Isolation and characterization of endothelial colony-forming cells from mononuclear cells of rat bone marrow

Author’s Accepted Manuscript Isolation and characterization of endothelial colonyforming cells from mononuclear cells of rat bone marrow Shukui Yu, Zhongxuan Li, Wei Zhang, Zhengde Du, Ke Liu, Denghua Yang, Shusheng Gong www.elsevier.com/locate/yexcr

PII: DOI: Reference:

S0014-4827(18)30343-4 https://doi.org/10.1016/j.yexcr.2018.06.013 YEXCR11077

To appear in: Experimental Cell Research Received date: 27 December 2017 Revised date: 12 June 2018 Accepted date: 13 June 2018 Cite this article as: Shukui Yu, Zhongxuan Li, Wei Zhang, Zhengde Du, Ke Liu, Denghua Yang and Shusheng Gong, Isolation and characterization of endothelial colony-forming cells from mononuclear cells of rat bone marrow, Experimental Cell Research, https://doi.org/10.1016/j.yexcr.2018.06.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Isolation and characterization of endothelial colony-forming cells from mononuclear cells of rat bone marrow Shukui Yu,a Zhongxuan Li,b Wei Zhang,a,c Zhengde Du,a Ke Liu,a Denghua Yang, a,d Shusheng Gong*a a

Department of Otolaryngology - Head and neck surgery, Beijing Friendship Hospital,

Capital Medical University, 95 Yong An Road, Beijing, 100050, China. b

Department of Cardiology, Chinese PLA General Hospital, 28 FuXing Road, Beijing,

100853, China c

Department of Otolaryngology - Head and neck surgery, Beijing YouAn Hospital,

Capital Medical University, 8 Xitoutiao, You An Men, Beijing, 100069, China d

Department of Otolaryngology - Head and neck surgery, Fuxing Hospital, Capital

Medical University, Beijing, 100038, China

*Corresponding author: Shusheng Gong E-mail: [email protected]

Abstract Transplantation of bone marrow-derived endothelial progenitor cells (BM-EPCs) has been used as a therapeutic strategy for vascular repair. However, it remains controversial whether BM-EPCs exhibit clonal endothelial colony-forming cell (ECFC) capacity, a characteristic of true EPCs. The aim of this study was to isolate and explore the cellular properties of BM-ECFCs. We isolated BM-ECFCs from rat bone marrow with high purity via an optimized method. This approach involved the removal of selective colonies based on the conventional differential adhesive culture method used to isolate ECFCs from peripheral and umbilical cord blood. Our results indicate that primary colony BMECFCs display a panel of surface antigen markers consistent with endothelial cells. These BM-ECFCs coexpress CD34, CD133, and VEGFR2 at high levels, and these levels decrease with passaging. These cells have high potential for proliferation, migration, and formation of capillary-like structures on Matrigel, and these abilities are retained during ex vivo expansion. Furthermore, BM-ECFCs cultured with 10% or 20% fetal bovine serum demonstrated two different patterns of spontaneous capillary-like structure formation. These results provide a foundation for isolation of ECFCs from human bone marrow for autologous cell transplantation and tissue engineering applications in the future.

Abbreviations: EPCs, endothelial progenitor cells; BM-EPCs, bone marrow derived endothelial

progenitor cells; ECFC, endothelial colony-forming cell; MACS, magnetic-activated cell sorting.

2

Keywords: endothelial progenitor cells, endothelial colony-forming cells, bone marrow, cell isolation, cell therapy

3

1.

Introduction

Since the discovery of endothelial progenitor cells (EPCs) in the peripheral circulation [1], their pathophysiological roles have been intensively investigated. EPCs participate in vasculogenesis during embryonic development and are required for postnatal angiogenesis [2,3]. In physiological conditions, EPCs are located

in

the

microenvironment

of

the

bone

marrow,

whereas

in

pathophysiological conditions, due to the presence of different cytokines and signals, EPCs leave the bone marrow and circulate in the peripheral blood [4]. Circulating

EPCs

are

capable

of

proliferation

and

endothelial-specific

differentiation [5,6]. Cumulative evidence suggests that bone marrow (BM)-derived EPCs play an important role in the formation of vessels during the wound healing process. EPCs can be mobilized into the blood circulation, and then directly recruited to pathological lesions. At the lesion, EPCs differentiate into mature endothelial cells to contribute to neovascularization at ischemia sites (vasculogenesis) [7], with the potential to engraft with the native vessels. Additionally, EPCs are capable of secreting a variety of angiogenic growth factors, and differentiated EPCs incorporate into the growing wall of pre-existing blood vessels to promote endogenous

revascularization

at

injury

sites

(angiogenesis)

[8].

These

physiological characteristics provide numerous therapeutic options for the use of EPCs to repair endothelial injury and facilitate the neovascularization of ischemic tissues. Studies have shown that intravenous transfusion or in situ transplantation of BM-EPCs increased vascular repair and promoted circulatory restoration of injured or ischemic tissues with subsequent improvement of organ function in 4

several vascular disease models [9–11]. Furthermore, the therapeutic potential of BM-EPCs can be enhanced by combining them with genetic modification or tissue engineering technologies, which may further contribute to applications for tissue remodeling [12,13]. Based on the cell population appearing within different cell culture periods, two major subpopulations of EPCs have been identified: early EPCs and late outgrowth EPCs [14]. Clonogenic and proliferative potential assays were used to characterize the late outgrowth EPCs, and then endothelial colony-forming cells (ECFCs) were identified via functional assays [15]. ECFCs can form capillaries spontaneously in vivo, which anastomose with the systemic vasculature of the host and are perfused [16,17]. Hence, ECFCs can be considered true EPCs and can be recommended for reparative therapy in cardiovascular disease [18]. To date, few reported studies have isolated ECFCs from bone marrow, and the major approved sources of ECFCs are peripheral and umbilical cord blood [19]. Here, we optimized the conventional ECFC culture method to isolate and expand ECFCs from rat bone marrow with high purity based on their clonogenic and proliferative potential. We explored the biological properties of BM-ECFCs with respect to proliferation potential, immunotype, migratory capacity, the ability to form capillary-like structures, and changes during ex vivo expansion. The findings of this study strengthen the current understanding of the ECFCs derived from bone marrow, which may play an important role during the revascularization process in pathological conditions, and may provide a preferred source of ECFCs for clinical applications.

2.

Material and methods 5

2.1. BM-derived ECFC isolation and expansion All procedures were performed according to the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study protocol was approved by the Institutional Animal Care and Use Committee of Capital Medical University, Beijing, China. Rat bone marrow was obtained by flushing the tibiae and femurs (4 long bones of the hind limbs) of one Sprague-Dawley rat (250–300 g), and mononuclear cells (MNCs) were isolated by density gradient centrifugation using Histopaque-1083 (Sigma-Aldrich, St. Louis, MO, USA). Mononuclear cells harvested from one rat were seeded on a 100-mm plate pre-coated with fibronectin (R&D Systems, Minneapolis, MN, USA) at a density of 2.5 × 106 cells/cm2. Cells were maintained in EGM-2MV (Lonza, Walkersville, MD, USA), which consists of EBM-2 basal medium, 5% FBS, and SingleQuots (Lonza) containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulinlike growth factor (IGF), endothelial growth factor (EGF), heparin, ascorbic acid, and the antibiotics gentamicin and amphotericin B. Non-adherent cells were discarded after 24 h, and the culture medium was changed every 2 days. Approximately 5 days later, cell clusters appeared, which were examined under the microscope daily (IX73, Olympus, Center Valley, PA, USA). Cells were purified following the morphological identification of ECFC colonies. According to our experience, ECFCs generally satisfy the following morphological criteria: (1) initial ECFC clusters consist of trigonal and polygonal cells; (2) early ECFC colonies form after 7-8 days of culture and the typical ECFC colonies appear after 10 days of culture; (3) ECFC colonies display a typical cobblestone-like morphology under high-power fields; and (4) along with the 6

extension of ECFC colonies, specific flat radial cells often appear at the periphery of colonies. During the primary culture process, the colonies without a cobblestone-like monolayer were selectively erased using a cell scraper in a timely manner before they infiltrated into ECFC colonies or became the predominant cell type, whereas the typical cobblestone-like colonies were grown until they were separately expanded after 14 days of culture (Fig. 1). The early EPCs removed during ECFC culture were harvested to use as control cells in the current study. ECFCs at passage five were obtained via clonal expansion.

2.2. Clonogenic assay and cell growth curve To determine the efficiency of the modified culture method, we performed BMECFCs colony-forming assays. BM-MNCs harvested from one rat were cultured for 14 days using the modified method in a 100-mm plate. BM-MNCs cultured for 14 days using the routine method (no removal of any cells or colonies during culture) served as a control. ECFC colonies were counted under an inverted microscope. Typical cobblestone-like colonies with a diameter of more than 1.5 mm were considered as ECFC colonies. Four replicates were performed. To measure the proliferative potential of BM-ECFCs cultured by the modified method, we generated growth curves to quantify population doubling (PD) with serial passage. After a period of latency (day 1 to day 5 of primary culture), the cells grew in clusters, which were counted under an inverted microscope and taken as the starting point for the ECFC growth curve. At day 14 of culture, cell colonies formed by clusters were separately trypsinized and propagated for 6 weeks. As a control, the early EPC colonies that appeared at day 7 of culture were also separately harvested and propagated for 4 weeks. At each subsequent passage, 7

cells were counted to generate the growth kinetic curve. PD was estimated using the following equation: PD = log2 (the number of cells harvested at the end of passage/the number of cells at the beginning of the assay). Four different primary cell colonies from ECFCs and early EPCs were included in this assay.

2.3. Immunocytochemistry With the aim of characterizing the BM-ECFCs in the early phase of primary culture, we performed immunofluorescence for endothelial antigens at 5 days of culture. Primary cells adherent on sterilized round glass cover slips in 24-well plates were fixed at 4°C for 3 h with 1 mL of 4% paraformaldehyde in phosphate buffered saline (PBS), and then gently washed twice with ice-cold PBS and incubated in 10% goat serum/PBS/0.1% Triton-X100 at room temperature for 1 h. Adhesive cells were incubated with the following primary antibodies overnight at 4°C: mouse anti-VE-cadherin (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-9989); mouse anti-eNOS (1:200; Santa Cruz Biotechnology, sc-376751); rabbit anti-vWF (1:400; Abcam, Cambridge, UK, ab-6994), rabbit anti-VEGFR-2 (1:400; Cell Signaling Technology, Danvers, MA, USA, #9698); and mouse antiCD31 (PECAM-1, 1:100; Abcam, ab-64543). After washing three times with PBS, goat anti-rabbit Alexa Fluor 594 and goat anti-mouse Alexa Fluor 488 or 594 were used as secondary antibodies (1:500; Invitrogen, Carlsbad, CA, USA) at room temperature for 1 h in darkness. After washing three times with PBS, stained cells were mounted in mounting medium containing DAPI. Stained cells were observed using confocal fluorescent microscopy (TCS SP5 II; Leica Microsystems, Wetzlar, Germany).

8

2.4. Incorporation of DiI-AcLDL and lectin binding To further identify the cultured cells as EPCs, primary cells adherent on sterilized round glass cover slips in 24-well plates were stained with 0.5 mL of 10% FBS/EGM-2 containing Ulex europaeus agglutinin I conjugated with FITC (UEA-I lectin-FITC, Vector Laboratories, Burlingame, CA, USA, FL-1061) and acetylated low density lipoprotein

conjugated

with

1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine

perchlorate (acLDL-DiI, Biomedical Technologies Inc., Stoughton, MA, USA, BT-902) at a final concentration of 4 μg/mL and 20 μg/mL, respectively. Cells were incubated at 37°C in a CO2 incubator for 4 h and subsequently fixed with 1 mL of ice-cold 4% paraformaldehyde in PBS at 4°C for 3 h. After fixation, the liquid content was gently removed using a 1-mL pipette. The adhesive cells were gently washed twice with 1 mL of ice-cold PBS. The cells were observed by confocal fluorescent microscopy (TCS SP5 II) after mounting in mounting medium containing DAPI.

2.5. Flow cytometry Single cell suspensions were prepared from adherent BM-MNCs cultured for 15 days using the routine method (no removal of any cells or colonies during culture); adherent BM-MNCs cultured for 15 days using the modified method; primary cobblestone-like colony ECFCs (cultured with the modified method and appearing at 15 days, ECFCs-P0); and ECFCs at passage five (clonal expansion from individual primary colonies, ECFCs-P5). The same number of cells (5 × 105) was used for each sample. Cells were resuspended in 100 μL of FACS buffer and incubated with Alexa Fluor® 647-conjugated rabbit anti-VEGFR2 (1:50; Cell Signaling Technology, Danvers, MA, USA, #12658), PE-conjugated mouse anti-CD34 (1:10; Thermo Fisher Scientific Inc., Waltham, MA, USA, MA1-10205), and FITC-conjugated 9

rabbit anti-CD133 (1:100; Bioss, Beijing, China, bs-4770R-FITC) for 1 h on ice in darkness. After staining, the cells were washed twice with PBS and resuspended in 500 μL of FACS buffer for analysis using a flow cytometer (BD LSRFortassa; Becton Dickinson, Franklin Lakes, NJ, USA) with analysis software (FACSDiva version 7.0; BD Biosciences). Samples without antibodies were used as negative controls to discard sample baseline fluorescence for gating. Single fluorochromestained cells were used as controls to set compensation, and appropriate fluorescence minus one stained samples were used to set gate boundaries for cell sorting (as shown in Supplemental Figure). All assays were performed on five independent cultures.

2.6. Cell migration assay To measure ECFC migration, a transwell chamber assay (8 μm pore size, 24 mm diameter; Corning/Costar, Corning, NY, USA) was performed. Early EPCs (control), ECFCs-P0, and ECFCs-P5 were seeded at a density of 1 × 104 cells per well in the upper transwell inserts in EGM-2 media. The lower chamber was filled with EGM-2 containing 5% FBS and stromal cell-derived factor 1α (SDF-1α, R&D Systems) at a concentration of 100 ng/mL. Cells seeded at the same density in the upper transwell inserts in EGM-2 media without SDF-1α in the lower chamber were used as a gravity control. The cells that had migrated through the pores were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet (Beyotime, Jiangsu, China) for 10 min after 6 and 12 h of incubation at 37 °C. Non-migrating cells were gently wiped from the upper surface. The cells that attached to the underside of the insert membrane were counted manually in

10

five random fields for each well under phase-contrast light microscopy (IX73, Olympus). Five independent wells were performed for all experiments.

2.7. Assay of tube formation To investigate the angiogenesis ability of ECFCs, cells were applied to the tube formation assay on Matrigel. Twenty-four-well plates were coated with Matrigel (150 μL/cm²; BD Biosciences, Billerica, MA, USA) and allowed to gel for 30 min at 37°C in a cell culture incubator. Early EPCs (control), ECFCs-P0, and ECFCsP5 were trypsinized, washed, and collected. Then, nearly 3.5 × 104 cells were suspended in 200 μL/cm² EGM-2, applied to pre-incubated Matrigel in 24-well plates, and placed in an incubator (37°C, 20% O2, 5% CO2) to allow tube formation. Tube formation was followed-up each hour and documented by phasecontrast light microscopy (IX73, Olympus) after 12 h of culture. Images were taken in four different fields per well, and the total area of complete tubes (mm2) and total cumulative tube length (mm) formed by cells per unit area (1 mm2) were measured using ImageJ software (National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/index.html). Assays were performed in triplicate. Additionally, the in vitro angiogenic capability of ECFCs was examined in Matrigel-free conditions in the presence of higher concentrations of FBS. When cobblestone-like cell colonies appeared after 7 days of primary culture of ECFCs with EGM-2 media containing 5% FBS, the concentration of FBS was increased to 10% or 20%, and the colony cells were continually cultured for 7 days and passaged individually in these culture conditions. Tube formation of ECFC at passage 3 was documented daily by phase-contrast light microscopy.

11

2.8. Statistical analysis Data are expressed as the mean ± standard deviation (SD) and were analyzed using SPSS 19.0 (IBM, Armonk, NY, USA). Independent-samples t‑test or one-way analysis of variance (ANOVA) followed by the post-hoc Scheffé test was used to determine the statistical significance between groups. The significance level for all tests was set at P < 0.05.

3.

Results

3.1. Morphology of BM-ECFC colonies Using the modified adhesive culture method, ECFCs were isolated as cell colonies from rat BM-MNCs and clonally expanded in vitro, achieving successful isolation in 16 out of 17 experiments in this study. Adherent cells were observed in clusters after 5 days of culture (Fig. 2A), and generally took 3 to 4 days to grow into colonies (Fig. 2B). Colonies gradually extended along with rapid proliferation of ECFCs (Fig. 2C), reaching a diameter of nearly 3 mm after 2 weeks of culture (Fig. 2D). Interestingly, a specific flat radial cell often appeared at the periphery of ECFC colonies (Fig. 2D and 2F), which was also observed in the progeny of these colonies. Colonies without cobblestone-like morphology, such as those of early EPCs (central cell core surrounded by spindle-shaped cells radiating into the periphery, appearing at 4-5 days of culture, Fig. 2G), HSCs (central cluster of rounded cells surrounded by elongated sprouting cells, appearing at 1-3 days of culture, Fig. 2H), MSCs (fibroblast-like morphology or vortex-like pattern, appearing at 5-6 days of culture, Fig. 2I), or other unidentified cells were removed in a timely manner throughout the primary culture process. 12

3.2. Clonogenic capacity and proliferation potential of BM-ECFCs Clonogenic and proliferative capacity is an essential characteristic of ECFCs and could be used to validate the effectiveness of the modified method. BM-MNCs cultured by the modified method generated significantly more colonies (27.77 ± 3.1) than BM-MNCs cultured by the routine method (0.50 ± 0.02, P < 0.01) (Fig. 3A). After 14 days of primary culture, it was difficult to distinguish ECFC colonies from cultured BM-MNCs using the routine method, in which various cells reached approximately full confluence. Moreover, the growth curve demonstrates that BMECFCs had a remarkable potential for clonal expansion (Figure 3B). Primary ECFC clusters (38.55 ± 5.12 cells per cluster) appeared at 5 days of culture and formed colonies (7.69 ± 0.25 × 103 cells per colony) at 14 days of culture, which were serially passaged. The number of ECFCs rapidly increased to 2.46 ± 0.19 × 1011, corresponding to up to 32 population doublings without obvious senescent features at 56 days of culture. In contrast, early EPCs showed low proliferative potential, although cells started proliferating immediately after seeding, and their number reached 5.12 ± 0.24 × 103 at 7 days of culture. After 24 days of culture, early EPCs proliferated to 8.74 ± 0.35 × 105, corresponding to 7 population doublings, and then the cells subsequently died out. Under the same culture conditions, early EPCs could only be cultured for 6 weeks.

3.3. Characterization of BM-ECFCs Adhesive clusters were characterized in the early phase of primary culture using several endothelial markers, including VEGF receptor 2, CD31, VE-cadherin, eNOS, and von Willebrand factor. Cells within the clusters expressed VEGF receptor 2 in the paranuclear Golgi region. CD31 expression was only observed at 13

partial cell membranes, suggesting that endothelial-junctions would be established at the CD31-positive areas between local cells. Positive expression of VE-cadherin was not visualized at cell membranes, perhaps because intercellular adherent junctions had not formed in the early phase of primary culture. Expression of eNOS was observed in the cytoplasm, and expression of von Willebrand factor was in intracellular Weibel-Palades bodies (Fig. 4). Additionally, these cell colonies showed intracellular uptake of DiI-AcLDL and cell surface binding of UEA-IFITC (Fig. 4), which are biological features of the endothelial lineage. Thus, these data suggest that the BM-ECFCs in the early phase of primary culture cannot express all markers of mature endothelial cells, which may still retain the properties of immature cells.

3.4.

Purity of isolated BM-ECFCs and their phenotypic changes during in vitro

expansion To confirm that our modified culture method improved BM-ECFC isolation and to analyze phenotypic changes in expanded cells, we performed polychromatic flow cytometry analysis. The percentage of CD34+/VEGFR2+ cells was higher in BM-MNCs cultured by the modified method (80.16 ± 13.4%) than in BM-MNCs cultured by the routine method (4.38 ± 2.7%, P < 0.01), suggesting that the modified culture method could produce ECFCs of high purity (Fig. 5A and B). The percentage of CD34+/CD133+/VEGFR2+ cells was higher in ECFCs-P0 (72.31 ± 12.1%) than in ECFCs-P5 (49.43 ± 13.9%, P < 0.05) (Fig. 5C and D). Flow cytometry analysis revealed that the simultaneous expression of VEGFR2 and CD133 with CD34 decreased with passaging, but was still maintained at a high level at the fifth passage. 14

3.5. Migratory capacity of BM-ECFCs Cellular migration is required for the formation of the vascular tube network. Therefore, we evaluated the migratory capacity of BM-ECFCs using a chemotaxis experiment. Transwell migration assay data indicated that the number of successfully migrated ECFCs (260 ± 58) was significantly greater than the number of successfully migrated early EPCs (202 ± 45, P < 0.05) at 12 hours, although no significant difference was observed at 6 hours (Fig. 6B). In addition, we found no significant difference between the migrated cell numbers of ECFCs-P5 and ECFCs-P0, suggesting that BM-ECFCs retained their migratory capacity during ex vivo expansion. There was no significant difference among migrated ECFCs at 12 hours of gravity control.

3.6. BM-ECFCs exhibited remarkable potential for forming tubular structures in vitro Angiogenesis is one of the essential capacities of ECFCs. Using the in vitro model of angiogenesis, we assessed the capacity of ECFCs to form capillary-like structures. Early EPCs cultured on Matrigel exhibited almost no tube formation, whereas ECFCs formed significantly more complete tubes (Fig. 7A). The tube areas measured were 0.23 ± 0.03 mm2 for early EPCs, 0.65 ± 0.05 mm2 for ECFCs-P0 (versus early EPCs, P < 0.01; Fig. 7B), and 0.78 ± 0.04 mm2 for ECFCs-P5 (versus ECFCs-P0, P < 0.05; Fig. 7B). The measured tube lengths were 5.47 ± 1.1 mm for early EPCs, 26.96 ± 2.7 mm for ECFCs-P0 (versus early EPCs, P < 0.01; Fig. 7C) and 34.18 ± 3.4 mm for ECFCs-P5 (versus ECFCs-P0, P < 0.05; Fig. 7C). Our findings show that ECFCs did not form any tubes during ex vivo expansion in the presence of 5% FBS (a low concentration), but they formed extensive capillary-like vascular tube networks after three passages in the presence 15

of 10% FBS (Fig. 8), which did not require the Matrigel matrix. In contrast, another pattern of spontaneous capillary-like structure formation, mediated by the specific flat radial cells, was observed upon increasing the concentration of FBS to 20% (Fig. 8). Collectively, these data suggest that BM-ECFCs had a marked angiogenic potential on Matrigel, which was not attenuated in the expanded ECFCs. Importantly, the intrinsic angiogenic capability of BM-ECFCs might be induced by high concentrations of FBS in vitro.

4.

Discussion

EPCs were first isolated by Asahara et al. from human peripheral blood [1]. Although similar populations have been isolated from other tissues, including bone marrow [9], umbilical cord blood [17], placenta [20] and spleen [21], peripheral blood-derived EPCs are still used widely in therapeutic angiogenesis trials, because peripheral blood is more accessible through less invasive procedures in clinical practice. Nevertheless, the number of circulating EPCs is extremely low, even in healthy individuals. Circulating EPCs account for < 0.02% of circulating cells in the peripheral blood [22], and their quantity and function are impaired by multiple cardiovascular risk factors, including hypertension, hypercholesterolemia, aging, and smoking [23]. The in vivo vasculogenic potential of EPCs derived from umbilical cord blood is superior to that of EPCs derived from peripheral blood [15], and adequate cell numbers can be achieved for EPC-based therapy. However, autologous transplantation of cord blood EPCs cannot be applied to patients who have not stored cord blood. Therefore, approaches aimed at obtaining a sufficient amount of highly purified autologous cells are pivotal in the therapeutic application of 16

EPC transplantation. Additionally, it is important to maintain the characteristic phenotype and proangiogenic activity of purified EPCs without allowing them to differentiate into mature endothelial cells during ex vivo expansion. Adult bone marrow is a reservoir of various functional stem and progenitor cell populations that provide a rich and reliable source for autologous cell therapy. However, the specific subset of EPCs, namely ECFCs, which display high proliferative potential and angiogenic properties both in vitro and in vivo [24,25], has been difficult to obtain from bone marrow [26,27]. Therefore, several reports have proposed that circulating ECFCs might derive from tissue-resident ECFCs rather than from the bone marrow [28]. Currently, putative EPCs have been isolated using either magnetic-activated cell sorting (MACS) or in vitro differential adhesive culture [29]. To our knowledge, commercial magnetic beads are designed to purify ECFCs derived from peripheral or cord blood. However, the surface antigenic markers of BM-ECFCs are poorly defined, limiting the utility of useful markers such as CD31, CD34, and CD144 [28]. In addition, the complicated BM niche provides a protected compartment where stem and progenitor cells can maintain their integrity and stemness [30]. This microenvironment is absent after MACS, which negatively influences the function of purified BM stem and progenitor cells, and may inhibit the colony-forming capacity of ECFCs. Therefore, the method of MACS is unsuitable for the isolation of BM-ECFCs. In this study, EPCs were separated from MNCs by in vitro differential adhesive culture, in which various EPC subpopulations, with different appearances and features, can be harvested. This is possible because different cell isolation procedures and culture conditions lead to growth variations during the transition from isolated 17

independent cells to adhesive colonies [29]. Importantly, the BM niche, containing crucial autologous components, could be relatively well preserved by this method. We speculate that the various cytokines and unidentified molecules secreted by BM-MNCs, including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), and multipotent adult progenitor cells (MAPCs), and their synergistic interactions play a significant role in promoting the initial fast adherence of BMECFCs. Moreover, these factors facilitate the survival, proliferation, and differentiation of BM-ECFCs and induce them to form cell colonies at the later stage of primary culture. However, the processes of cell culture should be monitored closely, and in the present study colonies without cobblestone morphology were removed in a timely manner, thereby ensuring that they could not become the predominant cell type. This could explain why MSC outgrowth, rather than that of ECFCs, was obtained from BM-MNCs when using the conventional method of differential adhesive culture that is used for other cell sources [26,27]. Thus, applying our modified culture method, we have successfully achieved clonal expansion and harvested ECFCs with a high proliferative potential from rat BM-MNCs. A consensus on the immunophenotype of ECFCs has been reached recently [19], including endothelial antigens, such as VEGFR2, CD31, VE-Cadherin, and von Willebrand factor. Here, we demonstrated the expression of these endothelial markers in isolated cells in the early phase of primary culture. These cells also exhibited biological features of the endothelial lineage, that is, intracellular uptake of DiI-AcLDL and cell surface binding of UEA-I-FITC [3], which identify ECFCs. The combination phenotype of CD34+/VEGFR2+ is widely employed as a 18

definition for EPCs [31], and previous studies have established that ECFCs derived from human peripheral or umbilical cord blood express the CD34 and VEGFR2 markers [25,29,32]. Therefore, to evaluate the efficiency of our modified culture method, we quantified the cells expressing CD34 and VEGFR2 as a percentage of the cultured cells using flow cytometry. In BM-MNCs cultured using the routine method, we found that CD34+/VEGFR2+ cells accounted for only 4.38% of the total population of adherent cells, whereas this fraction reached 80.16% in BMMNCs cultured by our modified method, and was higher than the proportion previously reported [33]. Given that typical ECFC colonies with a cobblestone appearance are formed in a later phase of primary culture (at least after 10 days of culture), there would be limited potential for ECFC growth if other cell types predominated in the culture plate before the emergence of ECFCs. Further, because the exact origin of ECFC remains unclear, it is imperative to identify the antigen profile of BM-ECFCs. Case and colleagues previously showed that purified CD34+/CD133+/VEGFR2+ cells isolated from adult peripheral blood co-expressed CD45 and could not give rise to endothelial colonies in vitro [34]. In contrast, CD34+/CD133+/VEGFR2+/CD45dim-neg cells have been detected in BM samples from human adults, which also express high levels of CD31 [35]. Based on this report, it is reasonable to speculate that CD34+/CD133+/VEGFR2+/CD45dim-neg might be used to identify BM-ECFCs. Indeed, in this study a large proportion of CD34+/CD133+/VEGFR2+ cells (72.31%) was detected in primary colony BMECFCs. This proportion decreased with passaging because CD34 and CD133 are highly expressed by immature stem cells and early progenitor cells, but their expression is gradually lost during differentiation and in vitro expansion [19,36,37]. 19

Similarly, Ingram et al. reported that only a small percentage of cells expressed CD34 and CD133 during passage of ECFCs derived from human peripheral or umbilical cord blood [24]. Previous studies revealed that changes in surface marker expression occur not only in differentiation of progenitor cells (internal factors) but also during mature EC culture [38], because these phenotypes are strongly influenced by culture conditions (external factors) as well [38,39]. Successful revascularization in injury sites via transplantation of ex vivo expanded EPCs strongly depends on their ability to migrate to ischemic tissues and form a vascular network. Expression of stromal-derived factor 1 (SDF-1), an important chemoattractant for EPCs expressing abundant CXCR4 receptors, is significantly increased at ischemic lesions, which mobilizes EPCs from the bone marrow to be incorporated into the angiogenic processes [40,41]. Our data indicate that BM-ECFCs possess a high capability of migrating towards SDF-1 in vitro, and this capability is retained during BM-ECFC expansion. In addition, we show that BM-ECFCs can form extensive capillary networks on Matrigel, exhibiting pronounced angiogenic properties in vitro. It should be noted that spontaneous formation of capillary-like structures could be observed independent of Matrigel. These structures exhibited different patterns during the ex vivo propagation of BMECFCs under different high concentrations of FBS. Additionally, capillary-like vascular tube networks appeared at passage 3 of BM-ECFCs culture, which is consistent with findings of published studies [42]. Although spontaneous formation of capillaries by transplanted ECFCs has been confirmed in vivo [16,17], the exact mechanism through which cultured ECFCs spontaneously form capillary-like structures in vitro is still unclear. We presume that this activity could be attributed 20

to the angiogenic sprouting ability of ECFCs [39,43]. Furthermore, BM-ECFCs cultured in 20% FBS show a different pattern of spontaneous formation of capillary-like structures. The morphological features of these structures indicate they are most likely mediated by the specific flat radial cells that are always situated in the center of the tube-like structures. Previous reports indicated that some as-yet unidentified nutrient component in FBS significantly promoted angiogenic activities of EPCs, such as proliferation, migration, and tube formation [44,45]. Although high concentrations of FBS augment the differentiation capacity of EPCs [45], they could accelerate cellular senescence of highly proliferative ECFCs. Therefore, a low concentration of FBS is more suitable for in vitro expansion of ECFCs. In conclusion, we have isolated ECFCs with a high proliferative potential from the rat bone marrow via our modified culture method and achieved extensive expansion at the level of ECFC colonies. Our results demonstrate that the BM CD34+/CD133+/VEGFR2+ fraction contains ECFCs that express some endothelial markers and possess pronounced angiogenic abilities, such as proliferation, migration, and tube network formation. Further studies are required to elucidate the therapeutic potential of BM-ECFCs in vascular disease using experimental animal models and cell transplantation or tissue engineering techniques. Finally, we expect that human bone marrow cells may be a feasible and efficient source of ECFCs suitable for therapeutic studies and clinical applications of autologous transplantation in the future. Acknowledgments

21

This work was supported by Beijing Natural Science Foundation (7174291), China Postdoctoral Science Foundation (2016M601067), Science and Technology Development Foundation of Shenzhen, China (JCYJ20160429181957912), and Science and Technology Development Foundation of Shenzhen Nanshan District, China (2016004).

Disclosures The authors have no conflicts of interest to disclose.

22

Figure legends

Fig. 1 Methods of rat BM-ECFC isolation and expansion. Rat bone-marrow-derived MNCs were seeded on fibronectin-coated plate and the non-adherent cells were removed. Colonies without cobblestone-like morphology appeared early and were removed in a timely manner to prevent them from becoming the predominant cells. The typical cobblestone-like colonies appeared after 9-11 days of culture, and were separately expanded after 14 days of culture. After 4-5 days of cell seeding, BM-ECFCs at passage 1 reached confluence, and were then serially passaged 1:3 every 3-4 days.

Fig. 2 Isolation of ECFC colonies from rat bone marrow. (A-F) Representative photomicrographs of primary ECFC colony formation during the culture process. (A) Adherent cells formed a cluster after 5 days of culture. (B) The cell cluster grew into a colony after 9 days, and this was often accompanied by the growth of other cell types (below the white dotted lines), which were then removed in a timely fashion. (C) After removal of the unwanted cells, the colony continued to grow and appeared as an endothelial colony. (D) The colony gradually extended and reached a diameter of nearly 3 mm, finally forming an ECFC colony after 15 days of culture. The white arrow indicates the specific flat radial cell at the periphery of the ECFC colony. (E) Higher-magnification image of the area in the center of the ECFC colony displaying typical cobblestone-like morphology. (F) Higher-magnification image of the area at the periphery of the ECFC colony displaying specific flat radial cells (white arrows). (G-I) Representative photomicrographs of colonies lacking cobblestone-like morphology and appearing before ECFC colony formation, including the following: (G) early EPC colonies; 23

(H) HSC colonies; and (I) MSC colonies. These colonies were removed in a timely fashion. Scale bar = 200 μm in A-D and 100 μm in E-I.

Fig. 3 Clonogenic activity and proliferative capacity of rat BM-ECFCs. (A) The number of emerging cobblestone-like colonies per 100-mm plate after 14 days of primary culture. The number of colonies was quantified; **P < 0.01 when compared to BM-MNCs cultured by the routine method. (B) Growth curves for the ex vivo expansion of ECFCs and early EPCs. Apart from ECFC clusters, cells were enumerated at each passage.

Fig. 4 Rat bone marrow-derived adherent cells were identified as ECFCs. Immunofluorescence staining demonstrated that BM-ECFCs expressed the endothelial cell markers VEGF receptor 2 (red), CD31 (red), VE-cadherin (green), eNOS (green), and von Willebrand factor (green). Testing of DiI-AcLDL (red) uptake and UEA-I-FITC (green) binding showed that the ECFCs were doublepositive. Nuclei in all samples were stained with DAPI (blue). Scale bar = 50 μm.

Fig. 5 Purity of isolated rat BM-ECFCs and phenotypic changes during in vitro expansion. (A) BM mononuclear cells were cultured for 15 days using the routine method or the modified method, labeled with fluorescent antibodies to CD34 and VEGFR2, and analyzed by FACS. Non-stained samples were used as a negative control. (B) The mean percentage of CD34+/VEGFR2+ cells (BM-ECFCs) among total BM-MNCs was quantified; **P < 0.01 when comparing BM-MNCs cultured by the routine method or modified method. (C) ECFCs-P0 and ECFCs-P5 were labeled with fluorescent antibodies to CD34, CD133, and VEGFR2 and analyzed by FACS. Non-stained samples were used as a negative control. (D) The mean 24

percentage of CD34+/CD133+/VEGFR2+ cells among total BM-ECFCs was quantified; *P < 0.05 for the comparison of ECFCs-P0 and ECFCs-P5.

Fig. 6 Rat BM-ECFC migration assay. (A) Representative phase-contrast photomicrographs of early EPCs (control), ECFCs-P0 and ECFCs-P5 that attached to the underside of the transwell insert membrane 12 h (left) of gravity control, 6 h (middle) and 12 h (right) under chemotaxis after being seeded. Scale bar = 100 μm. (B) Quantification of the number of early EPCs, ECFCs-P0 and ECFCs-P5 that had migrated; no significant difference was observed among the groups after 12 h of gravity control and 6 h under chemotaxis. After 12 h, the migration observed for early EPCs and ECFCs-P0 was significantly different, *P < 0.05. Data are expressed as the mean ± SD of five independent experiments.

Fig. 7 Capillary-like tube formation assay of rat BM-ECFCs. (A) Representative phase-contrast photomicrographs of the tubes formed in early EPCs (control), ECFCs-P0, and ECFCs-P5 12 h after being seeded on Matrigel. Scale bar = 200 μm. (B) Quantification of the formed tube area in the early EPCs, ECFCs-P0, and ECFCs-P5. Total tube area was measured as percentage of area covered by tubes per mm2 (four different fields per well); **P < 0.01 for the comparison of early EPCs and ECFCs-P0; *P < 0.05 for the comparison of ECFCs-P0 and ECFCs-P5. (C) Quantification of formed tube length in the early EPCs, ECFCs-P0, and ECFCs-P5. Total tube length was measured as mm/mm2 (four different fields per well); **P < 0.01 for the comparison of early EPCs and ECFCs-P0; *P < 0.05 for the comparison of ECFCs-P0 and ECFCs-P5. Data are expressed as the mean ± SD of three independent experiments. 25

Fig. 8 Spontaneous capillary-like structure formation of rat BM-ECFCs at passage 3. After 7 days of primary culture, ECFC colonies were cultured with EGM-2 medium containing 5% FBS, 10% FBS, or 20% FBS. ECFCs cultured with 5% FBS did not form any tubes during ex vivo expansion. ECFCs cultured with 10% FBS spontaneously formed capillary-like structures upon expansion to passage 3, independent of the presence of Matrigel. In contrast, ECFCs cultured with 20% FBS spontaneously formed capillary-like structures in another pattern at passage 3, which was mediated by the specific flat radial cells (white arrows). Scale bar = 100 μm.

26

References: [1]

T. Asahara, T. Murohara, A. Sullivan, M. Silver, R. van der Zee, T. Li, B. Witzenbichler, G. Schatteman, J.M. Isner, Isolation of putative progenitor endothelial cells for angiogenesis, Science 275 (1997) 964-967.

[2] A. Schmidt, K. Brixius, W. Bloch, Endothelial precursor cell migration during vasculogenesis, Circ. Res. 101 (2007) 125-136. [3] C. Urbich, S. Dimmeler, Endothelial progenitor cells: characterization and role in vascular biology, Circ. Res. 95 (2004) 343-353. [4] T. Takahashi, C. Kalka, H. Masuda, D. Chen, M. Silver, M. Kearney, M. Magner, J.M. Isner, T. Asahara, Ischemia and cytokine-induced mobilization of bone marrowderived endothelial progenitor cells for neovascularization, Nat. Med. 5 (1999) 434438. [5] M.

Hristov,

C.

Weber,

Endothelial

progenitor

cells:

characterization,

pathophysiology, and possible clinical relevance, J. Cell Mol. Med. 8 (2004) 498508. [6] T. Asahara, T. Takahashi, H. Masuda, C. Kalka, D. Chen, H. Iwaguro, Y. Inai, M. Silver, J.M. Isner, VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells, EMBO J. 18 (1999) 3964-3972. [7] E. Sbaa, J. Dewever, P. Martinive, C. Bouzin, F. Frerart, J.L. Balligand, C. Dessy, O. Feron, Caveolin plays a central role in endothelial progenitor cell mobilization and homing in SDF-1-driven postischemic vasculogenesis, Circ. Res. 98 (2006) 12191227. [8] M. Potente, H. Gerhardt, P. Carmeliet, Basic and therapeutic aspects of angiogenesis, Cell 146 (2011) 873-887. 27

[9] J.O. Jeong, M.O. Kim, H. Kim, M.Y. Lee, S.W. Kim, M. Ii, J.U. Lee, J. Lee, Y.J. Choi, H.J. Cho, N. Lee, M. Silver, A. Wecker, D.W. Kim, Y.S. Yoon, Dual angiogenic and neurotrophic effects of bone marrow-derived endothelial progenitor cells on diabetic neuropathy, Circulation 119 (2009) 699-708. [10] E. Park, K. Park, E. Liu, R. Jiang, J. Zhang, A.J. Baker, Bone-Marrow-Derived Endothelial Progenitor Cell Treatment in a Model of Lateral Fluid Percussion Injury in Rats: Evaluation of Acute and Subacute Outcome Measures, J. Neurotrauma 34 (2017) 2801-2811. [11] R. Zhang, J. Yang, J. Yuan, B. Song, Y. Wang, Y. Xu, The Therapeutic Value of Bone

Marrow-Derived

Endothelial

Progenitor

Cell

Transplantation

after

Intracerebral Hemorrhage in Rats, Front. Neurol. 8 (2017) 174. [12] J.Y. Xu, Y.K. Lee, Y. Wang, H.F. Tse, Therapeutic application of endothelial progenitor cells for treatment of cardiovascular diseases, Curr. Stem Cell Res. Ther. 9 (2014) 401-414. [13] M. Herrmann, A. Binder, U. Menzel, S. Zeiter, M. Alini, S. Verrier, CD34/CD133 enriched bone marrow progenitor cells promote neovascularization of tissue engineered constructs in vivo, Stem Cell Res. 13 (2014) 465-477. [14] C.H. Yoon, J. Hur, K.W. Park, J.H. Kim, C.S. Lee, I.Y. Oh, T.Y. Kim, H.J. Cho, H.J. Kang, I.H. Chae, H.K. Yang, B.H. Oh, Y.B. Park, H.S. Kim, Synergistic neovascularization by mixed transplantation of early endothelial progenitor cells and late outgrowth endothelial cells: the role of angiogenic cytokines and matrix metalloproteinases, Circulation 112 (2005) 1618-1627. [15] P. Au, L.M. Daheron, D.G. Duda, K.S. Cohen, J.A. Tyrrell, R.M. Lanning, D. Fukumura, D.T. Scadden, R.K. Jain, Differential in vivo potential of endothelial 28

progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels, Blood 111 (2008) 1302-1305. [16] J.M. Melero-Martin, Z.A. Khan, A. Picard, X. Wu, S. Paruchuri, J. Bischoff, In vivo vasculogenic potential of human blood-derived endothelial progenitor cells, Blood 109 (2007) 4761-4768. [17] M. Nagano, T. Yamashita, H. Hamada, K. Ohneda, K. Kimura, T. Nakagawa, M. Shibuya, H. Yoshikawa, O. Ohneda, Identification of functional endothelial progenitor cells suitable for the treatment of ischemic tissue using human umbilical cord blood, Blood 110 (2007) 151-160. [18] E.B Peters, Endothelial Progenitor Cells for the Vascularization of Engineered Tissues, Tissue Eng. Part B Rev. (2017). http://dx.doi:10.1089/ten.teb.2017.0127. [19] R.J. Medina, C.L. Barber, F. Sabatier, F. Dignat-George, J.M. Melero-Martin, K. Khosrotehrani, O. Ohneda, A.M. Randi, J.K.Y. Chan, T. Yamaguchi, V.W.M. Van Hinsbergh, M.C. Yoder, A.W. Stitt, Endothelial Progenitors: A Consensus Statement on Nomenclature, Stem Cells Transl. Med. 6 (2017) 1316-1320. [20] J. Patel, E. Seppanen, M.S. Chong, J.S. Yeo, E.Y. Teo, J.K. Chan, N.M. Fisk, K. Khosrotehrani, Prospective surface marker-based isolation and expansion of fetal endothelial colony-forming cells from human term placenta. Stem Cells Transl. Med. 2 (2013) 839–847. [21] N. Werner, S. Junk, U. Laufs, A. Link, K. Walenta, M. Bohm, G. Nickenig, Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury, Circ. Res. 93 (2003) e17-24. [22] G. Galasso, S. Schiekofer, K. Sato, R. Shibata, D.E. Handy, N. Ouchi, J.A. Leopold, J. Loscalzo, K. Walsh, Impaired angiogenesis in glutathione peroxidase-1-deficient 29

mice is associated with endothelial progenitor cell dysfunction, Circ. Res. 98 (2006) 254-261. [23] E. Shantsila, T. Watson, G.Y. Lip, Endothelial progenitor cells in cardiovascular disorders, J. Am. Coll. Cardiol. 49 (2007) 741-752. [24] D.A. Ingram, L.E. Mead, H. Tanaka, V. Meade, A. Fenoglio, K. Mortell, K. Pollok, M.J. Ferkowicz, D. Gilley, M.C. Yoder, Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood, Blood 104 (2004) 2752-2760. [25] E. Pelosi, G. Castelli, U. Testa, Endothelial progenitors, Blood Cells Mol. Dis. 52 (2014) 186-194. [26] G.R. Barclay, O. Tura, K. Samuel, P.W. Hadoke, N.L. Mills, D.E. Newby, M.L. Turner, Systematic assessment in an animal model of the angiogenic potential of different human cell sources for therapeutic revascularization, Stem Cell Res. Ther. 3 (2012) 23. [27] O. Tura, E.M. Skinner, G.R. Barclay, K. Samuel, R.C. Gallagher, M. Brittan, P.W. Hadoke, D.E. Newby, M.L. Turner, N.L. Mills, Late outgrowth endothelial cells resemble mature endothelial cells and are not derived from bone marrow, Stem Cells 31 (2013) 338-348. [28] M.C. Yoder, Is endothelium the origin of endothelial progenitor cells? Arterioscler. Thromb. Vasc. Biol. 30 (2010) 1094-1103. [29] K.K. Hirschi, D.A. Ingram, r M.C. Yode, Assessing identity, phenotype, and fate of endothelial progenitor cells, Arterioscler. Thromb. Vasc. Biol. 28 (2008) 1584-1595. [30] E. Rurali, B. Bassetti, G.L. Perrucci, M. Zanobini, C. Malafronte, F. Achilli, E. Gambini, BM ageing: Implication for cell therapy with EPCs, Mech. Ageing Dev. 30

159 (2016) 4-13. [31] M.C. Yoder, L.E. Mead, D. Prater, T.R. Krier, K.N. Mroueh, F. Li, R. Krasich, C.J. Temm, J.T. Prchal, D.A. Ingram, Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals, Blood 109 (2007) 18011809. [32] F. Timmermans, J. Plum, M.C. Yoder, D.A. Ingram, B. Vandekerckhove, J. Case, Endothelial progenitor cells: identity defined? J. Cell Mol. Med. 13 (2009) 87-102. [33] M. Ikutomi, M. Sahara, T. Nakajima, Y. Minami, T. Morita, Y. Hirata, I. Komuro, F. Nakamura, M. Sata, Diverse contribution of bone marrow-derived late-outgrowth endothelial progenitor cells to vascular repair under pulmonary arterial hypertension and arterial neointimal formation, J. Mol. Cell Cardiol. 86 (2015) 121-135. [34] J. Case, L.E. Mead, W.K. Bessler, D. Prater, H.A. White, M.R. Saadatzadeh, J.R. Bhavsar, M.C. Yoder, L.S. Haneline, D.A. Ingram, Human CD34+AC133+VEGFR2+ cells are not endothelial progenitor cells but distinct, primitive hematopoietic progenitors, Exp. Hematol. 35 (2007) 1109-1118. [35] P. Lanuti, G. Rotta, C. Almici, G. Avvisati, A. Budillon, P. Doretto, N. Malara, M. Marini, A. Neva, P. Simeone, G.E. Di, A. Leone, A. Falda, R. Tozzoli, C. Gregorj, C.M. Di, V. Trunzo, V. Mollace, M. Marchisio, S. Miscia, Endothelial progenitor cells, defined by the simultaneous surface expression of VEGFR2 and CD133, are not detectable in healthy peripheral and cord blood, Cytometry A 89 (2016) 259-270. [36] M.C. Yoder, Human endothelial progenitor cells, Cold Spring Harb. Perspect. Med. 2 (2012) a006692. [37] M. Peichev, A.J. Naiyer, D. Pereira, Z. Zhu, W.J. Lane, M. Williams, M.C. Oz, D.J. Hicklin, L. Witte, M.A. Moore, S. Rafii, Expression of VEGFR-2 and AC133 by 31

circulating human CD34(+) cells identifies a population of functional endothelial precursors, Blood 95 (2000) 952-958. [38] S. Amatschek, E. Kriehuber, W. Bauer, B. Reininger, P. Meraner, A. Wolpl, N. Schweifer, C. Haslinger, G. Stingl, D. Maurer, Blood and lymphatic endothelial cellspecific differentiation programs are stringently controlled by the tissue environment, Blood 109 (2007) 4777-4785. [39] D. Tasev, L.S. Konijnenberg, J. Amado-Azevedo, M.H. van Wijhe, P. Koolwijk, V.W. van Hinsbergh, CD34 expression modulates tube-forming capacity and barrier properties of peripheral blood-derived endothelial colony-forming cells (ECFCs), Angiogenesis 19 (2016) 325-338. [40] D.J. Ceradini, A.R. Kulkarni, M.J. Callaghan, O.M. Tepper, N. Bastidas, M.E. Kleinman, J.M. Capla, R.D. Galiano, J.P. Levine, G.C. Gurtner, Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1, Nat. Me d. 10 (2004) 858-864. [41] J. Yamaguchi, K.F. Kusano, O. Masuo, A. Kawamoto, M. Silver, S. Murasawa, M. Bosch-Marce, H. Masuda, D.W. Losordo, J.M. Isner, T. Asahara, Stromal cellderived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization, Circulation 107 (2003) 1322-1328. [42] C.M. Kahler, J. Wechselberger, W. Hilbe, A. Gschwendtner, D. Colleselli, H. Niederegger, E.M. Boneberg, G. Spizzo, A. Wendel, E. Gunsilius, J.R. Patsch, J. Hamacher, Peripheral infusion of rat bone marrow derived endothelial progenitor cells leads to homing in acute lung injury, Respir. Res. 8 (2007) 50. [43] E.M. Anderson, D.J. Mooney, The Combination of Vascular Endothelial Growth Factor and Stromal Cell-Derived Factor Induces Superior Angiogenic Sprouting by 32

Outgrowth Endothelial Cells, J. Vasc. Res. 52 (2015) 62-69. [44] Y. Yu, Y. Gao, H. Wang, L. Huang, J. Qin, R. Guo, M. Song, S. Yu, J. Chen, B. Cui, P. Gao, The matrix protein CCN1 (CYR61) promotes proliferation, migration and tube formation of endothelial progenitor cells, Exp. Cell Res. 314 (2008) 3198–3208. [45] T. Shumiya, R. Shibata, Y. Shimizu, M. Ishii, R. Kubota, S. Shintani, T. Murohara, Evidence for the therapeutic potential of ex vivo expanded human endothelial progenitor cells using autologous serum, Circ. J. 74 (2010) 1006‑1013.

33

34

35

36

37

38

Highlights

-

BM-ECFCs were isolated from rat bone marrow with high purity by an optimized method, which might be also suitable for isolation of ECFCs from other tissue sources or other species. Primary colony BM-ECFCs highly display CD34+/CD133+/VEGFR2+, which could be used as a panel of surface antigen markers for identification of ECFCs from bone marrow. BM-ECFCs have a high potential for migration and form capillary-like structures, and these abilities are retained during their ex vivo expansion, providing a preferred source of ECFCs for clinical applications in the future.

39