Jagged-1 Signaling in the Bone Marrow Microenvironment Promotes Endothelial Progenitor Cell Expansion and Commitment of CD133+ Human Cord Blood Cells for Postnatal Vasculogenesis

Mika Ishige-Wada; Sang-Mo Kwon; Masamichi Eguchi; Katsuto Hozumi; Hideki Iwaguro; Taro Matsumoto; Noboru Fukuda; Hideo Mugishima; Haruchika Masuda; Takayuki Asahara

doi:10.1371/journal.pone.0166660

Abstract

Notch signaling is involved in cell fate decisions during murine vascular development and hematopoiesis in the microenvironment of bone marrow. To investigate the close relationship between hematopoietic stem cells and human endothelial progenitor cells (EPCs) in the bone marrow niche, we examined the effects of Notch signals [Jagged-1 and Delta-like ligand (Dll)-1] on the proliferation and differentiation of human CD133⁺ cell-derived EPCs. We established stromal systems using HESS-5 murine bone marrow cells transfected with human Jagged-1 (hJagged-1) or human Dll-1 (hDll-1). CD133⁺ cord blood cells were co-cultured with the stromal cells for 7 days, and then their proliferation, differentiation, and EPC colony formation was evaluated. We found that hJagged-1 induced the proliferation and differentiation of CD133⁺ cord blood EPCs. In contrast, hDll-1 had little effect. CD133⁺ cells stimulated by hJagged-1 differentiated into CD31⁺/KDR⁺ cells, expressed vascular endothelial growth factor-A, and showed enhanced EPC colony formation compared with CD133⁺ cells stimulated by hDll-1. To evaluate the angiogenic properties of hJagged-1- and hDll-1-stimulated EPCs in vivo, we transplanted these cells into the ischemic hindlimbs of nude mice. Transplantation of EPCs stimulated by hJagged-1, but not hDll-1, increased regional blood flow and capillary density in ischemic hindlimb muscles. This is the first study to show that human Notch signaling influences EPC proliferation and differentiation in the bone marrow microenvironment. Human Jagged-1 induced the proliferation and differentiation of CD133⁺ cord blood progenitors compared with hDll-1. Thus, hJagged-1 signaling in the bone marrow niche may be used to expand EPCs for therapeutic angiogenesis.

Citation: Ishige-Wada M, Kwon S-M, Eguchi M, Hozumi K, Iwaguro H, Matsumoto T, et al. (2016) Jagged-1 Signaling in the Bone Marrow Microenvironment Promotes Endothelial Progenitor Cell Expansion and Commitment of CD133⁺ Human Cord Blood Cells for Postnatal Vasculogenesis. PLoS ONE 11(11): e0166660. https://doi.org/10.1371/journal.pone.0166660

Editor: Gangjian Qin, University of Alabama at Birmingham, UNITED STATES

Received: August 1, 2016; Accepted: November 1, 2016; Published: November 15, 2016

Copyright: © 2016 Ishige-Wada et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported in part by a a grant from the Ministry of Education, Science, Sports, and Culture of Japan 19790542 to Dr Mika Ishige-Wada; a grant from the Ministry of Education, Science, Sports, and Culture of Japan 15K09603 to Dr Mika Ishige-Wada; a grant from the Ministry of Education, Science, Sports, and Culture of Japan 25461091 to Dr Haruchika Masada; a grant from Japan Agency for Medical Research and Development 15bk0104012h0003 to Dr Takayuki Asahara. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Notch signaling plays a crucial role in cell fate determination of a variety of cell types during development and postnatal tissue organization, including murine vascular development and angiogenesis [1, 2]. Mutations of Notch receptors and ligands in mice cause abnormal organization of vascular and hematopoietic systems with severe hemorrhaging, which is embryonic lethal in the Notch signaling null mouse [3]. Two human diseases, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADSIL) and Alagille Syndrome exhibit vascular system abnormalities caused by Notch pathway mutations [4, 5]. Notch signaling is initiated by interactions between Notch receptors and their ligands expressed on cells. Mammals express four Notch receptors (Notch 1–4) and five Notch ligands [Jagged-1, -2, and Delta-like ligand (Dll)-1, -2, and -4]. Interactions of Notch receptors with the membrane-bound ligands of Delta and Jagged gene families are critical for Notch activation. Ligand binding induces γ-secretase-mediated cleavage and translocation of the Notch intracellular domain into the nucleus where it interacts with DNA-binding protein RBP-Jk to induce downstream target genes [1]. Primitive human CD34⁺ bone marrow cells express all Notch receptors [6]. Furthermore, primary cells and cultured stromal cells derived from the aorta-gonad-mesonephros, fetal liver, bone marrow, and osteoblasts express Jagged-1, Dll-1, and Dll-4 [7–9]. Therefore, these ligands expressed on stromal cells might interact with their respective Notch receptors on primitive hematopoietic cells in hematopoietic stem cell niches.

Endothelial progenitor cells (EPCs) identified as CD34⁺ mononuclear cells isolated from adult peripheral blood are a functional angiogenetic modulator of postnatal neovascularization [10]. Peripheral blood EPCs were originally derived from bone marrow, but are also present in cord blood (CB) as CD34⁺ and CD133⁺ cells [11]. Cytokines induce EPCs to proliferate and differentiate in the bone marrow, mobilize to systemic circulation, migrate to ischemic sites, differentiate into mature endothelial cells, and secrete angiogenic factors [12–14]. In the bone marrow microenvironment, the quantitative and qualitative features of EPCs might be regulated by several molecular mechanisms, which is similar to hematopoietic stem cells. We have demonstrated that Jagged-1 in the bone marrow niche is required for EPC development of neovascularization in mice [15]. Furthermore, Jagged-1 and Notch1 are critical for the role of EPCs in a mouse injury model [16, 17]. However, little is known about human EPCs and their development in the bone marrow niche.

In this study, we established an in vitro co-culture system similar to the bone marrow niche using HESS-5 bone marrow stromal cells to investigate the functional importance of Notch signals for human EPC-mediated neovascularization and the proliferation and differentiation of human CB-derived EPCs in vitro and in vivo.

Materials and Methods

CD133⁺ cell preparation

Human umbilical CB samples (50–100 mL) were collected in sterile blood packs (SC-200; Terumo Corp., Tokyo, Japan) containing a citrate-dextrose solution as the anticoagulant. Written informed consent was obtained from all mothers before labor and delivery. Protocols for sampling human umbilical CB were approved by our Institutional Review Board, the Clinical Investigation Committee at Tokai University School of Medicine. Mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation. The mononuclear cell layer was collected, washed twice with 2 mM ethylenediaminetetraacetic acid (EDTA) in PBS, and resuspended in degassed PBS with 0.5% bovine serum albumin (BSA) and 2 mM EDTA. CD133⁺ CB cells were separated from 1 × 10⁸ mononuclear cells by a magnetic bead separation method (Miltenyi Biotec, Gladbach, Germany). In brief, CD133⁺ cells were labeled with a hapten-conjugated monoclonal antibody (mAb) against human CD133 (clone AC133: Miltenyi Biotec), followed by microbeads coupled with an anti-hapten mAb. The bead-positive cells were enriched on positive selection columns twice in a magnetic field. Flow cytometric analysis of purified cells using a phycoerythrin (PE)-conjugated anti-CD133 mAb of a different clone (clone 293C3; Miltenyi Biotec) showed that 95% of the selected cells were positive for CD133.

Flow cytometry analysis

Flow cytometric profiles were obtained with a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson Immunocytometry Systems, Mountain View, CA). Briefly, 1 × 10⁴ magnetically separated CD133⁺ CB cells or 1 × 10⁵ cultured CB cells collected from each transfected HESS-5 stroma were washed twice with PBS containing 1% fraction V BSA (Sigma, St Louis, MO), preincubated with 10 μL FcR blocking regent (Miltenyi Biotec) to block nonspecific binding for 30 minutes at 4°C, and then incubated with mAbs for 30 minutes at 4°C. For kinase-insert domain containing receptor (KDR) staining, biotinated anti-mouse IgG₁ (BD Biosciences, San Jose, CA) and allophycocyanin (APC)-conjugated streptavidin (BD Biosciences) were used as secondary and tertiary antibodies. The stained cells were washed three times with PBS containing 1% BSA, resuspended in 0.5 mL PBS/1% BSA/propidium iodide (PI; Sigma), and analyzed by the FACSCalibur flow cytometer. Dead cells were excluded from the plots based on positive PI staining. The following mAbs were used to characterize the cultured CD133⁺ CB cells: CD34-fluorescein-5-isothiocyanate (FITC) (clone 581), CD34-PE (clone 581), CD45-FITC (clone HI30), CD31-FITC (clone WM59) (BD Biosciences), AC133-APC (clone AC133) (Miltenyi Biotec), and KDR (Sigma). For negative isotype controls, mouse IgG₁-FITC (clone MOPC-21), mouse IgG₁-PE (clone MOPC-21), mouse IgG₁-APC (clone MOPC-21), and unlabeled mouse IgG₁ (clone MOPC-21) (BD Biosciences) were used.

HESS-5 cell line

We used the murine bone marrow-derived stromal cell line HESS-5 (provided by Dr. K. Ando, Tokai University, Kanagawa, Japan), which maintains the reconstitution ability of ex vivo-generated human hematopoietic stem cells and does not express Jagged-1 or Dll-1 [18]. Originally, HESS-5 cells were grown in minimal essential medium (MEM; Gibco, Grand Island, NY) supplemented with 10% horse serum (Gibco) and penicillin/streptomycin (Gibco).

Retroviruses and producer cell lines

We established three types of feeder cells: control (HESS-5 cells transfected with an empty vector), hJagged-1 (HESS-5 cells transfected with human Jagged-1), and hDll-1(HESS-5 cells transfected with human Dll-1). Full-length cDNAs encoding human Jagged-1 and Dll-1 (provided by Dr. K. Hozumi and Dr. G. Ando, Tokai University, Kanagawa, Japan) were cloned into the NotI and XhoI restriction sites of GCDNsamI/N retrovirus vectors. These vectors were transfected transiently into PLAT-E packaging cells (provided by Dr. T. Kitamura, The University of Tokyo, Tokyo, Japan) using Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA). The culture supernatants were harvested as the source of the retrovirus as described previously [19]. HESS-5 cells were grown in MEM supplemented with 10% horse serum and penicillin/streptomycin. The day before transfections, 5 × 10³ HESS-5 cells were seeded in a 24-well plate. The cells were then incubated in 0.1 mL of retroviral supernatant containing either human Jagged-1, human Dll-1, or empty viruses in the presence of 8 μg/mL polybrene (Sigma) for 16 hours at 37°C with 5% CO₂. The transfection mixture was removed, α-MEM with 10% horse serum was added, and the cells were cultured for 24 hours. Then, the cells were transferred to a 6-well plate for further culture in α-MEM with 10% horse serum and penicillin/streptomycin. At 48 hours after culture, the transfected HESS-5 cells were stained with human nerve growth factor receptor (NGFR)-PE (clone CD40-1457) (BD Biosciences) and analyzed by flow cytometry (Fig 1A). The transduced cells were subsequently sorted using a FACS Vantage cell sorter (Becton Dickinson Immunocytometry Systems). More than 99% of the cells expressed NGFR, which were used for co-culture experiments with CB CD133⁺ cells.

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Fig 1. Expression and promoter activity of Notch signals in HESS-5 cells transfected with Notch ligand genes.

(A) Flow cytometric analysis of NGFR expression in control vector- (C), human Jagged-1 (J1)- and human Dll-1 (D1)-transfected HESS-5 cells, and the sorting gate (R2) for NGFR⁺ transfected cells. (B) Western blot analysis of hJagged-1 and hDll-1 proteins in HESS-5 cells. Actin was used as an internal control. (C) Notch1 and Notch2 reporter cell lines transfected with RBP-Jk-luc were co-cultured with HESS-5 stromal cells. Luciferase activity (relative light units) was normalized to the activity of Renilla luciferase. Data are expressed as means ± standard deviation (SD) (n = 3). *P < 0.01 between the indicated values.

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Western blotting

For western blot analysis, cell membrane proteins prepared from cultured retroviral transduced HESS-5 cells were separated on a 7.5% polyacrylamide gel (10 μg protein/lane). The proteins were transferred to nitrocellulose membranes (Hybond-ECL; Amersham Pharmacia Biotech, Piscataway, NJ), and incubated overnight at 4°C with primary antibodies: goat polyclonal IgG against human Jagged-1 (C-20) (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal IgG against human Dll-1 (H-265) (Santa Cruz Biotechnology), or rabbit polyclonal IgG against actin (Sigma). The membranes were washed three times and incubated with horseradish peroxidase-conjugated donkey anti-goat (Santa Cruz Biotechnology) or goat anti-rabbit (GE Healthcare, Buckinghamshire, England) IgG for 2 hours at room temperature. Antibody-labeled proteins were detected using an enhanced chemiluminescence detection system (PIERCE, Rockford, IL). hJagged-1 or Dll-1 proteins was detected only in each transduced HESS-5 cell line, but not in the empty vector-transduced control (Fig 1B).

Luciferase assays

Luciferase reporter assays were performed as described previously [20]. In brief, 2.5 × 10⁵ NIH/3T3 cells stably expressing Notch1 or 8 × 10⁵ CHO cells stably expressing Notch2 (provided by Dr. Hozumi K. Tokai University, Kanagawa, Japan) were transfected with RBP-Jk-luc (five RBP-J-binding sites) and pTK-Renilla plasmids (constitutive expression of Renilla luciferase for transfection efficiency control) by Transfast Transfection Reagent (Promega, Madison, WI). At 1 day after transfection, the cells were collected, and 5 × 10⁴ of cells were co-cultured with 5 × 10⁴ of each transduced HESS-5 stromal cell line for 48 hours. Then, luciferase activities were measured using a Dual Luciferase Kit (Promega) according to the manufacturer’s instructions. hJagged-1- and hDll-1-transduced HESS-5 cells were activated by RBP-Jk, a major Notch target. Control HESS-5 cells were not activated (n = 3) (Fig 1C).

Co-culture assays

To evaluate the effects of Notch ligand-expressing stromal cells on the progeny of EPCs, the human EPC fraction of CD133⁺ CB cells, which mostly overlap with CD34⁺ cells, were plated onto the various stroma. Briefly, at 24 hours before co-culture experiments, hJagged-1, hDll-1, and control HESS-5 cells (1 × 10⁴ in 0.5 mL medium) were plated in flat-bottomed, collagen-I-coated 24-well plates. CD133⁺ CB cells (1 × 10⁴) were plated onto irradiated HESS-5 cell layers in 24-well plates containing Stem Span SFEM (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 5% fetal bovine serum (FBS) (JRH Bioscience, Lenexa, KA) and cytokines including human stem cell factor (SCF) (100 ng/mL), interleukin (IL)-6 (20 ng/mL), thrombopoietin (TPO) (20 ng/mL), Flt-3 ligand (100 ng/mL), and vascular endothelial growth factor (VEGF) (50 ng/mL). Human SCF, IL-6, and TPO were a generous gift from Kirin Brewery Co. Ltd. (Tokyo, Japan). Flt-3 ligand and VEGF were purchased from PeproTech (London, UK). All cytokines were pure human recombinant molecules. At 7 days after incubation at 37°C with 5% CO₂, the cells were harvested, counted, and used for subsequent analyses and experimental assays. In some cultures, 10 μg/mL γ-secretase inhibitor IX (GSI IX: Calbiochem, Darmstadt, Germany) was added to the culture medium to inhibit Notch signal transduction.

Reverse transcription-polymerase chain reaction (RT-PCR) analysis

Total RNA was obtained from cultured CD133⁺ CB cells using an RNeasy Micro Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. First-strand DNA was synthesized from 100 ng RNA with random primers by a First Strand cDNA Synthesis Kit (Invitrogen) and amplified with specific primer pairs by Taq DNA polymerase (Takara, Otsu, Japan). The human specific primer pairs, PCR conditions, and products sizes are shown in S1 Table. Human umbilical cord vein endothelial cells were used as a positive control. PCR products were visualized in 2% ethidium bromide-containing agarose gels. To quantify vasculogenic gene expression in cultured CD133⁺ human CB cells, we measured the band intensities in gel images. After the images were recorded in a computer, the band intensities were processed with Image J software (http://imagej.en.softonic.com/). Specific mRNA expression levels were normalized to the intensities of human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH).

EPC colony-forming assay

Vasculogenic methylcellulose culture for the EPC colony-forming assay was performed in triplicate for each sample as described previously [21]. Briefly, 1 mL aliquots of a culture mixture containing 5 × 10² cultured CD133⁺ CB cells, Iscove’s modified Dulbecco’s medium (IMDM) (Gibco), Methocult H4236 (Stem Cell Technologies), 30% FBS (JRH Bioscience), penicillin/streptomycin, and cytokines, including human SCF (100 ng/mL), VEGF (50 ng/mL), basic fibroblast growth factor (FGF) (50 ng/mL), epidermal growth factor (EGF) (100 ng/mL), insulin-like growth factor (IGF)-1 (100 ng/mL), IL-3 (20 ng/mL), and heparin (2 IU/mL), were plated in 35-mm Primaria culture dishes (BD Falcon, Franklin Lakes, NJ). All cytokines were pure human recombinant molecules. Human IL-3 was a generous gift from Kirin Brewery Co. Ltd. Basic FGF, EGF, IGF-1 (PeproTech), and heparin (LEO Pharma, Ballerup, Denmark) were purchased. Cultures were incubated at 37°C with 5% CO₂ for 18 days, and then the number of cells was counted by microscopy. For flow cytometric analysis and the cell adhesion assay, EPC colonies were stained with EPC fluorescent chemical markers [22]. Briefly, EPC colonies were incubated with 2 μg/mL DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL) (Biomedical Technologies, Stoughton, MA) at 37°C for 1 hour. Methylcellulose was removed, and the cells were washed twice with PBS to remove non-adherent colonies. The remaining attached EPC colonies were harvested by pipetting 2 mM EDTA in PBS. For flow cytometric analysis, EPC colonies were fixed with 1% paraformaldehyde for 10 minutes and then reacted with 10 μg/mL FITC-labeled Ulex europaeus lectin type 1 (UEA-1) (Vector Laboratories Inc., Burlingame, CA) for 1 hour at 4°C, followed by flow cytometry. For the cell adhesion assay, stained and harvested cells from EPC colonies were counted, and then 2 × 10⁴ cells were incubated at 37°C in 0.1% BSA/IMDM with 100 ng stromal derived factor-1 (PeproTech) on 0.1% gelatin-coated (Sigma) 24-well plates. After 20 minutes, non-adherent cells were removed by washing with PBS, and adherent cells were counted by fluorescence microscopy.

Transplantation of EPCs experienced with Notch ligands into ischemic hindlimb in vivo

All experimental procedures were conducted in accordance with the national and institutional guidelines. The protocols were approved by the Institutional Animal Care and Use Committee of the Isehara Campus, Tokai University School of Medicine, based on the Guide for the Care and Use of Laboratory Animals (National Research Council). Sixteen male athymic nude mice (CLEA Japan, Inc., Tokyo, Japan), 8 to 9 weeks old and 17 to 20 grams in weight, were separated into four groups (Control, hJagged-1, hDll-1 and IMDM) and used. Unilateral hindlimb ischemia was induced in the mice by ligating and excising the left femoral artery, as described previously [23, 24]. All animals were housed in a room under a 12-h light and 12-h dark cycle and controlled humidity and temperature, with free access to food and water. The experimental animal protocols for making ischemic models were performed under adequate anesthesia using 1.5% to 2.0% isoflurane (Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan) to minimize pain in the mice in line with the 3Rs (replacement, reduction and refinement). After surgery, the mice were monitored twice a day and subcutaneously injected with buprenorphine (Repetan, 0.1 mg/kg body weight; Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) once a day for 3 days to relieve pain or discomfort. At sacrifice, pentobarbital sodium (Somnopentyl, 100 mg/kg body weight; Kyoritsu Seiyaku Co., Ltd., Tokyo, Japan) was intraperitoneally injected.

Cultured CB CD133⁺ cells were harvested and washed three times with a sufficient amount of IMDM to remove the culture medium completely. Then, 1 × 10⁵ cells in 50 μL of fresh unused IMDM were injected into the ischemic limb muscle of the mice immediately after the ligation procedure. As additional control animals, mice with hindlimb ischemia were identically injected with only medium of IMDM.

In vivo physiological and histological assessment

Regional blood flow in ischemic hind limbs was recorded and analyzed by laser Doppler perfusion imaging (LDPI) at 4, 7, 14 and 28 days after transplantation as described previously [15]. In the digital color-coded images, the red hue indicated regions of maximum perfusion, while medium perfusion levels were shown as yellow and low levels as blue. The resulting images also displayed absolute values in readable units. For quantification, the ratio of readable units was determined between ischemic and nonischemic hind limbs. All mice were euthanized at 28 days after transplantation by intraperitoneal administration. Our protocol included humane endpoints in cases in which food or water could not be consumed. However, these were not required in any cases and there were also no deaths prior to the experimental endpoint. Vascular density in sections from the ischemic hind limbs was evaluated at the microvascular level using a fluorescence microscope. Tissue sections from the lower calf muscles of ischemic limbs were obtained on day 28. Muscle samples were fixed with 4% paraformaldehyde at 4°C, embedded in OCT compound (Sakura Finetechnical, Tokyo, Japan), snap-frozen in liquid nitrogen, and cut into 5 μm-thick sections. Histological staining with isolectin B4 (Vector Laboratories) was performed, and capillary density was evaluated morphometrically by histological examination of 15 randomly selected fields. To detect transplanted human cells in mouse ischemic limb muscles, immunohistochemistry was performed with antibodies against human leukocyte antigen (HLA)-ABC (BD Biosciences) and human von Willebrand factor (vWF) (DAKO, Carpinteria, CA). First, HLA-ABC and vWF were labeled with a Zenon® Alexa Fluor® 594 Mouse IgG1 Labeling Kit and then an Alexa Fluor®488 Mouse IgG2a Labeling Kit (Molecular Probes, Karlsruhe, Germany), and then the labeled antibodies were applied for 2 hours. Nuclear counterstaining was performed with 4′-6-diamidino-2-phenylindole (DAPI; Vector Laboratories).

Statistical analysis

Statistical analysis was performed using StatView v5.0 (Abacus Concepts Inc., Berkeley, CA). All values are expressed as the mean ± standard deviation (SD). Statistical significance was evaluated by one-way analysis of variance. Differences of P < 0.05 were considered statistically significant.

Results

Effect of Notch ligands on CD133⁺ cell numbers

Numbers of hJagged-1-stimulated CD133⁺ cells significantly increased to 8.76 ± 2.07 × 10⁵ (P < 0.05) compared with control cells (6.09 ± 1.50 × 10⁵), whereas the number of hDll-1-stimulated CD133⁺ cells was reduced to 3.54 ± 1.07 × 10⁵ (P < 0.05) (Fig 2A). The expansion ratio to the control was 1.44-fold for hJagged-1-stimulated cells and 0.58-fold for hDll-1-stimulated cells. After blocking Notch signaling with GSI, the expansion ratios were similar to the control levels (n = 7 without GSI; n = 4 with GSI) (Fig 2B). No proliferation was observed in non-contact or contact co-culture with GSI (data not shown). Thus, hJagged-1 induces the expansion of CD133⁺ cells, whereas hDll-1 attenuates expansion.

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Fig 2. Effect of hJagged-1 and hDll-1 expression in HESS-5 stromal cells on nuclear cell proliferation.

CD133⁺ CB cells (1 × 10⁴) were co-cultured with control (C), hJagged-1 (J1)- or hDll-1 (D1)-expressing HESS-5 stromal cells. (A) Expansion of nuclear cells at 7 days after co-culture. Data are expressed as means ± SD. **P < 0.01, *P < 0.05 between the indicated values. (B) Expanded cell numbers were normalized to cell numbers activated by control HESS-5 stromal cells at day 7. Data are expressed as means ± SD (n = 7 without GSI; n = 4 with GSI). **P < 0.01, *P < 0.05. GSI: γ-secretase inhibitor.

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Effect of Notch ligands on EPC phenotypic differentiation

We next evaluated the effects of hJagged-1 and hDll-1 on the maintenance of EPCs and commitment to the endothelial lineage. Phenotypic analysis of collected cells co-cultured with stromal cells by flow cytometry revealed that the percentages of CD34⁺/CD38^- hematopoietic progenitor cells and CD31⁺/KDR⁺ endothelial lineage cells were decreased among hDll-1-stimulated CD133⁺ cells (Fig 3A). The percentages of CD34⁺/CD38^-, CD34⁺/CD133⁺, and CD31⁺/KDR⁺ cells among hJagged-1-stimulated CD133⁺ cells were increased slightly, but not significantly compared with the control (Fig 3A).

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Fig 3. Effect of hJagged-1- or hDll-1-expressing HESS-5 stromal cells on the differentiation of CD133⁺ CB cells.

(A) Flow cytometric analysis of CD31, CD34, CD38, CD133, and KDR expression on CD133+ cells co-cultured with control (C), hJagged-1 (J1)- or hDll-1 (D1)-expressing HESS-5 stromal cells at day 7. Numbers in the graph indicate the percentages of gate events within the quadrant. (B) CD34⁺/CD38^-, CD34⁺/CD133⁺, and CD31⁺/KDR⁺ cell numbers in 1 × 10⁴ CD133+ cells at 7 days after co-culture with the three types of HESS-5 stromal cells. Data are expressed as means ± SD (n = 5). **P < 0.01, *P < 0.05 between the indicated values. (C) Expanded cell numbers were normalized to the cell numbers activated by control HESS-5 cells at day 7. Data are expressed as means ± SD (n = 5 without GSI; n = 3 with GSI). **P < 0.01, *P < 0.05 between the indicated values. GSI: γ-secretase inhibitor.

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The numbers of CD34⁺/CD38⁻ hematopoietic progenitor cells and CD34⁺/CD133⁺ immature EPCs were (P < 0.05) increased significantly among hJagged-1-stimulated CD133⁺ cells compared with the control (Fig 3B). Furthermore, the numbers of CD31⁺/KDR⁺ endothelial linage cells were increased significantly by 1.5-fold among hJagged-1-stimulated CD133⁺ cells (P < 0.01), but were decreased significantly by 0.5-fold among hDll-1-transfected CD133⁺ cells (P < 0.01). Suppression of Notch signaling by GSI reversed these cellular changes compared with the controls (n = 5 without GSI; n = 3 with GSI) (Fig 3C).

To confirm the EPC phenotype of Notch ligand-stimulated cells, we evaluated the expression of VEGF-A and endothelial nitric oxide synthase (eNOS) mRNAs by competitive RT-PCR analysis. All cell types examined expressed VEGF-A and eNOS mRNA. There was significantly higher VEGF-A and eNOS mRNA expression in hJagged-1-stimulated CD133⁺ cells compared with Dll-1-stimulated CD133⁺ cells (both P < 0.05) (Fig 4). HESS-5 stromal cells expressing hJagged-1 or hDll-1 differentially affected the proliferation and differentiation of CD133⁺ cells to EPC lineages. Thus, Jagged-1 and Dll-1 have opposite effects on the emergence of EPCs.

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Fig 4. Expression analysis of a human endothelial marker and angiogenic factor by RT-PCR.

Expression of human endothelial marker and angiogenic factor mRNAs by competitive RT-PCR analysis of CD133⁺ cells co-cultured with control (C), hJagged-1 (J1)- or hDll-1 (D1)- expressing HESS-5 stromal cells. Expression of VEGF-A (A) and eNOS (B) mRNAs were normalized to the mRNA expression level activated by control HESS-5 stromal cells at day 7. Data are expressed as means ± SD (n = 3). **P < 0.01, *P < 0.05 between the indicated values.

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Effect of Notch ligands on EPC colony formation

Isolated CD133⁺ cells were stimulated with each stromal cell type at day 7 for EPC colony forming assays. EPC colonies expressed endothelial differentiation markers KDR, eNOS, and VE cadherin (data not shown). Compared with controls, the number of EPC colonies were significantly higher in hJagged-1-stimulated CD133⁺ cell cultures (P < 0.05) and significantly lower in hDll-1-stimulated CD133⁺ cell cultures (P < 0.05) (Fig 5A). Compared with controls, EPC colonies derived from hJagged-1-stimulated CD133⁺ cells were enriched with Ac-LDL/UEA-1 double-positive cells and showed significantly increased adhesive activity (P < 0.05), whereas hDll-1-stimulated cells showed a lower adhesive activity (Fig 5B and 5C).

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Fig 5. Formation and properties of EPC colonies.

Formation and properties of EPC colonies derived from CB CD133⁺ cells co-cultured with control (C), hJagged-1 (J1)- or hDll-1 (D1)-expressing HESS-5 stromal cells. (A) Numbers of EPC colony forming units (EPC-CFU) were determined at 18 days after co-culture. Data are expressed as means ± SD (n = 3). **P < 0.01, *P < 0.05 between the indicated values. Flow cytometric analysis of DiI-Ac-LDL uptake and UEA-1-FITC expression as markers of EPCs (B) and the adhesive ability (C) of EPC colonies derived from 5 × 10² CD133⁺ CB cells stimulated with each HESS-5 stromal cell type for 7 days. Numbers in the graph indicate the percentages of gate events within the quadrant of DiI-Ac-LDL+/UEA-1+. Data are expressed as means ± SD (n = 3). **P < 0.01, *P < 0.05 between the indicated values.

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Thus, HESS-5 bone marrow stromal cells expressing hJagged-1 enhanced EPC colony formation of CD133⁺ cells.

Transplantation of hJagged-1-stimulated CD133⁺ cells into mouse ischemic hindlimbs

Serial examination of hindlimb perfusion by LDPI was performed at several time points from day 0 to 28. Representative images of each group at day 14 are shown in Fig 6A. Regional blood flow in ischemic hindlimbs of mice was increased after transplantation of the three types of stimulated CD133⁺ cells. Blood flow was significantly increased at 28 days after injection of hJagged-1-stimulated CD133⁺ cells compared with hDll-1-stimulated cells or control cells (P < 0.05) (Fig 6B). Capillary density in ischemic hindlimbs of mice was significantly higher after transplantation of hJagged-1-stimulated cells compared with hDll-1-stimulated cells or control cells (P < 0.05) (Fig 7A and 7B).

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Fig 6. In vivo effects of transplanted EPCs stimulated by Notch ligands in mouse ischemic hindlimbs.

CD133⁺ cells (1 × 10⁵) co-cultured with control (C), hJagged-1 (J1)- or hDll-1 (D1)-expressing HESS-5 stromal cells for 7 days or fresh unused medium only (IMDM) were injected into ischemic limb muscles immediately after femoral artery and vein ligation. LDPI was performed before and at days 4, 7, 14 and 28. (A) Representative LDPI images of each group at day 28. (B) Color-coded recordings were analyzed by calculating the mean perfusion for each foot (ischemic and non-ischemic) from day 0 to 28. Perfusion is expressed as the ratio of left (ischemic) and right (non-ischemic) hindlimbs. Data are expressed as means ± SD (n = 4 in each group). *P < 0.05 between the indicated groups.

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Fig 7. Histological evaluation of neovascularization at day 28 after EPC transplantation.

(A) Representative isolectin B4 staining (red) in each group at day 28 after transplantation (×100 magnification). (B) Capillary density in hindlimbs injected with CD133⁺ CB cells (1 × 10⁵) co-cultured with control (C), hJagged-1 (J1)- or hDll-1 (D1)-expressing HESS-5 stromal cells or medium only (IMDM). Data are expressed as means ± SD (n = 4 in each group). **P < 0.01, *P < 0.05 between the indicated groups.

https://doi.org/10.1371/journal.pone.0166660.g007

Differentiation of transplanted cells stimulated by Notch ligands in ischemic muscles in vivo

Human endothelial cells derived from transplanted CD133⁺ cells co-cultured with Notch ligand-transduced stromal cells were identified in the vasculature by double staining of human HLA-ABC and human vWF. Human endothelial cells were identified in mice that received all three cell types, indicating that the transplanted human CD133⁺ cells had differentiated into endothelial cells in mouse ischemic muscles (S1 Fig).

Discussion

This study demonstrated that bone marrow stromal cell environments expressing hJagged-1 or hDll-1 have different effects on CD133⁺ CB cell proliferation and differentiation into EPCs in vitro and in vivo.

Hemangioblasts in developing embryos and adults are common precursor cells for hematopoietic and endothelial cells, and express cell surface markers such as CD34, CD133, and Tie-2 [25, 26]. Nevertheless, although many investigators have expanded hematopoietic stem/progenitor cells ex vivo by bone marrow niche Notch signaling, the results are controversial. The apparent discordant biological activities of soluble Notch ligands in liquid and solid phase hematopoiesis have been assessed [27]. It was established that soluble Notch ligands exert greater biological activities under solid-phase conditions by immobilizing signaling antibodies. While the mechanisms of the different activities of Notch ligands in liquid and solid phases are unclear, soluble ligands are not physiological products and the immobilized form might function similarly to the membrane-bound forms. To reproduce the bone marrow microenvironment in vitro with cytokines, we used membrane-bound forms of Notch ligands on HESS-5 stromal cells that have the reconstituting ability of ex vivo-generated human hematopoietic stem cells. Our system was considered feasible as an in vivo bone marrow niche compared with previously reported systems [28].

To investigate whether these phenotypic and functional differences were associated with differences in Notch ligand signal transduction, Delaney et al. demonstrated dose-dependent effects of Dll-1 on the growth and differentiation of hematopoietic cells [29]. In this study, we demonstrated that, although human Jagged-1- and human Dll-1-transduced HESS-5 stromal cells activated RBP-Jk Notch transcription factors at similar levels, the proliferation and differentiation of CD133⁺ cells stimulated by each stroma were different. These results suggested that the different effects of Jagged-1 and Dll-1 on EPCs were not caused by the dose-dependency of Notch signaling, but rather the different roles of Jagged-1 and Dll-1.

Targeted mutagenesis and transgenic studies have reported the specific roles of Notch receptors as well as Jagged and Delta ligands [30–32]. Although Notch signaling has different roles in embryonic vascular development of mutant mice, most mutants exhibit a similar phenotype characterized by the absence of angiogenic vascular remodeling in utero and abnormalities of arterio-vein differentiation [30–32]. Notch signaling, such as Jagged-1/Notch4, Dll-1, and RBP-Jk, have important roles in embryogenesis and the post-natal vasculature [33, 34]. Sainson et al., showed that the Notch pathway regulates blood vessel sprouting and branching in adult endothelial tip cells, and high expression of Jagged-1 in these tip cells [35]. Buchler et al., suggested up-regulation of tumor angiogenesis by Jagged-1/Notch1 and Dll-4 [36]. Thus, Notch signaling plays multiple roles during vascular development and post-natal angiogenesis. However, some of these roles are controversial. It is thought that specific combinations of Notch ligands and receptors might be involved in different aspects of endothelial cell biology [37], and each Notch signal might influence other Notch signals as lateral inhibition [38]. We have demonstrated that downregulation of mouse Jagged-1 significantly reduces EPC commitment in bone marrow and impairs blood vessel regeneration by decreasing EPCs [15]. In this study, hJagged-1 expressed in bone marrow microenvironmental stromal cells induced the proliferation and differentiation of EPCs. These results suggest that the Jagged-1/Notch axis is critical for EPC development in the bone marrow niche.

Jagged-1 and Dll-1 pathways might be different in EPCs, although the underlying mechanisms remain to be elucidated. It is possible that different Notch receptors or different intracellular signal pathways are present in EPCs. Notch signaling is initiated by interactions between Notch receptors and their ligands on cells. Four Notch receptors (Notch 1–4) have been identified in mammalian systems. Previously, we found that CD133⁺ CB cells, which are similar to CD34⁺ cells, express all Notch receptors [6]. It has been reported that Notch1 signaling is involved in development and postnatal angiogenesis, although its role in EPCs is unclear [39]. Among the intracellular signaling pathways, mitogen-regulateactivated protein kinase and phosphatidylinositol 3-kinase/protein kinase B pathways are downstream of Notch signals for the proliferation of endothelial cells [40]. Choi et al., demonstrated that inhibition of glycogen synthase kinase-3β enhances EPC proliferation [41]. Not only ligand-specific signals but also expressional profiles of receptor/ligand on EPCs are additionally discussed. It is indicated that Dll-1 stimulation may down-regulate Jag-1 expression or signaling via a negative feedback effect on Notch receptors, possibly lateral inhibition [35], while Jagged-1/Notch3 and Dll-1/Notch1 positive feedback systems were reported [37, 42]. Thus, the identification and characterization of receptor/ligand and intracellular signaling pathways of Notch signaling that are crucial in EPC functions require further investigation.

VEGF is a potent promoter of angiogenesis and regulator of blood vessel homeostasis. Furthermore, VEGF contributes to postnatal neovascularization by mobilizing EPCs [12]. In previous studies of mammalian endothelial cells, Notch signals were induced by downstream signals of the VEGF receptor [43]. In contrast, it was recently reported that Jagged-1 and Jagged-2 increase VEGF secretion, and that Notch1 activates hypoxia-inducible factor-1α, the main regulator of VEGF in cancer angiogenesis [36]. In the present study, VEGF expression was increased in EPCs stimulated by hJagged-1-expressing HESS-5 cells. Similar mechanisms of cancer angiogenesis might occur in the bone marrow niche and EPCs. Further studies are needed to clarify the relationship between Notch signals and VEGF in the bone marrow niche.

In this study, the transplantation of hJagged-1-stimulated EPCs recovered impaired regional blood flow and capillary density in vivo in mouse ischemic hindlimbs. Immunohistochemical analysis demonstrated that transplanted CD133⁺ CB cell-derived EPCs differentiated into endothelial cells in ischemic limb muscles, but a significant difference in the numbers of human endothelial cells was not found among the groups. It has been reported that the angiogenic potential of transplanted EPCs in ischemia is due to endothelial differentiation and the local production of angiogenic cytokines[14]. Thus, enhanced neovascularization induced by injection of hJagged-1-stimulated CD133⁺ CB cells might be caused by an enhancement of EPC potential (colony formation and release of angiogenic factors including VEGF). Although the proliferation rate was different, we obtained the largest number of ex vivo-expanded EPCs from hJagged-1-stimulated CD133⁺ cells. Thus, hJagged-1 signaling in the bone marrow niche might be effective to enhance the therapeutic potential of CD133⁺ CB cell-derived EPCs.

In conclusion, this study is the first report regarding the effect of human Notch ligand signaling on EPC proliferation and differentiation. hJagged-1-expressing bone marrow-derived stromal cells induced the proliferation and differentiation of CD133⁺ CB progenitors compared with hDll-1-expressing cells. Thus, hJagged-1 signaling in the bone marrow niche might aid the expansion of EPCs for therapeutic angiogenesis.

Supporting Information

S1 Fig. Histological evaluation of human endothelial cell development in vivo.

CD133⁺ CB cells (1 × 10⁵) co-cultured with control (C), hJagged-1 (J1)- or hDll-1 (D1)-expressing HESS-5 stromal cells for 7 days or fresh unused medium only (IMDM) were injected into mouse ischemic limbs. Representative double immunofluorescence staining of human vWF (red), HLA-ABC (green), and nuclear counterstaining with DAPI (blue) are shown for each group at 28 days after transplantation. All images show merged staining at ×400 magnification. Human endothelial cells were identified as vWF and HLA-ABC double-positive yellow cells (arrow). Scale bar represents 20 μm.

https://doi.org/10.1371/journal.pone.0166660.s001

(TIF)

S1 Table. Sequences of PCR primers for hVEGF-1, heNOS and hGAPDH, product sizes, and PCR conditions.

https://doi.org/10.1371/journal.pone.0166660.s002

(DOCX)

Acknowledgments

HESS-5 stromal cells were a generous gift from Kiyoshi Ando MD, PhD, at the Research Center for Regenerative Medicine, Tokai University School of Medicine, Kanagawa, Japan. We thank Tetsuro Tamaki PhD, for excellent technical advice and support, Yoshinori Okada PhD, Daisuke Suzuki PhD, Rie Ito, and Michiru Kobori for the preparation of this manuscript and excellent technical support, and Sachie Ota for secretarial assistance.

Author Contributions

Conceptualization: HMA TA.
Data curation: MI HMA TA.
Formal analysis: MI HMA TA.
Funding acquisition: TM HMA HMU TA.
Investigation: MI SMK ME HI.
Methodology: MI KH HMA.
Project administration: TM NF HMU TA.
Resources: KH HMA.
Software: MI ME HI.
Validation: MI SMK ME HI HMA.
Visualization: MI TA.
Writing – original draft: MI NF TA.
Writing – review & editing: MI TA.

References

1. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science. 1999;284(5415):770–6. pmid:10221902
- View Article
- PubMed/NCBI
- Google Scholar
2. Varnum-Finney B, Xu L, Brashem-Stein C, Nourigat C, Flowers D, Bakkour S, et al. Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nat Med. 2000;6(11):1278–81. pmid:11062542
- View Article
- PubMed/NCBI
- Google Scholar
3. Iso T, Hamamori Y, Kedes L. Notch signaling in vascular development. Arterioscler Thromb Vasc Biol. 2003;23(4):543–53. pmid:12615665
- View Article
- PubMed/NCBI
- Google Scholar
4. Joutel A, Corpechot C, Ducros A, Vahedi K, Chabriat H, Mouton P, et al. Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature. 1996;383(6602):707–10. pmid:8878478
- View Article
- PubMed/NCBI
- Google Scholar
5. Li L, Krantz ID, Deng Y, Genin A, Banta AB, Collins CC, et al. Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1. Nat Genet. 1997;16(3):243–51. pmid:9207788
- View Article
- PubMed/NCBI
- Google Scholar
6. Vercauteren SM, Sutherland HJ. Constitutively active Notch4 promotes early human hematopoietic progenitor cell maintenance while inhibiting differentiation and causes lymphoid abnormalities in vivo. Blood. 2004;104(8):2315–22. pmid:15231576
- View Article
- PubMed/NCBI
- Google Scholar
7. Walker L, Carlson A, Tan-Pertel HT, Weinmaster G, Gasson J. The notch receptor and its ligands are selectively expressed during hematopoietic development in the mouse. Stem Cells. 2001;19(6):543–52. pmid:11713346
- View Article
- PubMed/NCBI
- Google Scholar
8. Karanu FN, Murdoch B, Miyabayashi T, Ohno M, Koremoto M, Gallacher L, et al. Human homologues of Delta-1 and Delta-4 function as mitogenic regulators of primitive human hematopoietic cells. Blood. 2001;97(7):1960–7. pmid:11264159
- View Article
- PubMed/NCBI
- Google Scholar
9. Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, Knight MC, et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature. 2003;425(6960):841–6. pmid:14574413
- View Article
- PubMed/NCBI
- Google Scholar
10. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275(5302):964–7. pmid:9020076
- View Article
- PubMed/NCBI
- Google Scholar
11. Aoki M, Yasutake M, Murohara T. Derivation of functional endothelial progenitor cells from human umbilical cord blood mononuclear cells isolated by a novel cell filtration device. Stem Cells. 2004;22(6):994–1002. pmid:15536190
- View Article
- PubMed/NCBI
- Google Scholar
12. Asahara T, Takahashi T, Masuda H, Kalka C, Chen D, Iwaguro H, et al. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. Embo J. 1999;18(14):3964–72. pmid:10406801
- View Article
- PubMed/NCBI
- Google Scholar
13. Masuda H, Kalka C, Takahashi T, Yoshida M, Wada M, Kobori M, et al. Estrogen-mediated endothelial progenitor cell biology and kinetics for physiological postnatal vasculogenesis. Circ Res. 2007;101(6):598–606. pmid:17656679
- View Article
- PubMed/NCBI
- Google Scholar
14. Urbich C, Aicher A, Heeschen C, Dernbach E, Hofmann WK, Zeiher AM, et al. Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells. J Mol Cell Cardiol. 2005;39(5):733–42. pmid:16199052
- View Article
- PubMed/NCBI
- Google Scholar
15. Kwon SM, Eguchi M, Wada M, Iwami Y, Hozumi K, Iwaguro H, et al. Specific Jagged-1 signal from bone marrow microenvironment is required for endothelial progenitor cell development for neovascularization. Circulation. 2008;118(2):157–65. pmid:18591437
- View Article
- PubMed/NCBI
- Google Scholar
16. Ii M, Takeshita K, Ibusuki K, Luedemann C, Wecker A, Eaton E, et al. Notch signaling regulates endothelial progenitor cell activity during recovery from arterial injury in hypercholesterolemic mice. Circulation. 2010;121(9):1104–12. pmid:20176991
- View Article
- PubMed/NCBI
- Google Scholar
17. Kamei N, Kwon SM, Ishikawa M, Ii M, Nakanishi K, Yamada K, et al. Endothelial progenitor cells promote astrogliosis following spinal cord injury through Jagged1-dependent Notch signaling. J Neurotrauma. 2012;29(9):1758–69. pmid:22452482
- View Article
- PubMed/NCBI
- Google Scholar
18. Ando K, Nakamura Y, Chargui J, Matsuzawa H, Tsuji T, Kato S, et al. Extensive generation of human cord blood CD34(+) stem cells from Lin(-)CD34(-) cells in a long-term in vitro system. Exp Hematol. 2000;28(6):690–9. pmid:10880755
- View Article
- PubMed/NCBI
- Google Scholar
19. Morita S, Kojima T, Kitamura T. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 2000;7(12):1063–6. pmid:10871756
- View Article
- PubMed/NCBI
- Google Scholar
20. Schroeder T, Kohlhof H, Rieber N, Just U. Notch signaling induces multilineage myeloid differentiation and up-regulates PU.1 expression. J Immunol. 2003;170(11):5538–48. pmid:12759431
- View Article
- PubMed/NCBI
- Google Scholar
21. Masuda H, Alev C, Akimaru H, Ito R, Shizuno T, Kobori M, et al. Methodological development of a clonogenic assay to determine endothelial progenitor cell potential. Circ Res. 2011;109(1):20–37. pmid:21566217
- View Article
- PubMed/NCBI
- Google Scholar
22. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A. 2000;97(7):3422–7. pmid:10725398
- View Article
- PubMed/NCBI
- Google Scholar
23. Iwaguro H, Yamaguchi J, Kalka C, Murasawa S, Masuda H, Hayashi S, et al. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration. Circulation. 2002;105(6):732–8. pmid:11839630
- View Article
- PubMed/NCBI
- Google Scholar
24. Masuda H, Tanaka R, Fujimura S, Ishikawa M, Akimaru H, Shizuno T, et al. Vasculogenic conditioning of peripheral blood mononuclear cells promotes endothelial progenitor cell expansion and phenotype transition of anti-inflammatory macrophage and T lymphocyte to cells with regenerative potential. J Am Heart Assoc. 2014;3(3):e000743. pmid:24965023
- View Article
- PubMed/NCBI
- Google Scholar
25. Jaffredo T, Gautier R, Eichmann A, Dieterlen-Lievre F. Intraaortic hemopoietic cells are derived from endothelial cells during ontogeny. Development. 1998;125(22):4575–83. pmid:9778515
- View Article
- PubMed/NCBI
- Google Scholar
26. Wada M, Ebihara Y, Ma F, Yagasaki H, Ito M, Takahashi T, et al. Tunica interna endothelial cell kinase expression and hematopoietic and angiogenic potentials in cord blood CD34+ cells. Int J Hematol. 2003;77(3):245–52. pmid:12731667
- View Article
- PubMed/NCBI
- Google Scholar
27. Suzuki T, Chiba S. Notch signaling in hematopoietic stem cells. Int J Hematol. 2005;82(4):285–94. pmid:16298816
- View Article
- PubMed/NCBI
- Google Scholar
28. Lauret E, Catelain C, Titeux M, Poirault S, Dando JS, Dorsch M, et al. Membrane-bound delta-4 notch ligand reduces the proliferative activity of primitive human hematopoietic CD34+CD38low cells while maintaining their LTC-IC potential. Leukemia. 2004;18(4):788–97. pmid:14990974
- View Article
- PubMed/NCBI
- Google Scholar
29. Delaney C, Varnum-Finney B, Aoyama K, Brashem-Stein C, Bernstein ID. Dose-dependent effects of the Notch ligand Delta1 on ex vivo differentiation and in vivo marrow repopulating ability of cord blood cells. Blood. 2005;106(8):2693–9. pmid:15976178
- View Article
- PubMed/NCBI
- Google Scholar
30. Huppert SS, Le A, Schroeter EH, Mumm JS, Saxena MT, Milner LA, et al. Embryonic lethality in mice homozygous for a processing-deficient allele of Notch1. Nature. 2000;405(6789):966–70. pmid:10879540
- View Article
- PubMed/NCBI
- Google Scholar
31. Hrabe de Angelis M, McIntyre J 2nd, Gossler A. Maintenance of somite borders in mice requires the Delta homologue DII1. Nature. 1997;386(6626):717–21. pmid:9109488
- View Article
- PubMed/NCBI
- Google Scholar
32. Gale NW, Dominguez MG, Noguera I, Pan L, Hughes V, Valenzuela DM, et al. Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development. Proc Natl Acad Sci U S A. 2004;101(45):15949–54. pmid:15520367
- View Article
- PubMed/NCBI
- Google Scholar
33. Limbourg A, Ploom M, Elligsen D, Sorensen I, Ziegelhoeffer T, Gossler A, et al. Notch ligand Delta-like 1 is essential for postnatal arteriogenesis. Circ Res. 2007;100(3):363–71. pmid:17234965
- View Article
- PubMed/NCBI
- Google Scholar
34. Dou GR, Wang YC, Hu XB, Hou LH, Wang CM, Xu JF, et al. RBP-J, the transcription factor downstream of Notch receptors, is essential for the maintenance of vascular homeostasis in adult mice. Faseb J. 2008;22(5):1606–17. pmid:18096813
- View Article
- PubMed/NCBI
- Google Scholar
35. Sainson RC, Aoto J, Nakatsu MN, Holderfield M, Conn E, Koller E, et al. Cell-autonomous notch signaling regulates endothelial cell branching and proliferation during vascular tubulogenesis. Faseb J. 2005;19(8):1027–9. pmid:15774577
- View Article
- PubMed/NCBI
- Google Scholar
36. Buchler P, Gazdhar A, Schubert M, Giese N, Reber HA, Hines OJ, et al. The Notch signaling pathway is related to neurovascular progression of pancreatic cancer. Ann Surg. 2005;242(6):791–800, discussion -1. pmid:16327489
- View Article
- PubMed/NCBI
- Google Scholar
37. Liu H, Kennard S, Lilly B. NOTCH3 expression is induced in mural cells through an autoregulatory loop that requires endothelial-expressed JAGGED1. Circ Res. 2009;104(4):466–75. pmid:19150886
- View Article
- PubMed/NCBI
- Google Scholar
38. Bentley K, Gerhardt H, Bates PA. Agent-based simulation of notch-mediated tip cell selection in angiogenic sprout initialisation. J Theor Biol. 2008;250(1):25–36. pmid:18028963
- View Article
- PubMed/NCBI
- Google Scholar
39. Takeshita K, Satoh M, Ii M, Silver M, Limbourg FP, Mukai Y, et al. Critical role of endothelial Notch1 signaling in postnatal angiogenesis. Circ Res. 2007;100(1):70–8. pmid:17158336
- View Article
- PubMed/NCBI
- Google Scholar
40. Liu ZJ, Xiao M, Balint K, Soma A, Pinnix CC, Capobianco AJ, et al. Inhibition of endothelial cell proliferation by Notch1 signaling is mediated by repressing MAPK and PI3K/Akt pathways and requires MAML1. Faseb J. 2006;20(7):1009–11. pmid:16571776
- View Article
- PubMed/NCBI
- Google Scholar
41. Choi JH, Hur J, Yoon CH, Kim JH, Lee CS, Youn SW, et al. Augmentation of therapeutic angiogenesis using genetically modified human endothelial progenitor cells with altered glycogen synthase kinase-3beta activity. J Biol Chem. 2004;279(47):49430–8. pmid:15339925
- View Article
- PubMed/NCBI
- Google Scholar
42. Qian CF, Yan W, Zhang JX, Shi L, Qian J, Fu Z, et al. Notch1 induces enhanced expression of Delta-like-1 in the U251MG glioma cell line. Int J Mol Med. 2009;24(4):445–51. pmid:19724883
- View Article
- PubMed/NCBI
- Google Scholar
43. Lassaletta AD, Elmadhun NY, Burgess TA, Bianchi C, Sabe AA, Robich MP, et al. Microvascular notch signaling is upregulated in response to vascular endothelial growth factor and chronic myocardial ischemia. Circ J. 2014;78(3):743–51. pmid:24366099
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science. 1999;284(5415):770–6. pmid:10221902
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Varnum-Finney B, Xu L, Brashem-Stein C, Nourigat C, Flowers D, Bakkour S, et al. Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nat Med. 2000;6(11):1278–81. pmid:11062542
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Iso T, Hamamori Y, Kedes L. Notch signaling in vascular development. Arterioscler Thromb Vasc Biol. 2003;23(4):543–53. pmid:12615665
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Joutel A, Corpechot C, Ducros A, Vahedi K, Chabriat H, Mouton P, et al. Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature. 1996;383(6602):707–10. pmid:8878478
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Li L, Krantz ID, Deng Y, Genin A, Banta AB, Collins CC, et al. Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1. Nat Genet. 1997;16(3):243–51. pmid:9207788
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Vercauteren SM, Sutherland HJ. Constitutively active Notch4 promotes early human hematopoietic progenitor cell maintenance while inhibiting differentiation and causes lymphoid abnormalities in vivo. Blood. 2004;104(8):2315–22. pmid:15231576
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Walker L, Carlson A, Tan-Pertel HT, Weinmaster G, Gasson J. The notch receptor and its ligands are selectively expressed during hematopoietic development in the mouse. Stem Cells. 2001;19(6):543–52. pmid:11713346
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Karanu FN, Murdoch B, Miyabayashi T, Ohno M, Koremoto M, Gallacher L, et al. Human homologues of Delta-1 and Delta-4 function as mitogenic regulators of primitive human hematopoietic cells. Blood. 2001;97(7):1960–7. pmid:11264159
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, Knight MC, et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature. 2003;425(6960):841–6. pmid:14574413
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275(5302):964–7. pmid:9020076
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Aoki M, Yasutake M, Murohara T. Derivation of functional endothelial progenitor cells from human umbilical cord blood mononuclear cells isolated by a novel cell filtration device. Stem Cells. 2004;22(6):994–1002. pmid:15536190
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Asahara T, Takahashi T, Masuda H, Kalka C, Chen D, Iwaguro H, et al. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. Embo J. 1999;18(14):3964–72. pmid:10406801
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Masuda H, Kalka C, Takahashi T, Yoshida M, Wada M, Kobori M, et al. Estrogen-mediated endothelial progenitor cell biology and kinetics for physiological postnatal vasculogenesis. Circ Res. 2007;101(6):598–606. pmid:17656679
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Urbich C, Aicher A, Heeschen C, Dernbach E, Hofmann WK, Zeiher AM, et al. Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells. J Mol Cell Cardiol. 2005;39(5):733–42. pmid:16199052
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Kwon SM, Eguchi M, Wada M, Iwami Y, Hozumi K, Iwaguro H, et al. Specific Jagged-1 signal from bone marrow microenvironment is required for endothelial progenitor cell development for neovascularization. Circulation. 2008;118(2):157–65. pmid:18591437
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Ii M, Takeshita K, Ibusuki K, Luedemann C, Wecker A, Eaton E, et al. Notch signaling regulates endothelial progenitor cell activity during recovery from arterial injury in hypercholesterolemic mice. Circulation. 2010;121(9):1104–12. pmid:20176991
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. Kamei N, Kwon SM, Ishikawa M, Ii M, Nakanishi K, Yamada K, et al. Endothelial progenitor cells promote astrogliosis following spinal cord injury through Jagged1-dependent Notch signaling. J Neurotrauma. 2012;29(9):1758–69. pmid:22452482
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Ando K, Nakamura Y, Chargui J, Matsuzawa H, Tsuji T, Kato S, et al. Extensive generation of human cord blood CD34(+) stem cells from Lin(-)CD34(-) cells in a long-term in vitro system. Exp Hematol. 2000;28(6):690–9. pmid:10880755
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. Morita S, Kojima T, Kitamura T. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther. 2000;7(12):1063–6. pmid:10871756
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref20] 20. Schroeder T, Kohlhof H, Rieber N, Just U. Notch signaling induces multilineage myeloid differentiation and up-regulates PU.1 expression. J Immunol. 2003;170(11):5538–48. pmid:12759431
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref21] 21. Masuda H, Alev C, Akimaru H, Ito R, Shizuno T, Kobori M, et al. Methodological development of a clonogenic assay to determine endothelial progenitor cell potential. Circ Res. 2011;109(1):20–37. pmid:21566217
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref22] 22. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A. 2000;97(7):3422–7. pmid:10725398
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref23] 23. Iwaguro H, Yamaguchi J, Kalka C, Murasawa S, Masuda H, Hayashi S, et al. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration. Circulation. 2002;105(6):732–8. pmid:11839630
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref24] 24. Masuda H, Tanaka R, Fujimura S, Ishikawa M, Akimaru H, Shizuno T, et al. Vasculogenic conditioning of peripheral blood mononuclear cells promotes endothelial progenitor cell expansion and phenotype transition of anti-inflammatory macrophage and T lymphocyte to cells with regenerative potential. J Am Heart Assoc. 2014;3(3):e000743. pmid:24965023
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref25] 25. Jaffredo T, Gautier R, Eichmann A, Dieterlen-Lievre F. Intraaortic hemopoietic cells are derived from endothelial cells during ontogeny. Development. 1998;125(22):4575–83. pmid:9778515
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref26] 26. Wada M, Ebihara Y, Ma F, Yagasaki H, Ito M, Takahashi T, et al. Tunica interna endothelial cell kinase expression and hematopoietic and angiogenic potentials in cord blood CD34+ cells. Int J Hematol. 2003;77(3):245–52. pmid:12731667
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref27] 27. Suzuki T, Chiba S. Notch signaling in hematopoietic stem cells. Int J Hematol. 2005;82(4):285–94. pmid:16298816
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref28] 28. Lauret E, Catelain C, Titeux M, Poirault S, Dando JS, Dorsch M, et al. Membrane-bound delta-4 notch ligand reduces the proliferative activity of primitive human hematopoietic CD34+CD38low cells while maintaining their LTC-IC potential. Leukemia. 2004;18(4):788–97. pmid:14990974
View Article
PubMed/NCBI
Google Scholar

[110] View Article

[111] PubMed/NCBI

[112] Google Scholar

[ref29] 29. Delaney C, Varnum-Finney B, Aoyama K, Brashem-Stein C, Bernstein ID. Dose-dependent effects of the Notch ligand Delta1 on ex vivo differentiation and in vivo marrow repopulating ability of cord blood cells. Blood. 2005;106(8):2693–9. pmid:15976178
View Article
PubMed/NCBI
Google Scholar

[114] View Article

[115] PubMed/NCBI

[116] Google Scholar

[ref30] 30. Huppert SS, Le A, Schroeter EH, Mumm JS, Saxena MT, Milner LA, et al. Embryonic lethality in mice homozygous for a processing-deficient allele of Notch1. Nature. 2000;405(6789):966–70. pmid:10879540
View Article
PubMed/NCBI
Google Scholar

[118] View Article

[119] PubMed/NCBI

[120] Google Scholar

[ref31] 31. Hrabe de Angelis M, McIntyre J 2nd, Gossler A. Maintenance of somite borders in mice requires the Delta homologue DII1. Nature. 1997;386(6626):717–21. pmid:9109488
View Article
PubMed/NCBI
Google Scholar

[122] View Article

[123] PubMed/NCBI

[124] Google Scholar

[ref32] 32. Gale NW, Dominguez MG, Noguera I, Pan L, Hughes V, Valenzuela DM, et al. Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development. Proc Natl Acad Sci U S A. 2004;101(45):15949–54. pmid:15520367
View Article
PubMed/NCBI
Google Scholar

[126] View Article

[127] PubMed/NCBI

[128] Google Scholar

[ref33] 33. Limbourg A, Ploom M, Elligsen D, Sorensen I, Ziegelhoeffer T, Gossler A, et al. Notch ligand Delta-like 1 is essential for postnatal arteriogenesis. Circ Res. 2007;100(3):363–71. pmid:17234965
View Article
PubMed/NCBI
Google Scholar

[130] View Article

[131] PubMed/NCBI

[132] Google Scholar

[ref34] 34. Dou GR, Wang YC, Hu XB, Hou LH, Wang CM, Xu JF, et al. RBP-J, the transcription factor downstream of Notch receptors, is essential for the maintenance of vascular homeostasis in adult mice. Faseb J. 2008;22(5):1606–17. pmid:18096813
View Article
PubMed/NCBI
Google Scholar

[134] View Article

[135] PubMed/NCBI

[136] Google Scholar

[ref35] 35. Sainson RC, Aoto J, Nakatsu MN, Holderfield M, Conn E, Koller E, et al. Cell-autonomous notch signaling regulates endothelial cell branching and proliferation during vascular tubulogenesis. Faseb J. 2005;19(8):1027–9. pmid:15774577
View Article
PubMed/NCBI
Google Scholar

[138] View Article

[139] PubMed/NCBI

[140] Google Scholar

[ref36] 36. Buchler P, Gazdhar A, Schubert M, Giese N, Reber HA, Hines OJ, et al. The Notch signaling pathway is related to neurovascular progression of pancreatic cancer. Ann Surg. 2005;242(6):791–800, discussion -1. pmid:16327489
View Article
PubMed/NCBI
Google Scholar

[142] View Article

[143] PubMed/NCBI

[144] Google Scholar

[ref37] 37. Liu H, Kennard S, Lilly B. NOTCH3 expression is induced in mural cells through an autoregulatory loop that requires endothelial-expressed JAGGED1. Circ Res. 2009;104(4):466–75. pmid:19150886
View Article
PubMed/NCBI
Google Scholar

[146] View Article

[147] PubMed/NCBI

[148] Google Scholar

[ref38] 38. Bentley K, Gerhardt H, Bates PA. Agent-based simulation of notch-mediated tip cell selection in angiogenic sprout initialisation. J Theor Biol. 2008;250(1):25–36. pmid:18028963
View Article
PubMed/NCBI
Google Scholar

[150] View Article

[151] PubMed/NCBI

[152] Google Scholar

[ref39] 39. Takeshita K, Satoh M, Ii M, Silver M, Limbourg FP, Mukai Y, et al. Critical role of endothelial Notch1 signaling in postnatal angiogenesis. Circ Res. 2007;100(1):70–8. pmid:17158336
View Article
PubMed/NCBI
Google Scholar

[154] View Article

[155] PubMed/NCBI

[156] Google Scholar

[ref40] 40. Liu ZJ, Xiao M, Balint K, Soma A, Pinnix CC, Capobianco AJ, et al. Inhibition of endothelial cell proliferation by Notch1 signaling is mediated by repressing MAPK and PI3K/Akt pathways and requires MAML1. Faseb J. 2006;20(7):1009–11. pmid:16571776
View Article
PubMed/NCBI
Google Scholar

[158] View Article

[159] PubMed/NCBI

[160] Google Scholar

[ref41] 41. Choi JH, Hur J, Yoon CH, Kim JH, Lee CS, Youn SW, et al. Augmentation of therapeutic angiogenesis using genetically modified human endothelial progenitor cells with altered glycogen synthase kinase-3beta activity. J Biol Chem. 2004;279(47):49430–8. pmid:15339925
View Article
PubMed/NCBI
Google Scholar

[162] View Article

[163] PubMed/NCBI

[164] Google Scholar

[ref42] 42. Qian CF, Yan W, Zhang JX, Shi L, Qian J, Fu Z, et al. Notch1 induces enhanced expression of Delta-like-1 in the U251MG glioma cell line. Int J Mol Med. 2009;24(4):445–51. pmid:19724883
View Article
PubMed/NCBI
Google Scholar

[166] View Article

[167] PubMed/NCBI

[168] Google Scholar

[ref43] 43. Lassaletta AD, Elmadhun NY, Burgess TA, Bianchi C, Sabe AA, Robich MP, et al. Microvascular notch signaling is upregulated in response to vascular endothelial growth factor and chronic myocardial ischemia. Circ J. 2014;78(3):743–51. pmid:24366099
View Article
PubMed/NCBI
Google Scholar

[170] View Article

[171] PubMed/NCBI

[172] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

CD133+ cell preparation

Flow cytometry analysis

HESS-5 cell line

Retroviruses and producer cell lines

Western blotting

Luciferase assays

Co-culture assays

Reverse transcription-polymerase chain reaction (RT-PCR) analysis

EPC colony-forming assay

Transplantation of EPCs experienced with Notch ligands into ischemic hindlimb in vivo

In vivo physiological and histological assessment

Statistical analysis

Results

Effect of Notch ligands on CD133+ cell numbers

Effect of Notch ligands on EPC phenotypic differentiation

Effect of Notch ligands on EPC colony formation

Transplantation of hJagged-1-stimulated CD133+ cells into mouse ischemic hindlimbs

Differentiation of transplanted cells stimulated by Notch ligands in ischemic muscles in vivo

Discussion

Supporting Information

S1 Fig. Histological evaluation of human endothelial cell development in vivo.

S1 Table. Sequences of PCR primers for hVEGF-1, heNOS and hGAPDH, product sizes, and PCR conditions.

Acknowledgments

Author Contributions

References

CD133⁺ cell preparation

Effect of Notch ligands on CD133⁺ cell numbers

Transplantation of hJagged-1-stimulated CD133⁺ cells into mouse ischemic hindlimbs