Identification and characterization of a resident vascular stem/progenitor cell population in preexisting blood vessels
2011; Springer Nature; Volume: 31; Issue: 4 Linguagem: Inglês
10.1038/emboj.2011.465
ISSN1460-2075
AutoresHisamichi Naito, Hiroyasu Kidoya, Susumu Sakimoto, Taku Wakabayashi, Nobuyuki Takakura,
Tópico(s)Cardiac and Coronary Surgery Techniques
ResumoArticle16 December 2011free access Identification and characterization of a resident vascular stem/progenitor cell population in preexisting blood vessels Hisamichi Naito Hisamichi Naito Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Hiroyasu Kidoya Hiroyasu Kidoya Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Susumu Sakimoto Susumu Sakimoto Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Taku Wakabayashi Taku Wakabayashi Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Nobuyuki Takakura Corresponding Author Nobuyuki Takakura Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan JST, CREST, Sanbancho, Tokyo, Japan Search for more papers by this author Hisamichi Naito Hisamichi Naito Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Hiroyasu Kidoya Hiroyasu Kidoya Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Susumu Sakimoto Susumu Sakimoto Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Taku Wakabayashi Taku Wakabayashi Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Search for more papers by this author Nobuyuki Takakura Corresponding Author Nobuyuki Takakura Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan JST, CREST, Sanbancho, Tokyo, Japan Search for more papers by this author Author Information Hisamichi Naito1, Hiroyasu Kidoya1, Susumu Sakimoto1, Taku Wakabayashi1 and Nobuyuki Takakura 1,2 1Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan 2JST, CREST, Sanbancho, Tokyo, Japan *Corresponding author. Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: +81 6 6879 8312; Fax: +81 6 6879 8314; E-mail: [email protected] The EMBO Journal (2012)31:842-855https://doi.org/10.1038/emboj.2011.465 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Vasculogenesis, the in-situ assembly of angioblast or endothelial progenitor cells (EPCs), may persist into adult life, contributing to new blood vessel formation. However, EPCs are scattered throughout newly developed blood vessels and cannot be solely responsible for vascularization. Here, we identify an endothelial progenitor/stem-like population located at the inner surface of preexisting blood vessels using the Hoechst method in which stem cell populations are identified as side populations. This population is dormant in the steady state but possesses colony-forming ability, produces large numbers of endothelial cells (ECs) and when transplanted into ischaemic lesions, restores blood flow completely and reconstitutes de-novo long-term surviving blood vessels. Moreover, although surface markers of this population are very similar to conventional ECs, and they reside in the capillary endothelium sub-population, the gene expression profile is completely different. Our results suggest that this heterogeneity of stem-like ECs will lead to the identification of new targets for vascular regeneration therapy. Introduction Regeneration of the vasculature in ischaemic-injured organs is essential to ensure their integrity. Postnatal neovascular formation was originally thought to be mediated by angiogenesis, that is, the generation of new endothelial cells (ECs) from preexisting vessels, not by vasculogenesis, a process of blood vessel formation whereby the early vascular plexus forms from mesoderm by differentiation of angioblasts (Risau, 1997). However, accumulating evidence suggests that vasculogenesis persists into adult life, and contributes to the formation of new blood vessels (Asahara et al, 1997). It has been proposed that bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs) are important for promoting vascularization in many pathophysiological situations; several clinical trials are already ongoing based on this concept (Shantsila et al, 2007). However, some reports suggest that the contribution of EPCs to the neovascular ECs itself is not sufficient (Gothert et al, 2004; Peters et al, 2005). In the peripheral vasculature, there is considerable evidence that although preexisting ECs display many common features, they also represent a heterogeneous population. Transcriptional and antigenic differences in ECs from arteries and veins, and the morphological and functional characteristics referred to as continuous, fenestrated, and discontinuous are widely accepted (Risau, 1995). Recently, it has been shown that in response to angiogenic stimuli, a discrete population of cells, the so-called 'tip and stalk cells', lead and guide new sprouts and form additional ECs, respectively (Gerhardt et al, 2003). Furthermore, populations of ECs of another different phenotype, the so-called phalanx cells that generate stable blood vessels, have been reported (Mazzone et al, 2009). Additionally, the presence of stem/progenitor cells in the vessel wall has been proposed. Investigating adult vessels in mice revealed Sca1+ progenitor cells in the adventitia of large and medium-sized arteries and veins (Hu et al, 2004; Sainz et al, 2006; Passman et al, 2008). Similarly, CD34+ CD31− progenitor cells in the distinct zone between smooth muscle and the adventitial layer of the human adult vascular wall were identified (Zengin et al, 2006). These stem/progenitor cells were reported to have the ability to differentiate into ECs in culture and form capillary-like microvessels in ex-vivo assays. However, during angiogenic growth, microvascular ECs, rather than the ECs of the artery or vein which are completely covered by the vascular wall, are selected for neovascularization (Risau, 1995). Therefore, it is suggested that stem/progenitor cells in the vascular wall of larger blood vessels are not the main source of neovascular ECs. Haematopoietic cells (HCs) and ECs originate from common progenitors (Choi et al, 1998), with haemogenic ECs generating HCs during development (Nishikawa et al, 1998). Moreover, ECs support self-renewal of haematopoietic stem cells (HSCs; Hooper et al, 2009). We previously reported that HSCs also promote angiogenesis (Takakura et al, 2000), emphasizing the close developmental and functional relationships between HCs and ECs. Most BM HSCs appear dormant, and are characterized as side population (SP) cells effluxing Hoechst 33342 (Goodell et al, 1996). This staining method has been applied to explore stem cells of a wide range of tissues, including skin, lung, heart, mammary gland, muscle and testis (Challen and Little, 2006). It is possible that resident quiescent EC stem/progenitor cells in the preexisting blood vessels are also found within these SP cells. In this study, we examined the ECs residing in preexisting vessels precisely to identify the origin of neovascular ECs. Results Identification and characterization of endothelial SP cells Here, we analysed cells from hind limb muscle to identify endothelial SP cells. Among cells stained by the EC marker CD31, but not the HC marker, CD45 (CD31+ CD45− ECs) (Figure 1A), 1.15±0.14% were in the SP gate, confirmed by their disappearance with the drug efflux pump inhibitor, verapamil. They were distinct from the main population (MP) of cells (Figure 1B). Because the SP phenotype is a marker for quiescence in HSCs (Arai et al, 2004), we applied a method which identifies cells in G0 plus G1 phase by Hoechst 33342 distribution and assigns them to G0 or G1 by Pyronin Y RNA staining (Gothot et al, 1997). As shown in Figure 1C and D, 94.8±2.2% of endothelial SP (EC-SP) cells were in the PY− G0 fraction, clearly different from CD31+CD45− endothelial MP (EC-MP) cells. To confirm that EC-SP cells do reside in the blood vessel, we performed lectin perfusion assays. As shown in Figure 1E, ∼96% of CD31+CD45− cells were lectin positive, indicating that most of them were true ECs residing at the inner surface of vessels. The percentage of SP cells within the lectin+ EC population was approximately the same as the percentage of EC-SP cells identified in Figure 1B (see Figure 1F). On the other hand, ∼91% of EC-SP cells were lectin+, indicating that most of these cells reside at the inner surface of vessels (Figure 1G). Next, we characterized the phenotype of EC-SP cells. These were found to express the EC markers VE-cadherin, Flk-1, and Sca-1, but no haematopoietic lineage markers or the pericyte marker PDGFR-β. This phenotype is identical to the EC-MP cells. However, as with CD34-negative long-term repopulating HSCs (Osawa et al, 1996), EC-SP cells expressed little CD34, but CD133, a stem/progenitor cell marker in several tissues (Mizrak et al, 2008), was strongly expressed (Figure 2A). We confirmed that the EC-SP cell fraction was not contaminated with HCs, pericytes, or fibroblasts, by analysing lineage markers for those cell types in cells from the digested muscle sample (Supplementary Figures S1 and S2). Moreover, Notch4 mRNA levels were significantly lower in EC-SP than in EC-MP cells. In contrast, mRNA expression for ABCB1a (Multiple drug resistance 1a (MDR1a)) and ABCG2, a member of the ABC transporter gene family correlating with SP phenotype (Bunting et al, 2000), was higher in the EC-SP cells (Figure 2B). Furthermore, the expression of several other ABC transporters that are reported to correlate with SP phenotype was higher in the EC-SP cells (Supplementary Figure S3). Morphologically, the nuclear-to-cytoplasm (N/C) ratio of the EC-SP cells was higher than the EC-MP cells (Figure 2C). In addition, acetylated low-density lipoprotein (Ac-LDL) uptake that is functional property of ECs was observed by EC-SP cells but less than by EC-MP cells (Figure 2D and E). Taken together, we conclude that EC-SP cells are not pericytes, fibroblasts, or HCs but are true ECs already committed to the EC lineage and are phenotypically and morphologically different from EC-MP cells. Figure 1.Identification of endothelial side population cells. (A) Flow cytometric analysis of hind limb ECs from wild-type mice. (B) Hoechst 33342 staining of CD31+CD45− ECs gated as shown in (A). Note that verapamil selectively prevents Hoechst exclusion from EC-SP cells. (C) Incorporation of Pyronin Y (PY) in EC-SP (left-hand side) and EC-MP (right-hand side) cells. (D) Quantitative evaluation of PY− cells among EC-SP (SP) and EC-MP (MP) cells. Error bars are ±s.e.m. **P<0.01 (n=7). (E) Flow cytometric analysis of mouse hind limb ECs after in-vivo infusion of lectin. Lectin-positive cells among the CD31+CD45− cells are shown in the red gate and total CD31+CD45− cells are shown in the black gate. 95.9±0.2% (n=6) of the CD31+CD45− ECs were lectin positive. (F) Hoechst staining of lectin+ CD31+CD45− cells. (G) Representative flow cytometric plots of EC-SP cells (black dots). The lectin-positive population is shown in the red gate. 90.6±1.4% (n=4) of the EC-SP cells were lectin positive. Download figure Download PowerPoint Figure 2.Characterization of endothelial side population cells. (A) Histogram showing expression levels of surface markers in EC-SP, EC-MP cells and the negative control. (B) Quantitative RT–PCR analysis of mRNA as indicated in EC-SP and EC-MP cells, corrected for expression of the control gene GAPDH. Of the endothelial genes, Notch4 was significantly lower in EC-SP cells. Expression levels of the ABC transporter ABCG2 and ABCB1a (MDR1a) were higher in EC-SP cells. Expression of chemokine receptor CXCR4 and hypoxia-inducible factor (HIF1a) was higher in EC-SP cells. Error bars are ±s.e.m. **P<0.01, *P 6). (C) Haematoxylin and eosin staining of EC-SP and EC-MP cells isolated by FACS. (D) Freshly isolated cells from hind limb were stained with Ac-LDL and then Hoechst staining was performed to detect EC-SP cells. EC-SP cells were cytospun onto slides and Ac-LDL uptake was evaluated. Some of the EC-SP cells showed weak uptake of Ac-LDL, but all were positive. (E) Intensity of Ac-LDL uptake was evaluated by FACS analysis. As indicated, Ac-LDL uptake was observed in EC-SP cells but was lower than in EC-MP cells. Scale bars, 10 μm (C) and 100 μm (D). Download figure Download PowerPoint EC-SP cells are not derived from BM, are distinct from EPCs, and are distributed in the peripheral vessels To exclude the possibility that EC-SP cells are only found in the lower limb, we analysed different organs and confirmed that these cells are distributed all over the body, but are not detectable in some organs (Figure 3A–C). For example, we could not identify the EC-SP pattern in the brain, probably due to constitutively high ABC transporter expression (Miller, 2010; Figure 3A and C). In addition, we were unable to detect the SP pattern in cultured ECs (Figure 3A and C). Interestingly, EC-SP cells were also not detectable in peripheral blood or BM, suggesting an origin different from EPCs and that EC-SP cells are present in the peripheral blood vessels. Moreover, EC-SP cells are not present in the lymphatic endothelium (Supplementary Figure S4) and express lower levels of arterial markers but similar levels of venous markers compared with total ECs (Supplementary Figure S5). This indicates that EC-SP cells reside predominantly in veins and capillaries but not in the lymphatics. To confirm that EC-SP cells are not identical to EPCs, we transplanted BM cells from GFP mice into irradiated wild-type mice and assessed the presence of GFP-positive EC-SP cells. Flow cytometry showed that among CD31+CD45− ECs from the hind limb muscle of GFP BM-transplanted mice (Figure 3D), 3.75±0.13% were GFPdim (Figure 3E), but that none of these were EC-SP cells (Figure 3F). This was also confirmed in a BM transplantation model using neonates, in which BM cells were replaced by the injection of BM cells from GFP mice into the liver of wild-type neonates within 12 h after birth. This model allows us to ask whether EPCs derived from BM undergo EC transition at the growing stage and become EC-SP cells. However, among CD31+CD45− ECs from the hind limb muscle of GFP newborn BM-transplanted mice (Figure 3G), we could not detect any GFP-positive or GFP-dim ECs, suggesting that EC-SP cells do not originate from EPCs derived from BM (Figure 3H). It has been reported that EPCs express CXCR4 (Walter et al, 2005); accordingly, the BM CD34+ EPC cell fraction strongly expresses CXCR4. However, EC-SP cells were found to express CXCR4 at significantly lower levels (Figure 3I). Taking these data together, we conclude that EC-SP cells are not identical to EPCs. Figure 3.EC-SP cells are present in different organs and are not derived from BM in BM chimeric mice. (A) The ECs from several organs and cultured cell lines as indicated were stained with Hoechst. Percentages of the EC-SP cells are shown in the table. There were few CD31+CD45− ECs in the bone marrow and peripheral blood; SP cells were hardly detected at all. In the EC lines (HUVEC, HUAEC, bEnd3, and EOMA), SP cells were not detected. Of note, the EC-SP phenotype disappeared after culturing primary ECs from hind limbs. (B) One example showing EC-SP cells of lung that disappeared following verapamil treatment. (C) In the brain, a stereotypic EC-SP pattern is not observed and there are no EC-SP cells within the bEnd3 population. (D–F) BM cells from GFP mice were transplanted into lethally irradiated wild-type mice. Four weeks after transplantation, cells from hind limbs were analysed. (D) Representative flow cytometric plots of cells from hind limb muscle. CD31+CD45− EC fraction (red) and CD31−CD45+ peripheral blood fraction (green) are gated. (E) Histogram of CD31+CD45− ECs and CD31−CD45+ peripheral blood cells obtained from hind limbs. Almost all blood cells (green line) after transplantation were GFP positive. Approximately 4% of CD31+CD45− ECs (red line) were weakly GFP positive (GFPdim). GFPdim EC population is shown in arrowed region. (F) Hoechst staining of GFPdim ECs. The SP phenotype was not seen. (G, H) Analysis of hind limb muscle cells from newborn transplantation model. (G) Representative flow cytometric plots of cells from hind limb muscle of BM chimeric mice; CD31+CD45− EC fraction is gated (red). (H) Histogram showing GFP intensity of CD31+CD45− ECs obtained from hind limb and peripheral blood. In this model, GFP-positive CD31+CD45− ECs make up <0.01% of total CD31+CD45− ECs, suggesting no major contribution of BM cells to EC-SP cells. (I) Quantitative evaluation of CXCR4 mRNA expression in EC-SP cells and CD34+ bone marrow mononuclear cells (BMCs) by real-time PCR. Note that CXCR4 expression is 17 times higher in CD34+ BM cells (BMC) than in EC-SP cells (SP). Error bars are ±s.e.m. **P<0.01 (n=7). Download figure Download PowerPoint Proliferation and colony-forming capacity of EC-SP cells in vitro If EC-SP cells are indeed a stem/progenitor population, they must be able to generate large numbers of mature ECs and form colonies originating from a single EC. To explore this issue in vitro, sorted EC-SP cells were cultured on OP9 stromal cells which support EC growth (Takakura et al, 1998). After 10 days, EC-SP cells generated higher numbers of colonies with a 'cordlike' structure (Zhang et al, 2001), which formed a fine vascular network, as well as producing higher numbers of ECs than EC-MP cells (Figure 4A–D). It was estimated that 1.2±0.5% of EC-SP cells formed cobblestone-like (sheet-like) colonies (Supplementary Figure S6). To ensure that this degree of colony-forming ability was not a specific property only of ECs from hind limb muscle vessels, EC-SP cells from different organs were cultured on OP9 stromal cells. It was found that they also possessed greater colony-forming ability than EC-MP cells (Supplementary Figure S7). Moreover, we confirmed that these colony-forming cells are indeed ECs, because the colonies were positive for the EC markers CD34, CD105, Flk1, VE-cadherin, vWF, and ZO-1 (Supplementary Figure S8) but negative for the haematopoietic markers B220, CD4, CD8, Gr1, Mac1, Ter119, and CD45 (Supplementary Figure S9A). We excluded the possibility that a contaminating HSC population was giving rise to ECs in our culture system by demonstrating that CD31+ ECs could not be induced from BM-derived c-Kit+Sca-1+Lin− HSC populations (Supplementary Figure S9B). Moreover, VEGF blockade resulted in prevention of colony formation, indicating that expansion of ECs from EC-SP cells depended on VEGF-VEGFR signalling (Supplementary Figure S10). Matrigel plug assays carried out with GFP-positive cells showed that EC-SP cells formed entire vascular networks in the matrigel, but EC-MP cells only formed separate colonies with a small network (Figure 4E). Moreover, to compare the ability of single EC-SP or EC-MP cells to generate EC, numbers of cells in single colonies were counted. It was found that EC-SP cells have a greater capacity to produce ECs than do EC-MP cells (Figure 4F and G). To further clarify whether EC-SP cells are indeed committed to the EC lineage, we crossed endothelial-specific VE-cadherin-Cre-ERT mice with loxP-CAT-EGFP reporter mice and sorted GFP-positive EC-SP cells (Supplementary Figure S11A and B). In the GFP+ (VE-cadherin+) CD31+CD45− fraction, the percentage of EC-SP cells was comparable to wild-type mice. When cultured on OP9 cells for 10 days, GFP+ EC-SP cells generated colonies similar to those from wild-type mice (Figure 4H). Furthermore, EC-SP cells did not give rise to the mesenchymal and haematopoietic lineage in vitro (Supplementary Figures S12 and S13A). Next, to assess clonal expansion of ECs from single cells, we performed time-lapse analysis of EC-SP cells and found that a single EC-SP cell could form a colony (Figure 5A; Supplementary Movie S1). Moreover, to establish whether this EC-SP cell clonal expansion can occur in every colony, sorted EC-SP cells from normal mice and C57BL/6-Tg(CAG-EGFP) mice (EGFP mice) were mixed in equal proportions and cultured on OP9 stromal cells. As expected, colonies with 'cordlike' structures were generated from either GFP-positive or -negative ECs (Supplementary Figure S14), suggesting that a single EC-SP cell is able to generate a single colony. Limiting dilution analysis revealed that the frequency of cells with the capacity to form colonies was significantly higher in EC-SP cells than in EC-MP cells by a factor of 10 (1 in 6.6 and 1 in 66, respectively) (Figure 5B and C). Moreover, long-term culture-initiating cell (LTC-IC) assays revealed that ECs having higher proliferative potential were produced from EC-SP cells than could be produced by EC-MP cells (Figure 5D). These findings indicate that cells able to generate EC colonies are enriched within the EC-SP population. Figure 4.Endothelial SP cells have EC colony-forming ability. (A) EC-SP cells and EC-MP cells were cultured on OP9 feeder cells and stained with anti-CD31 antibody. (B) Colonies are shown in the low power field. The EC-SP cells form fine CD31-positive networks and many colonies compared with EC-MP cells. (C) The number of colonies stained with anti-CD31 antibody and (D) number of VE-cadherin+ ECs counted by flow cytometry in one well of a 6-well culture dish. Error bars are ±s.e.m. **P<0.01 (n=12). (E) EC-SP and EC-MP cells were sorted from EGFP mice and transplanted to wild-type mice with matrigel. Gated area is shown in higher magnification. (F, G) Nuclear staining of ECs forming colonies on OP9 cells for the evaluation of cell number. Representative image of an EC colony stained with anti-CD31 antibody and Hoechst (F) and quantification of the number of ECs composing one colony (G). (H) EC colonies derived from EC-SP cells from VE-cadherin promoter EGFP mice. Scale bars, 500 μm (A), 1 mm (E), 200 μm (F left panel and H), 50 μm (F right panel), and 5 mm (B). Download figure Download PowerPoint Figure 5.Single EC-SP cells form EC colonies. (A) Time-lapse analysis of EC-SP cell from EGFP mice. (B, C) Limiting dilution assay of EC-SP (B) and EC-MP (C) cells. EC-SP and EC-MP cells were cultured on OP9 feeder cells and titrated down to 20, 10, 5, 3, 1, 0 and 200, 100, 50, 30, 10, 0 cells, respectively. The number of colonies was counted after staining with anti-CD31 antibody and the frequency of colony-forming cells was calculated according to Poisson statistics. (D) Results of long-term culture-initiating assays. 5 × 102 primary EC-SP or EC-MP cells were cultured and the number of colonies counted (P0). Cells were harvested and 5 × 102 sorted ECs derived from the first or second rounds of culture were cultured again (P1 and P2, respectively). Note that the P2 assay using ECs from EC-MP cells could not be performed due to insufficient ECs in P1. **P 5). Scale bar, 100 μm (A). Download figure Download PowerPoint Angiogenic stimuli induced by ischaemia activate EC-SP cells To study the potential of the EC-SP cells to facilitate neovascularization in vivo, we first investigated their proliferative capacity using a hind limb ischaemia model (occlusion of the femoral artery). The percentage and absolute number of EC-SP cells increased 1 day after induction of ischaemia, peaked after 3 days at 4.03±1.44% and gradually declined again to the steady state after 2 weeks (Figure 6A, D and E). Addition of verapamil blocked the EC-SP cells, confirming their SP phenotype (Figure 6B). Sham operation on the other hind limb did not have any effect (Figure 6C). Cell-cycle analysis revealed that ∼40% of the EC-SP cells began to divide after induction of ischaemia (Figure 6F and G). The colony-forming ability of the ischaemic EC-SP and EC-MP cells was comparable with that of the same cell types in the steady state (Supplementary Figure S15). Next, we used a BM transplantation model to confirm that the EC-SP cells proliferating after the induction of ischaemia are not derived from BM cells. When hind limb ischaemia was induced in chimeric mice generated by transplanting BM cells from EGFP mice into wild-type mice, all CD31−CD45+ blood cells in the hind limb were positive for GFP, but CD31+CD45− cells within either the EC-MP or EC-SP populations were negative for GFP (Figure 6H–K). This implies that the increased EC-SP cells after induction of ischaemia were not derived from the BM. Taken together, these results suggest that EC-SP cells are quiescent in the steady state but actively proliferate in peripheral vessels when exposed to angiogenic stimuli induced by ischaemia. Figure 6.EC-SP cells proliferate under conditions of tissue hypoxia. Flow cytometric analysis of Hoechst 33342 staining of CD31+CD45− ECs from hind limbs in which ischaemia had been induced (A, B) and sham-operated hind limbs from the other side of the animal (C) with (B) or without (A, C) Verapamil treatment. Quantitative evaluation of the number of EC-SP cells (D) and the percentage of EC-SP cells (E) from one hind limb. Control in (D) indicates EC-SP cells in the sham-operated hind limb. Error bars are ±s.e.m. *P 10), **P 10). (F) Hoechst and PY emission pattern of EC-SP cells sorted from the hind limb 3 days after induction of ischaemia. (G) Percentage of PY-low G0 EC-SP cells under steady-state conditions or the ischaemic state as observed in (F). Error bars are ±s.e.m. **P<0.01 (n=6). (H–K) Excluding the possibility that proliferating EC-SP cells are derived from BM. Representative FACS analysis of cells from the hind limb (H) and BM (I) 3 days after induction of ischaemia in BM chimeric mice transplanted with BM cells derived from EGFP mice into wild-type mice. (J) Hoechst staining of the CD31+CD45− ECs (black gate in (H)). EC-SP cells (red gate) and EC-MP cells (green gate) are shown. (K) Histogram showing GFP positivity in the gated populations. Colours of lines are the same as the gated colours in (I) and (J). Only CD45+ BM cells (blue gate in (I)) are GFP positive but EC-SP cells and EC-MP cells are GFP negative. Download figure Download PowerPoint EC-SP cells contribute to the regeneration of vascular endothelium in vivo Next, we transplanted EC-SP or EC-MP cells into ischaemic limbs, observed their contribution to the neovasculature, and compared the effectiveness of restoration of the vasculature after ischaemia. To this end, we transplanted 3000 cells from EGFP mice and evaluated blood flow by laser Doppler perfusion image analyzer. After 14 days, the blood flow in the hind limbs of EC-SP-transplanted mice was completely restored, whereas transplantation of EC-MP cells resulted in congestion, with necrosis of the toes (Figure 7A and B). At the site of transplantation, blood vessel density was greater in the animals receiving EC-SP cells (Figure 7C and D). Stereomicroscopic observations on living EC-SP-transplanted mice 14 day after transplantation revealed many GFP-positive vessels on the hind limb muscle surface (Figure 7E). These newly formed GFP-positive vessels were filled with red blood cells, suggesting their connection to the systemic circulation. In contrast, blood vessels originating from EC-MP cells were very small, and even when cordlike, contained no erythrocytes (Figure 7E and F). Immunohistochemical analysis revealed that transplanted GFP-positive EC-SP cells gave rise to CD31-positive ECs but not to smooth muscle actin (SMA)-positive mural cells, connected to GFP-negative CD31-positive host ECs (Figure 7G; Supplementary Figure S16). Furthermore, we investigated the long-term contribution of transplanted EC-SP cells to blood vessel maintenance, and found that they still persisted 6 months after injection. Moreover, complete blood vessels could be generated solely from GFP-positive ECs derived from EC-SP cells, whereas ECs derived from EPCs made only a partial contribution and were unable by themselves to reconstitute vessels in their entirety (Takahashi et al, 1999; Figure 6E). Finally, to confirm that these newly developed blood vessels originate from cells already committed to ECs, we utilized GFP+EC-SP cells derived from VE-cadherin Cre mice crossed with lox-GFP reporter mice, as described in Supplementary Figure S11A and B. This revealed that GFP+EC-SP cells generated fine vascular colonies when transplanted into ischaemic limbs (Supplementary Figure S11C). Figure 7.Recovery from ischaemia and long-term incorporation of ECs from EC-SP cells into newly developed blood vessels. (A) Hind limb ischaemia was induced in wild-type mice and EC-SP or EC-MP cells sorted from EGFP mice were transplanted. Gross appearance, Laser Doppler image, and representative photographs of hind limb toes 14 day after transplantation. (B) Blood perfusion ratio of ischaemic hind limb measured by laser Doppler imaging at 3, 7, and 14 days after treatment. Error
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