow–derived cells to participate in vascular remodeling.
Here, we transplanted either total bone marrow (TBM) cells, KSL fraction cells, or a highly purified HSC into lethally irradiated wild-type mice . In all groups, peripheral blood cells were successfully reconstituted. However, bone marrow–derived cells were seldom detected in the injured artery when a single HSC was injected into irradiated mice. These results suggest that it is a rare property for a purified HSC to transdifferentiate into vascular cells.
MATERIALS AND METHODS
Significant Engraftment of a Single Tip-SP CD34–KSL Cell
Either TBM cells (1 x 106, TBM group), c-Kit+, Sca-1+, Lin– cells (3 x 103, KSL fraction), or a single Tip-SP CD34–KSL cell (1, HSC fraction) were injected into lethally irradiated wild-type mice. Consistent with our previous report , a single Tip-SP CD34–KSL cell showed significant donor cell engraftment for long term (Figs. 1A, 1B). Consistent with our previous study , both myelocytes/monocytes and T/B lymphocytes derived from a single Tip-SP cell were detected at 3, 6, and 12 months after transplantation. Flow cytometry at 16 weeks after bone marrow reconstitution revealed that peripheral blood cells had been reconstituted with the injected cells in TBM (79.6% ± 5.1%), KSL (68.4% ± 5.1%), and HSC (34.4% ± 6.5%) groups (Fig. 1B).
Figure 1. Successful engraftment of a single Tip-SP CD34–KSL cell, 1 x 106 total bone marrow (TBM group) cells (n = 4), 3 x 103 c-Kit+ Sca-1+ Lin– (KSL group) fraction cells (n = 6), or a single Tip-SP CD34–KSL cell (HSC group) (n = 7) harboring green fluorescent protein (GFP) were injected into lethally irradiated wild-type mice. (A): Proportion of the GFP-positive cells in peripheral blood after transplantation of a single Tip-SP CD34–KSL cell. Time courses of three representative mice are reported. (B): Representative flow cytometric histograms of peripheral leukocytes in TBM, KSL, and HSC groups at 16 weeks after transplantation. Abbreviations: HSC, hematopoietic stem cell; SP, side population.
Failure of a Single HSC-Derived Cell to Participate in Vascular Remodeling
Wire-mediated endovascular injury was induced to the femoral artery at 12 weeks after irradiation and injection. At 16 weeks after stem cell transplantation, the femoral arteries showed neointimal formation that mainly consisted of -SMA–positive cells in all groups (Fig. 2A). The neointima contained a significant number of GFP-positive cells in the TBM group (24.0% ± 7.2%; n = 4) and the KSL group (14.1% ± 6.1%; n = 6). On the other hand, GFP-positive cells were seldom detected in the neointima of the HSC group (0.2% ± 0.1%; n = 7). Similarly, the media contained a significant number of GFP-positive cells in the TBM group (31.1% ± 11.2%) and KSL group (16.8% ± 6.6%). In contrast, GFP-positive cells were rarely detected in the media in the HSC group (2.7% ± 1.0%) (Fig. 2B, Table 1).
Figure 2. Failure of a highly purified HSC to contribute to vascular remodeling after mechanical vascular injury. (A): Representative cross-sections of the vascular lesions. At 12 weeks after irradiation and stem cell transplantation, wire-mediated injury was induced in the femoral artery of the bone marrow chimeric mice. The injured arteries were harvested at 16 weeks, embedded in plastic resin, and observed under a confocal microscope (FLUOVIEW FV300; Olympus). Arrowheads indicate the internal elastic lamina. Arrow indicates a GFP-positive cell observed in adventitia in the HSC group. Bar = 50 μm. (B, C): Frequency of GFP-positive cells among the total cells in the(B) neointima and (C) media. *p <.05; **p < .01. Abbreviations: DIC, differential interference contrast; GFP, green fluorescent protein; HSC, hematopoietic stem cell; KSL, c-Kit+, Sca-1+, Lineage–; L, lumen; M, media; NI, neointima; TBM, total bone marrow.
Table 1. Frequency of green fluorescent protein (GFP)–positive cells in neointima and media per a cross-section 4 weeks after vascular injury in bone marrow chimeric mice
Next, we characterized the bone marrow–derived cells observed in the vascular lesions. In the KSL group as well as in the TBM group, many GFP-positive cells expressed -SMA in the neointima and media (Figs. 3A–3C). The bone marrow–derived cells on the luminal side were positive for endothelial markers (BS-lectin and CD31) (Figs. 3D, 3E), as previously reported . In the HSC group, very few GFP-positive cells were detected in the lesions in the HSC group (Fig. 3C). All of the GFP-positive cells were positive for CD45 (Fig. 3F). We could not find GFP-positive cells that expressed -SMA or endothelial markers.
Figure 3. Double immunofluorescent images of the injured arteries. Plastic-embedded sections were stained for (A, B, C) -smooth muscle actin (-SMA, red), (D, E) CD31 (red), and (F) CD45 (red) followed by counterstaining with Hoechst 33258 (blue). Arrowheads indicate the internal elastic lamina. Arrows indicate green fluorescent protein–positive cells that were positive for markers. Bar = 10 μm. Abbreviations: HSC, hematopoietic stem cell; KSL, c-Kit+, Sca-1+, Lineage–; L, lumen; M, media; NI, neointima; TBM, total bone marrow.
DISCUSSION
Our finding suggests that a highly purified murine HSC seldom transdifferentiates into vascular cells. Distinct cell populations other than hematopoietic cells may be responsible for most bone marrow–derived smooth muscle–like cells and endothelial like–cells that could be observed in vascular lesions after mechanical injury.
ACKNOWLEDGMENTS
Poulsom R, Alison MR, Forbes SJ et al. Adult stem cell plasticity. J Pathol 2002;197:441–456.
Matsuzaki Y, Kinjo K, Mulligan RC et al. Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells. Immunity 2004;20:87–93.
Krause DS, Theise ND, Collector MI et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369–377.
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.
Jang YY, Collector MI, Baylin SB et al. Hematopoietic stem cells convert into liver cells within days without fusion. Nat Cell Biol 2004;6:532–539.
Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–705.
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.
Wagers AJ, Sherwood RI, Christensen JL et al. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 2002;297:2256–2259.
Osawa M, Hanada K, Hamada H et al. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 1996;273:242–245.
Blau H, Brazelton T, Keshet G et al. Something in the eye of the beholder. Science 2002;298:361–362.
Tanaka K, Sata M, Hirata Y et al. Diverse contribution of bone marrow cells to neointimal hyperplasia after mechanical vascular injuries. Circ Res 2003;93:783–790.
Okada S, Yoshida T, Hong Z et al. Impairment of B lymphopoiesis in precocious aging (klotho) mice. Int Immunol 2000;12:861–871.
Sata M, Maejima Y, Adachi F et al. A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia. J Mol Cell Cardiol 2000;32:2097–2104.
Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.
Vassilopoulos G, Wang PR, Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature 2003;422:901–904.
Campbell JH, Tachas G, Black MJ et al. Molecular biology of vascular hypertrophy. Basic Res Cardiol 1991;86:3–11.(Makoto Saharaa, Masataka )
b Advanced Clinical Science and Therapeutics, University of Tokyo Graduate School of Medicine, Tokyo, Japan;
c Department of Physiology, Keio University School of Medicine, Tokyo, Japan;
d PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan;
e CREST, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan
Key Words. Endothelial cells ? Hematopoietic stem cells ? Progenitor ? Smooth muscle cells ? Transdifferentiation
Correspondence: Masataka Sata, M.D., Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Telephone: 81-3-3815-5411; Fax: 81-3-3814-0021; e-mail: msata-circ@umin.ac.jp
ABSTRACT
Recent evidence suggests that bone marrow–derived cells may participate in regeneration of remote organs . Bone marrow contains both hematopoietic and nonhematopoietic cells. Hematopoietic stem cells (HSCs) are defined as having the capacity for self-renewal and the ability to differentiate into all mature hematopoietic lineages . Although it was assumed that HSCs give rise to hematopoietic cells, recent reports proposed the possibility that HSCs may have the broader potential to differentiate into nonhematopoietic cells, including epithelial cells , hepatocytes , cardiomyocytes , and vascular cells . In contrast, others cast doubt on the pluripotency of adult HSCs under physiological conditions by analyzing uninjured organs of bone marrow chimeric mice .
There might be two possibilities that account for the discrepancy. First, the HSCs used differ in their purity . Most of the studies analyzed the CD34–, c-Kit+, Sca-1+, Lineage– (CD34–KSL) bone marrow cells , which have been assumed as the most primitive HSCs . However, even in the best case series reported , only one in five recipients showed successful engraftment after single-cell transplantation, indicating that the CD34–KSL fraction represents a heterogeneous population containing nonhematopoietic cells. It is possible that nonhematopoietic cells among the CD34–KSL cells might be responsible for the pluripotency. Second, the apparent discrepancy could merely derive from the analysis of noninjured versus injured tissues . We reported that the mode of injury is crucial for the recruitment of bone marrow–derived cells to vascular remodeling . Thus, it remains unclear whether a highly purified single HSC can contribute to vascular remodeling after severe vascular injuries, which are essential for bone marrow–derived cells to participate in vascular remodeling.
Here, we transplanted either total bone marrow (TBM) cells, KSL fraction cells, or a highly purified HSC into lethally irradiated wild-type mice . In all groups, peripheral blood cells were successfully reconstituted. However, bone marrow–derived cells were seldom detected in the injured artery when a single HSC was injected into irradiated mice. These results suggest that it is a rare property for a purified HSC to transdifferentiate into vascular cells.
MATERIALS AND METHODS
Significant Engraftment of a Single Tip-SP CD34–KSL Cell
Either TBM cells (1 x 106, TBM group), c-Kit+, Sca-1+, Lin– cells (3 x 103, KSL fraction), or a single Tip-SP CD34–KSL cell (1, HSC fraction) were injected into lethally irradiated wild-type mice. Consistent with our previous report , a single Tip-SP CD34–KSL cell showed significant donor cell engraftment for long term (Figs. 1A, 1B). Consistent with our previous study , both myelocytes/monocytes and T/B lymphocytes derived from a single Tip-SP cell were detected at 3, 6, and 12 months after transplantation. Flow cytometry at 16 weeks after bone marrow reconstitution revealed that peripheral blood cells had been reconstituted with the injected cells in TBM (79.6% ± 5.1%), KSL (68.4% ± 5.1%), and HSC (34.4% ± 6.5%) groups (Fig. 1B).
Figure 1. Successful engraftment of a single Tip-SP CD34–KSL cell, 1 x 106 total bone marrow (TBM group) cells (n = 4), 3 x 103 c-Kit+ Sca-1+ Lin– (KSL group) fraction cells (n = 6), or a single Tip-SP CD34–KSL cell (HSC group) (n = 7) harboring green fluorescent protein (GFP) were injected into lethally irradiated wild-type mice. (A): Proportion of the GFP-positive cells in peripheral blood after transplantation of a single Tip-SP CD34–KSL cell. Time courses of three representative mice are reported. (B): Representative flow cytometric histograms of peripheral leukocytes in TBM, KSL, and HSC groups at 16 weeks after transplantation. Abbreviations: HSC, hematopoietic stem cell; SP, side population.
Failure of a Single HSC-Derived Cell to Participate in Vascular Remodeling
Wire-mediated endovascular injury was induced to the femoral artery at 12 weeks after irradiation and injection. At 16 weeks after stem cell transplantation, the femoral arteries showed neointimal formation that mainly consisted of -SMA–positive cells in all groups (Fig. 2A). The neointima contained a significant number of GFP-positive cells in the TBM group (24.0% ± 7.2%; n = 4) and the KSL group (14.1% ± 6.1%; n = 6). On the other hand, GFP-positive cells were seldom detected in the neointima of the HSC group (0.2% ± 0.1%; n = 7). Similarly, the media contained a significant number of GFP-positive cells in the TBM group (31.1% ± 11.2%) and KSL group (16.8% ± 6.6%). In contrast, GFP-positive cells were rarely detected in the media in the HSC group (2.7% ± 1.0%) (Fig. 2B, Table 1).
Figure 2. Failure of a highly purified HSC to contribute to vascular remodeling after mechanical vascular injury. (A): Representative cross-sections of the vascular lesions. At 12 weeks after irradiation and stem cell transplantation, wire-mediated injury was induced in the femoral artery of the bone marrow chimeric mice. The injured arteries were harvested at 16 weeks, embedded in plastic resin, and observed under a confocal microscope (FLUOVIEW FV300; Olympus). Arrowheads indicate the internal elastic lamina. Arrow indicates a GFP-positive cell observed in adventitia in the HSC group. Bar = 50 μm. (B, C): Frequency of GFP-positive cells among the total cells in the(B) neointima and (C) media. *p <.05; **p < .01. Abbreviations: DIC, differential interference contrast; GFP, green fluorescent protein; HSC, hematopoietic stem cell; KSL, c-Kit+, Sca-1+, Lineage–; L, lumen; M, media; NI, neointima; TBM, total bone marrow.
Table 1. Frequency of green fluorescent protein (GFP)–positive cells in neointima and media per a cross-section 4 weeks after vascular injury in bone marrow chimeric mice
Next, we characterized the bone marrow–derived cells observed in the vascular lesions. In the KSL group as well as in the TBM group, many GFP-positive cells expressed -SMA in the neointima and media (Figs. 3A–3C). The bone marrow–derived cells on the luminal side were positive for endothelial markers (BS-lectin and CD31) (Figs. 3D, 3E), as previously reported . In the HSC group, very few GFP-positive cells were detected in the lesions in the HSC group (Fig. 3C). All of the GFP-positive cells were positive for CD45 (Fig. 3F). We could not find GFP-positive cells that expressed -SMA or endothelial markers.
Figure 3. Double immunofluorescent images of the injured arteries. Plastic-embedded sections were stained for (A, B, C) -smooth muscle actin (-SMA, red), (D, E) CD31 (red), and (F) CD45 (red) followed by counterstaining with Hoechst 33258 (blue). Arrowheads indicate the internal elastic lamina. Arrows indicate green fluorescent protein–positive cells that were positive for markers. Bar = 10 μm. Abbreviations: HSC, hematopoietic stem cell; KSL, c-Kit+, Sca-1+, Lineage–; L, lumen; M, media; NI, neointima; TBM, total bone marrow.
DISCUSSION
Our finding suggests that a highly purified murine HSC seldom transdifferentiates into vascular cells. Distinct cell populations other than hematopoietic cells may be responsible for most bone marrow–derived smooth muscle–like cells and endothelial like–cells that could be observed in vascular lesions after mechanical injury.
ACKNOWLEDGMENTS
Poulsom R, Alison MR, Forbes SJ et al. Adult stem cell plasticity. J Pathol 2002;197:441–456.
Matsuzaki Y, Kinjo K, Mulligan RC et al. Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells. Immunity 2004;20:87–93.
Krause DS, Theise ND, Collector MI et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369–377.
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.
Jang YY, Collector MI, Baylin SB et al. Hematopoietic stem cells convert into liver cells within days without fusion. Nat Cell Biol 2004;6:532–539.
Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–705.
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.
Wagers AJ, Sherwood RI, Christensen JL et al. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 2002;297:2256–2259.
Osawa M, Hanada K, Hamada H et al. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 1996;273:242–245.
Blau H, Brazelton T, Keshet G et al. Something in the eye of the beholder. Science 2002;298:361–362.
Tanaka K, Sata M, Hirata Y et al. Diverse contribution of bone marrow cells to neointimal hyperplasia after mechanical vascular injuries. Circ Res 2003;93:783–790.
Okada S, Yoshida T, Hong Z et al. Impairment of B lymphopoiesis in precocious aging (klotho) mice. Int Immunol 2000;12:861–871.
Sata M, Maejima Y, Adachi F et al. A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia. J Mol Cell Cardiol 2000;32:2097–2104.
Jiang Y, Jahagirdar BN, Reinhardt RL et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:41–49.
Vassilopoulos G, Wang PR, Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature 2003;422:901–904.
Campbell JH, Tachas G, Black MJ et al. Molecular biology of vascular hypertrophy. Basic Res Cardiol 1991;86:3–11.(Makoto Saharaa, Masataka )