Identification of mouse colony-forming endothelial progenitor cells for postnatal neovascularization: a novel insight highlighted by new mouse colony-forming assay
- Shizu Tsukada†1,
- Sang-Mo Kwon†2,
- Takenori Matsuda3,
- Seok-Yun Jung2,
- Jun-Hee Lee2,
- Sang-Hun Lee2,
- Haruchika Masuda1 and
- Takayuki Asahara1, 3Email author
© Tsukada et al.; licensee BioMed Central Ltd. 2013
Received: 14 June 2012
Accepted: 9 January 2013
Published: 28 February 2013
Endothelial progenitor cells (EPCs) play a critical role in restoration of ischemic diseases. However, the actual status of EPC development and the mechanisms of EPC dysfunctions in patients with various ischemic diseases remain unknown.
To investigate the detailed function of EPCs in experimental murine models, we have established an EPC colony forming assay (EPC-CFA) in murine EPCs. The abilities of murine EPCs in differentiation, adhesive capacity, proliferative potency, and transplantation in vitro and in vivo were then examined.
Peripheral blood mononuclear cells (PB-MNCs), bone marrow mononuclear cells (BM-MNCs) or bone marrow c-Kit+/Sca-1+ lineage negative (BM-KSL) cells differentiated into two types of EPC colony forming units (EPC-CFUs), large sized EPC (large-EPC)-CFUs and small sized EPC (small-EPC)-CFUs. Gene expression analysis demonstrated that both EPC-CFU-derived cells expressed eNOS, Flk-1 and VE-cadherin, markers of endothelial cells (ECs), although the small-EPCs derived from small-EPC-CFU were higher in number and showed more immature features (higher population of KSL cells). Functionally, the large-EPCs derived from large-EPC-CFU had higher adhesive capacity but lower proliferative potency than small-EPCs, showing improved tubular forming capacity and incorporation potency into primary EC-derived tube formation. Importantly, hindlimb ischemia increased the frequencies of large-EPC-CFUs differentiated from PB-MNCs and bone marrow. Actually, transplantation of large-EPCs into ischemic hindlimb enhanced neovascularization in hindlimb ischemia model, although small-EPCs or murine ECs did not, suggesting that large-EPC-CFUs might play an important role in restoration of ischemic diseases.
We demonstrated, using a murine ischemia model, that the EPC-CFA could be a useful way to investigate the differentiation levels of murine EPCs, further providing a crucial clue that large-EPC-CFU status may be more functional or effective EPCs to promote neovascularization.
Endothelial progenitor cells (EPCs) [1–3] play an important role in the restoration of ischemic vascular diseases [2–5]. Recently, several independent groups have shown that transplantation of EPCs into ischemic hindlimb or myocardial tissue improves organ function following the growth of new vessels [6–11]. In clinical aspects, the frequency of circulating EPCs may also serve as a biomarker for vascular function, and the number of circulating EPCs has been reported to be reduced in patients with diabetes mellitus or risk factors for coronary artery disease and to negatively correlate with the Framingham cardiovascular risk score [12–15]. However, the actual mechanical status of EPC development and the 'evaluation system' for EPC dysfunctions in patients with various ischemic diseases remain to be disclosed.
Because EPCs accumulate in ischemic injured tissues and repair injured tissue following cluster formation [1, 2, 9], not only the number of EPCs identified by uptake of acetylated-low density lipoprotein (acLDL) and lectin reactivity but also the colony-forming potential of EPCs is important for angiogenic therapy. Thus, the assay system in which colony-forming potential of EPCs can be assessed is important. EPCs should encompass a group of cells existing in a variety of stages, ranging from hemangioblastic hematopoietic stem cells to fully differentiated endothelial cells (ECs), and EPCs can be classified into stages according to differentiation levels in each circulating EPC and tissue EPC . Recently, the methods to culture colony-forming unit-endothelial cells (CFU-ECs)  or to culture endothelial colony-forming cells (ECFCs) were established on mononuclear cells from peripheral blood or cord blood [17–20]. However, it was reported that CFU-ECs were not EPCs but were myeloid cells that differentiate into phagocytic macrophages and that T cells could mimic the morphology of CFU-ECs [19, 21]. Besides, the culture of ECFCs enables us to evaluate the EPC colony-forming potential change as EPCs differentiated during culture in vitro. In these assay systems, each EPC at different differentiation levels could not be discriminated at the same time, and the differentiation capacities of immature stem cells could not be tested. In our laboratory, EPC-CFA, a novel method to assess the colony-forming potential of EPCs at different differentiation levels, was recently established and enables us to investigate the commitment of each cell [22–24].
In the present study, we aimed to methodologically establish the murine EPC-CFA on PB-MNCs, BM-MNCs, or BM-KSL cells by analyzing the functions of each EPC-CFU at different differentiation levels and to clarify the roles of each EPC-CFU at different differentiation levels in vivo by using hindlimb ischemic mice. By EPC-CFA, we investigated the status of EPC differentiation in response to ischemic signals and the effects of two types of EPC-CFUs - small-EPC-CFUs or large-EPC-CFUs - in a hindlimb ischemia model on in vivo neovascularization.
Materials and methods
Experiments were performed on male 8- to 10-week-old C57BL/6J mice and BALB/CA-nu/nu mice (Japan Clea, Tokyo, Japan) maintained under a 12-hour light/dark cycle and in accordance with the regulations of Tokai University. Standard laboratory chow and water were available ad libitum. The protocols were approved by guidelines of 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) (Institutional Review Board ID number 083005).
Peripheral blood was obtained from the heart immediately before sacrifice and was separated by Histopaque-1083 (Sigma-Aldrich, St. Louis, MO, USA) density gradient centrifugation, as previously described . Briefly, low-density mononuclear cells were harvested and washed twice with Dulbecco's phosphate-buffered saline (PBS) supplemented with 2 mmol/L ethylenediaminetetraacetic acid (EDTA). Contaminated red blood cells were hemolyzed by using ammonium chloride solution. BM-MNCs were obtained by flushing the femurs and tibias and reacted with a mixture of biotinylated monoclonal antibodies against B220 (RA3-6B2), CD3 (145-2C11), CD11b (M1/70), TER-119 (Ly-76), and Gr-1 (RB6-8C5) (all from BD Pharmingen, San Diego, CA, USA) as lineage markers to deplete lineage-positive cells from BM-MNCs by using AutoMACS (Becton Dickinson, Franklin Lakes, NJ, USA). Lineage-negative bone marrow cells (BM-LNneg) were incubated with saturating concentrations of directly labeled anti-c-Kit (at 1:25 dilution) (BD Biosciences, Franklin Lakes, NJ, USA) and anti-Sca-1 antibodies (at 1:25 dilution) (BD Biosciences) for 30 minutes on ice, and then the c-Kit+/Sca-1+ lineage-negative cells (BM-KSL) were isolated with live sterile cell sorting (FACSVantage SE; Becton Dickinson).
Endothelial progenitor cell colony-forming assay
Various cells were cultured in methylcellulose-containing medium M3236 (StemCell Technologies, Vancouver, BC, Canada) with 20 ng/mL stem cell-derived factor (Kirin, Tokyo, Japan), 50 ng/mL vascular endothelial (VE) growth factor (R&D Systems, Minneapolis, MN, USA), 20 ng/mL interleukin-3 (Kirin), 50 ng/mL basic fibroblast growth factor (Wako, Osaka, Japan), 50 ng/mL epidermal growth factor receptor (Wako), 50 ng/mL insulin-like growth factor-1 (Wako), 2 U/mL heparin (Ajinomoto, Tokyo, Japan), and 10% fetal bovine serum (FBS) on a 35-mm dish for 8 days. Cell densities for each sample were as follows: PB-MNCs 7 × 105 cells per dish, BM-MNCs 1 × 104 cells per dish, BM-LNneg 2.5 × 103 cells per dish, and BM-KSL 500 cells per dish. The EPC-CFUs were identified as large-EPC-CFUs or small-EPC-CFUs by visual inspection with an inverted microscope under 40× magnification. Large-EPC-CFUs were composed of spindle-shaped cells, and small-EPC-CFUs were composed of round adhesive cells.
Endothelial progenitor cell-colony-forming unit staining
After 8 days in culture, the EPC-CFU cultures were treated with 0.4 μg/mL 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate-labeled acLDL (acLDL-DiI; Biomedical Technologies Inc., Stoughton, MA, USA) for 1 hour and fixed by application of 1 mL of 2% paraformaldehyde (PFA) for 1 hour at room temperature. After a wash of the methylcellulose-containing medium with PBS, the cultures were reacted with fluorescein isothiocyanate (FITC)-conjugated BS-1 lectin (Sigma-Aldrich) for 1 hour at room temperature. After a wash with PBS, the cultures were observed under a fluorescence microscope (IX70; Olympus, Tokyo, Japan).
Large-endothelial progenitor cell or small-endothelial progenitor cell isolation
Cells composed of small-EPC-CFUs were collected with a pipette under a microscope as small-EPCs. Then the cultures were washed with PBS, and large-EPCs were harvested after treatment with 2 mmol/L EDTA/PBS. For the purpose of cell transplantation into a hindlimb ischemia model, non-attached cells were isolated as small-EPCs by washing with PBS, whereas attached cells were harvested as large-EPCs by treatment with EDTA/PBS (5 mmol/L) for 5 minutes at 37°C.
Culture plates (24-well) were coated with human fibronectin (100 μg/mL; Gibco, now part of Invitrogen Corporation, Carlsbad, CA, USA). Large-EPCs or small-EPCs (2 × 104 cells per well) were allowed to attach in EGM-2 (Cambrex Bio Science Walkersville, Walkersville, MD, USA) for 20 minutes at 37°C, and the non-adherent cells were aspirated. The adherent population was fixed with 1% PFA for 20 minutes and stored in PBS. The numbers of adherent cells were quantified from counts in six random microscopic fields per well.
At day 7, EPC-CFU cultures were treated with 10 μmol/L bromodeoxyuridine (BrdU) (Sigma-Aldrich) and incubated for 24 hours. BrdU positivities of large-EPCs or small-EPCs were analyzed by using BrdU flow kits (BD Pharmingen) and a fluorescence-activated cell sorter, as previously described .
Tubular formation assay
Two-week derived CD133- mononuclear cells of human cord blood were used as ECs. These cells were confirmed to be ECs by tubular formation and immunocytochemistry of endothelial nitric oxide synthase (eNOS), kinase insert domain receptor (KDR), and VE-cadherin (data not shown) . Each small-EPC or large EPC was labeled with acLDL-DiI for 1 hour. After washing of the labeled small-EPCs or large-EPCs with PBS, the 1 × 103 cells were mixed together with 1.2 × 104 ECs in 50 μL of 2% FBS/EBM-2. Cell suspension (50 μL) was applied onto 50 μL of Matrigel (BD, Franklin Lakes, NJ, USA) per well of a 96-well plate (BD Falcon, Franklin Lakes, NJ, USA) and then incubated for 8 hours. After incubation, the numbers of tubular formation were counted on a display of Photoshop software (Adobe, San Jose, CA, USA) after a picture per well was taken at 40× magnification under a light microscope (Eclipse TE300; Nikon, Tokyo, Japan). The numbers of incorporated labeled cells into tubes were also counted on a display of Photoshop software after a picture per well was taken at 100× magnification under a fluorescence microscope.
Isolated small-EPCs (5 × 104) were suspended in 50 μL of Iscove's modified Dulbecco's medium (IMDM) (Gibco) and applied onto 100 μL of methylcellulose-containing medium per well of a 96-well plate (BD Falcon). After 2 days of incubation, methylcellulose-containing medium was changed to IMDM containing acLDL-DiI and BS-1 lectin-conjugated FITC and then incubated for 1 hour. After a wash with PBS, cultures were observed under a fluorescence microscope.
Reverse transcription-polymerase chain reaction
Total RNA of small-EPCs or large-EPCs was prepared with an RNeasy Micro/Mini kit (Qiagen, Valencia, CA, USA). Reverse transcription-polymerase chain reaction (RT-PCR) was performed by using Superscript III Reverse Transcriptase (Invitrogen Corporation) with 1 μg of total RNA. PCR amplification was then performed with synthetic gene-specific primers for eNOS (forward primer, 5'-GGATTGTGTCACTTCGTTCGGT-3'; reverse primer, 5'-CAGCAGGATGCCCTAACTACCA-3'; product length, 183 base pairs (bp)), Flk-1 (forward primer, 5'-AAAGAGAGGAACGTCGGCAGA-3'; reverse primer, 5'-AAGCACACAGGCAGAAACCAGT-3'; product length, 376 bp), VE-cadherin (forward primer, 5'-AGATTCACGAGCAGTTGGTCA-3'; reverse primer, 5'-GATGTCAGAGTCGGAGGAATT-3'; product length, 355 bp), and β-actin (forward primer, 5'-AACACCCCAGCCATGTACGTA-3'; reverse primer, 5'-AAGGAAGGCTGGAAAAGAGCC-3'; product length, 416 bp) by using exTaq polymerase (Takara, Kyoto, Japan). To quantify transcripts, semi-quantitative RT-PCRs were performed and normalized to Actb, which encodes β-actin. PCRs were performed at 94°C for 45 seconds, 64°C for 1 minute, and 72°C for 1 minute for 35 or 33 or 22 cycles and analyzed on 2% agarose gels.
For flow cytometry analysis, we used monoclonal antibodies specific to Sca-1 and c-Kit. BM-LNneg- or EPC-CFU-derived cells were incubated with directly labeled anti-Sca-1 (at 1:100 dilution) and anti-c-Kit (at 1:100 dilution) antibodies for 30 minutes on ice. The cells were analyzed by two-color flow cytometry by using a FACS caliber (Becton Dickinson).
Animal model of ischemic hindlimb
Unilateral hindlimb ischemia was created in C57BL/6J mice or BALB/CA-nu/nu as previously described . Briefly, the animals were anesthetized with Nembutal (60 mg/kg intraperitoneally; Dainippon Sumitomo Pharma Co., Osaka, Japan) and then an incision in the skin overlying the middle portion of the left hindlimb was performed. After ligation of the proximal end of the femoral artery, the distal portion of the saphenous artery was ligated and the artery, as well as all side branches, was dissected free and excised. The skin was closed by using a surgical stapler.
Monitoring of hindlimb blood flow
After anesthesia, hindlimb perfusion was measured by using a laser Doppler perfusion imaging system (LDPI; Moor Instruments, Wilmington, DE, USA). The stored perfusion values behind the color-coded pixels representing the microvascular blood flow distribution are available for analysis. Color photographs were recorded and analysis performed by calculating the average perfusion of the ischemic and non-ischemic foot. To account for variables such as ambient light and temperature, the results are expressed as the ratio of perfusion in the left (ischemic) versus right (normal) limb. In the EPC transplantation experiment, isolated small-EPCs, large-EPCs, or murine ECs (2.5 × 105) derived from the aorta of C57BL/6J were transplanted into hindlimb induced nude mice by intramuscular injection, respectively (n = 8).
Measuring of capillary density
Twenty-eight days after ischemia, capillary density was determined in tissue sections from the lower calf muscles of ischemic and healthy limbs by expressed as number of CD31+ cells as ECs per myocyte. To stain the capillary, we performed a staining procedure with rat anti-mouse CD31 antibodies (BD Biosciences) or Alexa-fluor 594 (Molecular Probes, now part of Invitrogen Corporation) anti-iso-lectin B4 reagents (Sigma-Aldrich).
All data were presented as mean ± standard deviation. P values were calculated by using the unpaired Student t test. For the analysis of in vivo ischemia experiments, the Scheffe's test was performed for the multiple comparisons after analysis of variance between each group. A P value of less than 0.05 was considered statistically significant.
Development of murine endothelial progenitor cell colony-forming assay
Characterization of large endothelial progenitor cells or small endothelial progenitor cells
Importance of small-endothelial progenitor cells as large-endothelial progenitor cell-colony-forming unit sprouting cells
Kinetics of endothelial progenitor cell-colony-forming units in response to ischemia
Contribution of large-endothelial progenitor cells or small-endothelial progenitor cells to postnatal/adult neovascularization
EPCs can be classified into various differentiation levels in both circulating EPCs and tissue EPCs . Here, we first defined in vivo EPC status by establishing the novel murine EPC-CFA, in which the colony-forming potential of EPCs at different differentiation levels can be assessed. We demonstrated, for the first time, that hindlimb ischemia induced onsets of large-EPCs, which might be the accelerated differential status of EPCs. The observation was further supported by an in vivo experiment in which transplantation of more mature large-EPCs into a hindlimb ischemia model enhanced neovascularization, implying the contribution of large-EPC-CFUs in a pathogenic situation as 'cells ready to operate'.
Two types of EPC-CFUs represented distinct functional differences in both in vitro EPC colony study and in vivo EPC transplantation study. The adhesive potential and the incorporation into tubes formed by EC-like cells of large-EPCs were superior to those of small-EPCs, and small-EPCs had higher proliferation capacity than large-EPCs, which was consistent with the data on EPC-CFUs from BM-LNneg and BM-KSL (data not shown). In these points, definitive large-EPCs had similar functions to ECs compared with primitive small-EPCs. Besides, the secondary culture revealed that small-EPCs could differentiate into adherent cell, large-EPCs; in contrast, large-EPCs could not differentiate into round cell, small-EPCs (data not shown). These data showed that definitive large-EPCs are well-differentiated EPCs compared with primitive small-EPCs (Figure 1e). VE-cadherin is specifically expressed in adherent junctions of ECs and exerts important functions in cell-cell adhesion . The different expression level of VE-cadherin between large-EPCs and small-EPCs might explain the better potential of adhesion, incorporation into tubes, and migration (data not shown) of definitive large-EPCs than those of primitive small-EPCs, which were consistent with our recent findings using human cord blood AC133+ cells . Gene expression profiles revealed that both EPC-CFUs were committed to endothelial lineage because both definitive large-EPCs and primitive small-EPCs expressed eNOS, Flk-1, and VE-cadherin, which are EC-specific markers . However, both EPC-CFUs would be different from mature ECs in terms of colony formation capacity, tubular formation ability, and contribution of in vivo neovascularization, demonstrated by ischemia model, because ECs could not form colonies and did not have an effect on the restoration of blood vessels, and EPC-CFU-derived cells could not form tubes on Matrigel in a culture without ECs.
In this EPC-CFA, to compare the potentials to produce EPC-CFUs of three populations in BM (BM-MNCs, BM-LNneg, and BM-KSL), we calculated the numbers of cells producing one EPC-CFU in BM-MNC, BM-LNneg, and BM-KSL populations. It was revealed that one large-EPC-CFU was derived from 1.1 × 103 ± 0.2 × 103 BM-MNCs or 3.6 × 102 ± 1.1 × 102 BM-LNneg or 57 ± 34 BM-KSL (Figure 1b). One small-EPC-CFU was derived from 5.5 × 102 ± 0.7 × 102 BM-MNCs or 1.2 × 102 ± 0.2 × 102 BM-LNneg or 28 ± 3 BM-KSL (Figure 1b). These data demonstrated that BM-LNneg had 3- or 4.6-fold higher potential to produce large- or small-EPC-CFUs than BM-MNCs, respectively, and this suggested that more immature EPCs were contained mainly in the BM-LNneg population. In addition, it was demonstrated that BM-KSL had the highest potential to produce EPC-CFUs in any other populations in BM, and those potentials to produce large- or small-EPC-CFUs were 6.3- or 4.3-fold higher than BM-LNneg, respectively, and this suggested that immature EPCs were highly enriched in the BM-KSL population. In this study, using EPC-CFA, we determined that BM-KSL was the major population which highly enriched immature EPCs. We concluded, in this paper, that small-EPCs differentiated into large-EPCs because BM-KSL grew into small-EPCs about 5 days after plating and then those small-EPCs derived from BM-KSL could differentiate into large-EPCs in further culture. In our study, it remained unclear which niche component does small- or large-EPC differentiate from'. This should be definitely addressed in further issues.
In a clinical setting, the frequency of circulating EPCs serves as a biomarker for vascular function, and the number of circulating EPCs has been reported to be reduced in patients with diabetes mellitus or risk factors for coronary artery disease and to negatively correlate with the Framingham cardiovascular risk score [12–15]. Transplantation of EPCs into ischemic hindlimb or myocardial tissue improves organ function following new vessel growth [6–10]. Thus, EPCs play an important role in the restoration of ischemic vascular diseases. But essential molecular events that control the differentiation to EPCs and changes in EPCs in response to ischemia had not been clarified yet. Then we investigated the changes of EPCs in response to hindlimb ischemia in EPC-CFA and revealed that the population of KSL, which enriched immature EPC populations in BM, increased by ischemia. In previous studies, it was demonstrated that BM-derived EPCs were mobilized in response to tissue ischemia . In this study, we showed, for the first time, that the ischemic signals could promote the differentiation of PB-MNCs, BM-MNCs, or BM-KSL cells into mature EPC-CFUs. Ischemia-induced differentiation into large-EPC-CFUs suggested that definitive large-EPC-CFUs as more mature EPCs might play an important role in the restoration of ischemic tissue, and this possibility was supported by the recovery of limb perfusion by transplantation of BM-KSL-derived large-EPCs into a hindlimb ischemia model compared with small-EPCs. In ischemic tissue, the expression of stromal cell-derived factor-1 (SDF-1) was induced by transcription factor hypoxia-inducible factor-1 (HIF-1) according to hypoxic gradients [27, 28]. SDF-1 enhances differentiation of BM-derived c-Kit+ stem cells into EPCs . Thus, the EPC differentiation presented above might be promoted by SDF-1, which is induced by HIF-1 after ischemia.
Our novel findings highlighted the actual status of EPCs via a redefinition of the differential stages of EPCs through BM-derived stem cells using our established murine EPC-CFA. The understanding of molecular cascades of EPC development from primitive small-EPC-CFUs to definitive large-EPC-CFUs will provide us some useful therapeutic advantages to solve the quantitative or qualitative problems for EPCs therapy.
acetylated-low density lipoprotein
1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate-labeled acetylated-low density lipoprotein
bone marrow c-Kit+/Sca-1+ lineage-negative
lineage-negative bone marrow cell
bone marrow mononuclear cell
colony-forming unit-endothelial cell
endothelial colony-forming cell
endothelial nitric oxide synthase
endothelial progenitor cell
endothelial progenitor cell colony-forming assay
endothelial progenitor cell colony-forming unit
fetal bovine serum
Iscove's modified Dulbecco's medium
- large EPC-1:
large endothelial progenitor cell derived from small endothelial progenitor cell
peripheral blood mononuclear cell
polymerase chain reaction
reverse transcription-polymerase chain reaction
We thank Rie Ito and Michiru Kobori for providing technical assistance and Sachie Ota for secretarial assistance. We thank all of our colleagues for helpful discussions and encouragement. This work was supported by the Academic Frontier Promotion Program of the Ministry of Education, Culture, Sports, Science, and Technology in Japan; a National Research Foundation grant funded by the Korean government (MEST) (2010-0020260); and the Basic Science Research Program of the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (2010-0003132).
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