Morphology of endothelial progenitor cells separated from rat bone marrow and peripheral blood under microscope (Figure 1-5)

Immunofluorescence test results

The immunofluorescence test of the cells after cultured for 14 days showed the positive expression of surface markers CD34, CD133 and KDR (Figure 6-7).

Flow cytometry analysis results

The positive expression rates of endothelial progenitor cells CD34 and KDR (vascular endothelial growth factor-receptor) in the bone marrow were 69.0% and 84.8%, and in the peripheral blood were 5.4% and 32.7% (Figure 8-9).

Determination of ingest function

1,1'-dioctadecyl-3, 3, 3', 3'-tetramethylindo- carbocyanine perchlorate-acetylated-low density lipoprotein and FITC-labeled Ulex europaeus agglutinin 1 double fluorescent staining and the laser scanning confocal microscope showed that the cells could ingest acetylated-low density lipoprotein and combined with Ulex europaeus agglutinin 1 and then differentiate into endothelial progenitor cells, and 95% or more endothelial progenitor cells were double-stained positive (Figure10).

[1] Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275(5302):964-967. [2] Schatteman GC. Adult bone marrow-derived hemangioblasts, endothelial cell progenitors, and EPCs. Curr Top Dev Biol. 2004;64:141-180. [3] Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res. 1999;85(3):221-228. [4] Friedrich EB, Walenta K, Scharlau J, et al. CD34-/CD133+/ VEGFR-2+ endothelial progenitor cell subpopulation with potent vasoregenerative capacities. Circ Res. 2006;98(3): e20-25. [5] Chu K, Jung KH, Lee ST, et al. Circulating endothelial progenitor cells as a new marker of endothelial dysfunction or repair in acute stroke. Stroke. 2008;39(5):1441-1447. [6] Hirschi KK, Ingram DA, Yoder MC. Assessing identity, phenotype, and fate of endothelial progenitor cells. Arterioscler Thromb Vasc Biol. 2008;28(9):1584-1595. [7] Nakul-Aquaronne D, Bayle J, Frelin C. Coexpression of endothelial markers and CD14 by cytokine mobilized CD34+ cells under angiogenic stimulation. Cardiovasc Res. 2003;57(3):816-823. [8] Zhang ZG, Zhang L, Jiang Q, et al. Bone marrow-derived endothelial progenitor cells participate in cerebral neovascularization after focal cerebral ischemia in the adult mouse. Circ Res. 2002;90(3):284-288. [9] Besler C, Doerries C, Giannotti G, et al. Pharmacological approaches to improve endothelial repair mechanisms. Expert Rev Cardiovasc Ther. 2008;6(8):1071-1082. [10] Shirota T, Yasui H, Shimokawa H, et al. Abrication of endothelial progenitor cell (EPC)-seeded intravascular stent devices and in vitro endothelialization on hybrid vascular tissue. Biomaterials. 2003;24(13):2295-2302. [11] Au P, Daheron LM, Duda DG, et al. Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels. Blood. 2008;111(3): 1302-1305. [12] Huang WJ, Xiao FY, Zhang HQ, et al. Effects of endothelial progenitor cells and endothelial outgrowth cells in repair of injured carotid vessel: a comparative study with rabbits. Zhonghua Yi Xue Za Zhi. 2007;87(44):3143-3147. [13] Hur J, Yoon CH, Kim HS, et al. Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis. Arterioscler Thromb Vasc Biol. 2004;24(2):288-293. [14] Yang N, Li D, Jiao P, et al. The characteristics of endothelial progenitor cells derived from mononuclear cells of rat bone marrow in different culture conditions. Cytotechnology. 2011;63(3):217-226. [15] Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest. 1999;103(9):1231-1236.

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Endothelial progenitor cells are kinds of precursor cells that can differentiate into vascular endothelial cells-like cells. It is not only involved in embryonic blood vessel formation, but also found in adult individuals with angiogenesis and angiogenesis closely related to stem cells. They play an important role in repairing vascular and collateral vascular network, have functions in promoting angiogenesis in ischemic vascular regeneration and furthering nerve regeneration, predicting the development of cerebral ischemia.

MATERIALS AND METHODS
Design
An observational experiment.
Time and setting
The experiment was performed in Laboratory of Pathology, Department of Basic Medicine, Jilin University from January 2011 to June 2011.
Materials
Experimental animals
Healthy adult Wistar rats, weighing 250-300 g, were purchased from animal laboratory in Department of Basic Medicine, Jilin University, China.
The main instruments and reagents are as follows:

Methods
Isolation of monocytes from rat peripheral blood
A total of 15 mL peripheral blood was collected aseptically from healthy Wistar rats weighing 250-300 g. Anticoagulantted by Heparin, and diluted with an equal volume of phosphate buffered solution containing 0.02% ethylene diamine tetraacetic acid, and then the diluted blood was gently superimposed on the liquid in the cell layer by the ratio of 1:2, 1 500 r/min centrifugation for 20 minutes. After centrifugation, the mononuclear cell layer was sucked, and washed five times with volume phosphate buffered solution containing 0.02% ethylene diamine tetraacetic acid for twice, at room temperature centrifuged 10 minutes at 1 500 r/min; discarded the supernatant, M199 medium containing 20% fetal bovine serum and 10 μg/L vascular endothelial growth factor was used to adjust the cell concentration into 1×10⁶/cm².
Isolation of monocytes from rat bone marrow
The long bone was isolated aseptically, the muscles were broken away from both ends of the epiphysis, washed the bone marrow with a syringe pump PBS chamber from pink to white. The fluid was placed into the sterile 50 mL centrifuge tube, centrifuged with 1 000 r/min for 5 minutes, the supernatant was discarded. Learn 6 mL M199 medium re-suspended cells, wind and percussion mix. The wash medium was slowly added above rat lymphocyte separation medium with the ration of 2:1, so that to form a clear hierarchy, then centrifuged for 20 minutes with the speed of 1 500 r/min. After centrifugation, fuzzy filamentous or floc between the culture medium (red) and lymphatic separation medium (white) was obtained. Then the cells were mixed with 5 times volume of phosphate buffered solution by wind and percussion method, and centrifuged for 2 times of 20 minutes with the speed of 1 500 r/min. Discard the supernatant, then the concentration of the cells was adjusted into 1×106/cm2 with the M199 medium containing 20% fetal bovine serum and 10 μg/L vascular endothelial growth factors.
Primary culture of endothelial progenitor cells
Mononuclear cells were seeded on the 24-well plate which paved with FN in advance in accordance with 1×106/cm2, then added in the M199 medium and static cultured in the incubator at 37 ℃ with the fraction of 5% CO2 and the 100% humidity. Peripheral blood endothelial progenitor cells: the cells were standing for 3 days, then the non-adherent cells were washed away, and the medium was changed once every 3 days; bone marrow endothelial progenitor cells: the non-adherent cells were washed away after standing for 24 hours, the medium was changed for half every day, and then the medium was totally change and observed and recorded.
Immunofluorescence cytochemistry staining
The adherent cells were cultured with 4% paraformaldehyde for different times, then washed with phosphate buffered solution and closed. The cells were firstly dropped with anti-body of CD34, CD133 and KDR with the concentration 1:100, cultured under 4 ℃ for overnight, then washed with phosphate buffered solution for 5 minutes and for 3 times; then added with FITC-conjugated secondary antibody with the concentration of 1:50, cultured under dark incubation for 30 minutes; then washed with phosphate buffered solution for 5 minutes and for 3 times. The results were observed under inverted fluorescence microscope.
Flow cytometry
Primary cells were selected by flow cytometry for counting, then 2×105 adherent cells were mixed with CD34 and KDR monoclonal antibody respectively, incubated at room temperature for 30 minutes; cells were washed with phosphate buffered solution for 2 times, then added with FITC-conjugated secondary antibody and diluted in dark, kept at 4 ℃ in dark for 30 minutes; washed the cells with phosphate buffered solution, and the cells were detected on the machine after suspended with 300 μL phosphate buffered solution.
Determination of ingest function
The adherent cells were incubated with low density lipoprotein labeled with 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate (10 mg/L) under 37 ℃ for 4 hours, then the cells were fixed with 2% paraformaldehyde for 10 minutes. The cells were immersed with phosphate buffered solution, and the FITC-labeled Ulex europaeus agglutinin 1 (10 mg/L) was added to the samples and incubated at 37 ℃ for 1 hour. The results were observed under laser confocal microscope.
Main outcome measures
The morphology of the cells was observed under microscope; the expression of CD34, CD133 and KDR was detected with immunofluorescence and the positive rate of CD34 and KDR was detected with flow cytometry.

In 1997, Asahara et al ^[1] firstly proposed endothelial progenitor cells, the CD34⁺ mononuclear cells were isolated by magnetic sorting method from human peripheral blood, and the cells cultred in vitro were adherent and in spindle-shaped. The cells can be specifically homing in angiogenesis tissue, can express the endothelial progenitor cell-specific antigen, differentiate and proliferate into mature endothelial cells and form bureaucratic tone-like structure, the cells are the endothelial progenitor cells. Recent studies confirmed that both endothelial progenitor cells and hematopoietic stem cells are derived from a common blood-hemangioblastoma and settled in the bone marrow^[2]. In addition, Asahara et al[3] established transgenic mouse bone marrow transplantation model found that these cells were not only exited in the peripheral blood, but also in the bone marrow, spleen and cord blood. Endothelial progenitor cells cultured for different times are in oval, long spindle or spindle-shaped, the morphology of tge cells mainly identified by cell surface markers, but unified endothelial progenitor cells specific surface markers have not found yet. Currently, the blood cell differentiation CD34, CD133, vascular endothelial growth factor receptor 2, stem cell membrane receptor (cKit), stem cell antigen (Sca^-1), von Willebrand factor are the major cell surface markers of endothelial progenitor cells^[4]. CD133 is the hematopoietic stem cell marker earlier than CD34, and it has been regarded as a marker to distinguish immature of endothelial progenitor cells^[5]. Now most of the studies using CD34⁺/vascular endothelial growth factor 2⁺ double-positive cell markers or CD34⁺/vascular endothelial growth factor 2⁺/CD133⁺ three positive markers to identify endothelial progenitor cells^[6]. Therefore, we have chosen CD34, KDR and CD133 as the surface antibodies to identify endothelial progenitor cells.

Endothelial progenitor cells take part in angiogenesis after ischemic stroke. Endothelial progenitor cells gather at the injury and ischemia and hypoxia tissue, and plays an important role in endothelial repairing after vascular injury and establishment of collateral circulation vascular network, and has great application prospects in repairing damaged vascular endothelium, promoting nerve regeneration, promoting the establishment of collateral circulation and building microvascular tissue engineering networks.

Angiogenesis and angiogenesis are the two main forms of blood vessel growth. Various factors can give birth to new blood vessels through sprouting way under the existing vascular which called angiogenesis; angiogenesis is the formation of blood vessels in the avascular zone, that is endothelial progenitor cells raising and homing to the damaged or ischemic tissue area, and forming the new blood vessels or vascular network. Earlier, many scholars believe that angiogenesis only occurs in the embryonic period, and disappears after birth^[7]. The discovery and the research of endothelial progenitor cells confirmed that angiogenesis plays an important role in postnatal vascular formation and vascular damage repairing. Zhang et al ^[8] transplanted the bone marrow cells marked with integrated LacZ gene to the mouse model of focal ischemia, and finding that a large number of marked bone marrow cells could be seen around the infarction and the cells were involved in angiogenesis. Besler et al ^[9] found that the bone marrow cells were large gathered around the infraction after injected into the cerebral ischemia rat, indicating that bone marrow-derived cells can migrate and “homing” to the damaged brain tissue. Endothelial progenitor cells can promote angiogenesis and promote the repairing of endothelial damage. Shirota et al^[10] seeded the endothelial progenitor cells on the microvascular bracket, and found that a large number of endothelial progenitor cells proliferated in the vascular lumen and formed cobblestone and cobblestone-like cell layer which can improve microcirculation in the ischemic area and promote the nerve growth. Au et al ^[11] found that after endothelial progenitor cells transplantation, a large number of blood vessels or vascular network-like structures appeared, the perfusion could be seen in the cavity; Huang et al ^[12] transplanted the endothelial progenitor cells into the rabbits with carotid artery injury, and found that a large number of endothelial progenitor cells were gathered in the damaged blood vessels lumen or wall and on the surface of new blood vessels, the vascular re-endothelialization area was 91.6%, analysis results showed that endothelial progenitor cells can secrete the anti-thrombotic biologically active substances: nitric oxide, prostaglandins 1 and tissue-type plasminogen activator and other relevant factors, inhibit intimal hyperplasia, and endothelial progenitor cells transplantation can be considered as a treatment method to prevent thrombosis and restenosis after intervention.

In 2004, Hur et al ^[13] proposed two types of endothelial progenitor cells from adult peripheral blood, according to the significant differences of their sources, morphology, surface markers and proliferation, the cells were named as early endothelial progenitor cells and late endothelial progenitor cells. Early endothelial progenitor cells were separated from the peripheral blood, elongated spindle-shaped and bipolar needle shape, colony aggregation growth, appeared after primary cultured for 4 to 7 days, and after cultured for 3 to 4 weeks, the cells were in oval cobblestone morphology, the proliferation ability was decreased gradually and apoptosis; the early endothelial progenitor cells has relationship with the macrophages and hematopoietic stem cells co-expressed surface markers of CD34, CD45 and CD133. Late endothelial progenitor cells were separated from the bone marrow, the endothelial progenitor cells appeared after mesecnhymal stem cells adherent cultured for 2 to 3 weeks, sand can sustained and steady proliferate for more than 12 weeks and in typical cobblestone-like morphology, and the cells can express the cell-specific antigens. Our experiments found that the endothelial progenitor cells both from the bone marrow or peripheral blood can expressed CD34, CD133 and KDR; the early and late endothelial progenitor cells were identified according to the strong cloned proliferation ability of stem cells, and the endothelial progenitor cells identified with this method is called late endothelial progenitor cells or endothelial outgrowth cells, and the endothelial outgrowth cells can also express the specific surface markers of CD34, CD133 and KDR. In our experiments, we found that the endothelial progenitor cells cultured from the bone marrow samples have strong proliferative activity, and in the early stage, the cells were in long spindle-shaped and bipolar needle shape, the cells were in colony-like aggregation growth; after cultured for 7 days, the cells were in typical cobblestone and paving stone-like shape; after 1-2 months later, a large number of long spindle-shaped cells mass were reunion and connected, and formed a large number of cell colonies, and then the cells were sub-cultured for many times successfully and finding that the proliferative activity is still strong and the cells were in blood vessel-like growth.

We isolated a large number of late endothelial progenitor cells from bone marrow which having the strong proliferation activity, morphology and surface markers in line with the reported features. During culturing process, the cells were found with the characteristics of blood vessel activity, and the cells were in lumen and bridging-like growth. The cells were subcultured for many times successfully and finding that the proliferative activity is still strong, large numbers of island-like colony growth appeared. Therefore, we believe that the late endothelial progenitor cells with strong proliferative activity can be obtained from the bone marrow, and a better seed source of vascular stem cells was founded. Some literatures reported that^[14] the mononuclear cells isolated from the bone marrow were treated with EGM-2MV and M199 culture medium for different induction times and identified with different surface markers to identify the early and late endothelial progenitor cells, and the results found that bone marrow-derived mononuclear cells cultured in EGM-2MV medium can be differentiated into late endothelial progenitor cells, and the bone marrow-derived mononuclear cells cultured in M199 medium can be differentiated into long spindle-shaped early endothelial progenitor cells. The results indicated that the proliferative activity of the endothelial progenitor cells cultured in EGM-2MV medium was stronger than that cultured in the M199 medium, and the endothelial progenitor cells cultured in EGM-2MV medium have the angiogenesis capacity. Flow cytometry found that expression of cell surface markers CD133 and FLK-1 was lower than the previously reported. But in our experiment, we found a lot of late endothelial progenitor cells in M199 medium, surface markers CD34, CD133 and KDR of endothelial progenitor cells were positive, so the differences between early endothelial progenitor cells and late endothelial progenitor cells may come from the sources of specimen and the methods.

Isner et al^[15] the used exogenous angiogenic growth factor to promote the establishment of micro-circulation ischemia, proposed a “therapeutic angiogenesis” concept. With the development of endothelial progenitor cells, the physiological significance of angiogenesis and the high proliferative potential and oriented homing characteristics of endothelial progenitor cells have been gradually elucidated, providing a new idea for the treatment of ischemic cerebrovascular disease with revascularization. According to the characteristics of vascular targeting positioning of endothelial progenitor cells, endothelial progenitor cells will be amplified in vivo and in vitro for transplantation therapy, or through the endothelial progenitor cells gene therapy to promote the endothelial progenitor cells highly express angiogenesis inducing factor, promote angiogenesis in ischemic cerebrovascular disease, improve the local ischemia blood flow and establish the collateral circulation. The effect of stem cells combined gene therapy with the endothelial progenitor cells as seed cells for the treatment of angiogenesis is more significant. We isolated endothelial progenitor cells from bone marrow and peripheral blood in this experiment, and a large number of late endothelial progenitor cells from bone marrow which having the strong proliferation activity, morphology and surface markers in line with the reported features were isolated, and found a better seed source of vascular stem cells, in order to provide technical and theoretical support for endothelial progenitor cells in experimental research and clinical studies of ischemic cerebrovascular disease.

从大鼠骨髓和外周血中分离出大量增殖活性强，形态学及表面标志均符合文献报道的晚期内皮祖细胞，找到了更好的成血管干细胞的种子来源。

缺血性脑卒中是威胁人类健康和安全的疾病，目前主要依靠药物治疗，药物在急性期显著降低了死亡率，但在后期神经功能的恢复方面收效甚微，介入治疗严格的治疗时间窗和适应证使得仅有少部分患者获益。随着生命科学领域的发展，组织工程技术、细胞治疗、基因治疗已广泛应用于医学领域，干细胞治疗可能成为缺血性脑卒中最有希望的治疗方法之一。随着骨髓源性与外周血中的内皮祖细胞的发现，血管生成的生理、病理意义及内皮祖细胞的高增殖潜能和定向归巢特性逐渐被阐明，为血管重建治疗缺血性脑血管疾病提供了新思路。利用内皮祖细胞定向迁移及对内膜损伤血管靶向定位的特性，通过内皮祖细胞在体内外扩增进行移植治疗，或通过内皮祖细胞联合基因治疗使内皮祖细胞高表达血管新生诱导因子，促进缺血性脑血管病血管新生。内皮祖细胞的生物学特性预示其在缺血性疾病领域有广阔的潜力，本实验从大鼠骨髓和外周血中分离获得了内皮祖细胞，并分离出大量增殖活性强，形态学及表面标志均符合文献报道的晚期内皮祖细胞，以期为内皮祖细胞在缺血性脑血管病的实验研究和临床研究方面提供技术及理论支持。

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