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a Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan;
b R&D Laboratories,Asahi Kasei Medical Co., Ltd., Tokyo, Japan
Key Words. Angiogenesis • Cord blood • Endothelium • Progenitor cell
Correspondence: Toyoaki Murohara, M.D., Ph.D., Department of Cardiology, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan. Telephone: 81-52-744-2149; Fax: 81-52-744-2157; e-mail: muro-hara{at}med.nagoya-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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Transplantation of EPCs or bone marrow mononuclear cells (MNCs) containing EPCs into severely ischemic tissues has been shown to induce angiogenesis and to increase functional blood supply [7,9]. This strategy is termed therapeutic angiogenesis by cell transplantation. However, the method of the isolation of EPCs is still complicated. In particular, isolation of MNCs, the initial step of cell isolation, requires several centrifuge processes of blood samples through a Ficoll density gradient [7]. These methods are often time-consuming and are accompanied by a higher chance of contamination. To overcome these issues, we recently developed a novel MNC isolation device for the processing of human cord blood (StemQuickTME, Asahi Kasei Medical, Tokyo, Japan) [10,11]. This filter system was proven to be easy in handling, and the blood processing was performed within a completely closed system inside an ordinary clean bench. However, there has been no report demonstrating that any cell filter–processed human MNC could give rise to EPCs. Accordingly, we examined whether functional angiogenic EPCs would differentiate from culture of MNCs obtained by our new cell-filtration device, StemQuickTME.
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MATERIALS AND METHODS |
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Isolation of MNCs
We isolated MNCs from human cord blood using two different methods. First, we used the StemQuickTME filter. This device has a spike needle for connection to a primary blood bag containing the cord blood sample, resulting in the blood being processed within a completely closed system (Fig. 1
). The StemQuickTME comprises two closed bags with an integrated mononuclear cell filter for the enrichment of stem cells [10,11]. After connection of the primary cord blood bag to the system, the bag was hung 45 cm over the surface of the bench, and blood flowed by gravity down through the filter fibers to the drain bag. After rinsing with 40 ml saline, MNCs were first trapped within a filter during the filtration of the blood, whereas red blood cells (RBCs) and platelets passed through the filter pores into a drain bag. In the second step, MNCs trapped within the filter were recovered by a retrograde flushing with 19-ml solution supplemented with 20% human serum albumin (Aventis Pharma Japan, Tokyo, Japan) and 16 ml of Dextran 40 (Kobayashi Pharmaceutical, Osaka, Japan), along with 18 ml of sterile air rapidly through a port at the outlet of the filter to the recovery bag. For the elution procedure, a syringe filled with the recovery solution was pressed out at a constant speed and force by a special recovery device. Isolated cells were counted using an automatic cell counter SF-3000 (Sysmex, Tokyo, Japan).
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The preprocessing and postprocessing complete blood count and white blood cell (WBC) analysis were examined on five additional cord blood samples. We calculated the recovery rates of WBCs and MNCs of each method and compared them between the StemQuickTME filtration method and the Ficoll centrifuge method. The recovery rate was calculated by the following formula: recovery rate (%) = [total number of WBCs (MNCs) in final MNC suspension / total number of WBCs (MNCs) in original whole blood that was subjected to cell isolation] x 100. MNC fraction was defined as monocytes plus lymphocytes fractions after the composition analysis of WBCs.
We also calculated and compared the reduction rate of RBCs, platelets, and granulocytes between the two isolation methods. The reduction of these blood cells (RBCs, platelets, or granulocytes) was calculated by the following formula: cell reduction (%) = [(total number of cells in original whole blood) – (total number of cells in final MNC suspension)] / (total number of cells in original whole blood that was subjected to MNC isolation) x100.
Flow Cytometric Analysis
Recent studies suggested that circulating EPCs are likely present in a CD34+CD133+ fraction of circulating MNCs in human peripheral blood [12]. We therefore used flow cytometry to analyze the number of CD34+ and CD133+ cells with CD45low in cord blood MNCs isolated by the StemQuickTME filtration method and the Ficoll method. We additionally compared the recovery rates of CD34+ and CD133+ cells with CD45low between the two separation methods. In previous reports, circulating MNCs with CD45lowCD34+CD133+ were quantified as tentative progenitor cells (PCs) giving rise to EPCs [13]. Four milliliters of cord blood was obtained, and WBCs were stained with allophycocyanin-conjugated anti-CD45 monoclonal antibody (mAb) (Caltag Laboratories, Burlingame, CA), PC5-conjugated anti-CD34 mAb (Beckman Coulter, Fullerton, CA), and phycoerythrin (PE)–conjugated anti-CD133 mAb (Miltenyi Biotec, Bergisch Glad-bach, Germany). Samples were subjected to a 2D side-scatter fluorescence dot plot analysis (FACScan, Becton, Dickinson, Frankline Lakes, NJ). After appropriate gating with low cytoplasmic granularity and with low expression of CD45, the numbers of CD45lowCD34+, CD45lowCD133+, and CD45low CD34+CD133+ cells were quantified.
Cell Culture
The medium used for all cell-culture experiments was Medium-199 supplemented with 20% fetal bovine serum, endothelial cell growth supplement, heparin (100 µg/ml), and antibiotics (Life Technologies, Grand Island, NY) (standard medium), as described previously [5,7]. Total MNCs were cultivated on gelatin-coated plastic cell culture plates for 7 days.
Chemical Detection of EPCs
Fluorescent chemical detection of EPCs was performed on EPC-like AT cells after 7 days in culture. Fluorescent staining was used to detect binding of fluorescein isothiocyanate–labeled Ulex europaeus agglutinin (UEA)-1 (Sigma, St. Louis) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine–labeled acetylated low-density lipoprotein (DiI-acLDL; Molecular Probes, Eugene, OR); these are characteristic features of endothelial lineage cells [14]. EPC-like attaching cells were first incubated in medium containing DiI-acLDL (15 µg/ml) for 4 hours at 37°C and then fixed with 1% paraformaldehyde for 10 minutes. After washing, the cells were reacted with UEA-1 (10 µg/ml) for 1 hour. After the staining, cells were examined under fluorescence microscopy (Nikon, Kawasaki, Japan).
ReverseTranscription–Polymerase Chain Reaction
By reverse transcription–polymerase chain reaction (RT-PCR) analysis, we examined the expression of several endothelial lineage genes (i.e., CD31, Lox-1, endothelial nitric oxide synthase [eNOS], KDR, and CD133), as well as a reference housekeeping gene, GAPDH, during the course of the cord blood MNC culture differentiating into EPC-like AT cells. Total RNA was extracted from cells at each time point using a guanidium thiocyanate-phenol chloroform solution (TRIzol, Life Technologies Inc., Gaithersburg, MD), quantified by measuring absorption at 260 nm, and subjected to RT-PCR analysis. Total RNA was reverse transcribed using oligo dT primers and RNAse H–reverse transcriptase (Superscript II; Life Technologies Inc.) with 1 µg of total RNA per sample. Used primer sets and the sizes of produced fragments are listed in Table 1.
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In Vivo Angiogenesis Model: Unilateral Hindlimb Ischemia in Immunodeficient Nude Rats
We finally examined whether culture-expanded EPCs from MNCs isolated by the cell filtration device could participate in capillary formation in the ischemic hindlimb in vivo. For transplantation of human EPCs, immune-suppressed nude rats (F344/N rnu/rnu, Clea, Tokyo, Japan) were used to avoid potential graft-versus-host reactions. Male nude rats (n = 3) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and then the left femoral artery and vein were excised to induce hindlimb ischemia. Rats then received fluorescence (PKH2-GL, Sigma)–labeled EPCs (5 x105 cells/rat) intra-muscularly at the ischemic thigh muscles using 26-gauge needles. Two weeks after the surgery and cell transplantation, animals were euthanized with an overdose of pentobarbital. Ischemic skeletal muscle samples were isolated, embedded, and snap-frozen in OCT compound (Tissue-Tek, Miles, Vista, CA). Six-micrometer multiple frozen sections were prepared from each specimen so that the muscle fibers were oriented in a transverse fashion. Fluorescence microscopy was used to detect transplanted EPCs with fluorescence incorporated into the ischemic tissues.
We next investigated whether local tissue implantation of cord blood–derived EPCs could augment neovascularization in ischemic hindlimbs of nude rats (i.e., therapeutic vasculogenesis) as a functional evaluation. An additional seven nude rats were subjected to unilateral hindlimb ischemia. On the day of the induction of limb ischemia, rats were injected in their ischemic adductor muscle area with human cord blood–derived EPCs (5 x105 cells/animal, n = 3) or saline as a control (n = 4). Regional blood flow was evaluated thereafter using a laser Doppler blood flow (LDBF) analyzer (moorLDI, Moor Instruments Inc., Devon, U.K.). All animal experiments were approved by the Institutional Animal Care and Use Committee.
Laser Doppler Blood Flow Image
We measured the ratio of the ischemic (left) to normal (right) hindlimb blood flow using a LDBF analyzer (moorLDI) [7]. When laser scanning is performed, moving blood cells shift the frequency of the laser light according to the Doppler principle. Change in the frequency is displayed as a color-coded image representing blood flow (i.e., blood cell movement). Low to no flow is displayed as dark blue, whereas high flow is displayed as red to white. Before initiating laser scanning, rats were placed on a heating plate kept at 37°C to minimize data variations attributable to body temperature. At predetermined time points (before and on postoperative days 7, 14, and 21), we performed two consecutive scans over the same region of interest (legs and feet) in each animal and found essentially no difference between the two scans. After scanning them twice, the stored images were subjected to computer-assisted quantification of blood flow, and the average flow of the ischemic and nonischemic feet was calculated. To minimize data variables attributable to ambient light and temperature, the LDPI index was expressed as the ratio of left (ischemic) to right (nonischemic) limb blood flow.
Statistics
All values are presented as mean standard error. All data were subjected to unpaired Student’s t-test for comparison between two means. Probability values <.05 were considered to be statistically significant.
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RESULTS |
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). We then calculated percent reduction of RBCs, platelets, and granulocytes. Percent reductions of RBCs, platelets, and granulocytes were significantly lower in samples isolated by the StemQuickTME filtration method than in those isolated by the Ficoll centrifuge method (Fig. 2B). Therefore, the recovery rate of MNCs was superior, but the reduction rates of other cell types (i.e., RBCs, platelets, and granulocytes) were inferior in the StemQuickTME filtration method compared with the Ficoll centrifuge method.
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Differentiation of Spindle-Shaped Attaching EPC-Like Cells
When cord blood MNCs isolated by the StemQuickTME filter were cultured on gelatin-coated plastic plates, several cell clusters appeared within 48 hours, and numerous spindle-shaped attaching cells differentiated and sprouted (Fig. 3A). The spindle-shaped attaching cells were observed as isolated cells, cell clusters, or linear cord-like structures. Such morphological appearance resembled that of endothelial progenitor cells developed from the culture of adult human peripheral blood, as reported previously [5].
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), two characteristic features of cells in endothelial lineage.
RT-PCR Analysis of Endothelial Lineage-Related Genes
We performed RT-PCR to analyze the expression of endothelial lineage-related mRNAs in MNCs and attaching cells during the culture of MNCs differentiating into EPC-like attaching cells (Fig. 4). RT-PCR analysis showed that CD31, Lox-1, and eNOS were persistently expressed on these cells during culture for up to 21 days. The expression of CD31 and Lox-1 mRNA had a tendency to decrease during culture. In contrast, another stem cell marker, CD133 mRNA, was expressed early after starting cell culture (~day 7), but its expression was markedly suppressed thereafter. In contrast, KDR mRNA was little expressed initially and gradually appeared thereafter. Because attaching cells at days 7–14 of MNC culture had multiple endothelium-related markers and functions, we defined such spindle-shaped attaching cells as EPC fraction in the present study.
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). Within 24 hours, incorporation of labeled EPCs was identified within the angiogenesis-like HUVEC networks (Fig. 5B).
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).
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). Typical LDBF images obtained at day 14 of hindlimb ischemia are shown in Figure 6B. Therefore, the transplantation of EPCs (3 x 105 cells/animal) augmented ischemia-induced neovascularization and blood flow in vivo. |
DISCUSSION |
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Therapeutic angiogenesis is an effective means for treating patients with critical limb ischemia or end-stage ischemic heart diseases [13]. For this purpose, genes or proteins of angiogenic growth factors (e.g., vascular endothelial growth factor, basic fibroblast growth factor, and hemopoietic growth factor) have been used to enhance angiogenesis and collateral vessel formation in ischemic tissues [15,16]. Recently, we and other investigators reported that bone marrow–derived EPCs circulate in the peripheral blood of normal adults, and transplantation of culture-expanded EPCs augmented neovascularization in experimental animals in vivo [7,14]. Implantation of culture-expanded autologous EPCs from MNCs, therefore, is an attractive strategy for therapeutic angiogenesis in patients with severe ischemic diseases. However, Ficoll density centrifugation or buffy-coat isolation methods are commonly used to isolate MNCs from human blood samples. Indeed, functional EPCs could be culture-expanded from MNCs obtained with Histopaque/Ficoll- density centrifugation methods [7]. However, the centrifuge method is usually time consuming, and blood samples have a higher chance of being contaminated. To overcome these issues, we recently developed a novel cell-filtration device to isolate MNCs with a completely closed-circuit system named StemQuickTME filter [10,11]. In the present study, we used this filter device to isolate MNCs from cord blood samples and tested whether functional EPCs could differentiate from MNCs isolated by this filtration device.
In the present study, cord blood MNCs were successfully isolated by the StemQuickTME filter without exposure to the air. Moreover, the procedure was very simple and reproducible. The operation time of the filtration method (<10 minutes) was significantly shorter than that of the Ficoll density gradient centrifuge method (>1 hour). The recovery rates of WBCs and MNCs were significantly greater in samples isolated by the StemQuickTME method than in those isolated by the Ficoll centrifuge method. Consistently, the recovery rates of stem and progenitor cells (i.e., CD45lowCD34+ CD133+ and CD45lowCD34+CD133+) were also significantly greater in the StemQuickTME filtration method than in the Ficoll centrifuge method. The percent reduction of RBCs, platelets, and granulocytes was significantly lower in the StemQuickTME filtration method than in the Ficoll centrifuge method. We believe that the 75%–85% reduction rates of RBCs and platelets may be minimally effective for the preparation of stem and progenitor cells. Only the reduction rate of granulocytes was markedly lower (40%) in the StemQuick TME method than in the Ficoll method (80%). However, contaminated granulocytes do not adhere to culture dishes during the differentiation of EPCs from MNC culture, and thus granulocytes can be easily removed at the first replacement of culture medium. Therefore, remaining granulocytes would little affect the efficiency of EPC culture expansion. Taken together, the StemQuickTME filtration system would be an efficient and convenient method for processing EPC isolation from human cord blood samples.
During culture of MNCs isolated by the StemQuickTME filter, spindle-shaped attaching cells differentiated from primary culture, and these cells showed several typical features consistent with EPCs, as previously reported [7]. These cells incorporated DiI-acLDL and showed binding capacity to lectin. Furthermore, in vitro culture of EPCs participated in angiogenesis-like endothelial network formation with mature HUVECs on Matrigel, and in vivo transplanted EPCs were incorporated into the capillary structures in the ischemic hindlimb tissues of immunodeficient nude rats. Finally, local transplantation of EPCs significantly augmented functional blood flow in the ischemic hindlimb of nude rats in vivo. Thus, filter-isolated MNCs and subsequent culture-expanded EPCs were well functional, and these cells would become a useful cell source for therapeutic angiogenesis.
Another interesting finding of the present study is that several endothelium-related genes are expressed in a different time course during primary culture of MNCs differentiating into EPCs. CD31, Lox-1, and eNOS were persistently expressed on these cells during culture for 21 days. Among these, we for the first time showed that Lox-1 is expressed on the surface of EPCs during culture. Because Lox-1 is a receptor for oxidized LDL (oxLDL), a highly atherogenic lipoprotein, functions of EPCs could be potentially influenced by oxLDL [17]. In this regard, Chen et al. [18] showed that oxLDL significantly inhibited angiogenesis in experimental animal models. In this process, altered EPC functions elicited by oxLDL may possibly be involved.
RT-PCR analysis also revealed that another stem cell marker, CD133, was expressed at the early period of MNC culture but was markedly reduced thereafter, indicating that CD133 seems to be an early marker for identifying EPCs in peripheral blood. In this regard, several recent studies indicated that coexpression of CD34 and CD133 could serve as a tentative molecular marker for circulating EPCs in human peripheral blood [12,13]. KDR was little expressed initially (i.e., in freshly isolated circulating MNCs) but gradually appeared thereafter in attaching cells. These results suggest that CD133 seems to become a marker for early EPCs, whereas KDR will be used as a marker for more differentiated EPCs [19].
Conclusions
Therapeutic angiogenesis with stem cell transplantation for end-stage ischemic disease has gained great attention [9]. In this regard, culture expansion of autologous EPCs from peripheral blood is an ideal means to treat patients. The present method provides a unique strategy to isolate MNCs from human peripheral blood samples with a completely closed system, and EPCs could be isolated from culture of MNCs obtained with this filtration system. Unfortunately, this filtration system is not suitable for bone marrow MNC isolation, because the filter technology is not adjusted for the removal of marrow stromal cells and fat cells. However, we are developing a filter device suitable for marrow MNC isolation and a closed-culture bag system for EPC differentiation. These new methods will provide us with a more convenient and feasible way to obtain autologous EPCs for therapeutic angiogenesis in the future.
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ACKNOWLEDGMENTS |
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REFERENCES |
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