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

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.


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][8][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][13][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.

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 × 10 8 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.

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 × 10 3 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% CO 2 . 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.

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. Antibodylabeled 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).

Reverse transcription-polymerase chain reaction (RT-PCR) analysis
Total RNA was obtained from cultured CD133 + CB cells using an RNeasy Micro Kit (QIA-GEN 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).

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 × 10 5 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 Zenon1 Alexa Fluor1 594 Mouse IgG1 Labeling Kit and then an Alexa Fluor1488 Mouse IgG2a Labeling Kit (Molecular Probes, Karlsruhe, Germany), and then the labeled antibodies were applied for 2 hours. Nuclear counterstaining was performed with 4 0 -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.

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 + /CD38hematopoietic 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).
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.

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).

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).

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. Jagged-1 Promotes EPC Expansion and Commitment of CB CD133 + Cells  Fig 6. In vivo effects of transplanted EPCs stimulated by Notch ligands in mouse ischemic hindlimbs. CD133 + cells (1 × 10 5 ) 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. 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][31][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][31][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 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 × 10 5 ) 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. doi:10.1371/journal.pone.0166660.g007 Jagged-1 Promotes EPC Expansion and Commitment of CB CD133 + Cells 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.