Preparation of human cerebral endothelial cells
This study was conducted using our previous research method [5].
HEN6 cells were prepared from human fetal cerebral vasculature. The Institutional Animal Care and Use Committee of the hospital granted approval for study procedures, which were carried out in line with the guidelines of the National Institute of Health Guide for the Care and Use of Laboratory Animals. The periventricular area of telencephalic tissues of 11 to 14 weeks human fetal cadavers was used. We initially established primary cell cultures from the cerebral vasculature of these human fetuses [5].
After careful separation from the brain, the fetal cerebral vasculature was dissected into small fragments and then subjected them to homogenization with four strokes of a 1 mL Teflon-glass homogenizer (Wheaton, Millville, NJ, USA). Subsequently, the tissue debris was subjected to centrifugation at 600 g for five minutes at 4 °C. The next step was resuspension of the homogenate in 1 mL of 0.1% Collagenase/Dispase® (Boehringer Mannheim, Mannheim, Germany) in Ca2- and Mg2-free HBSS (Hanks Balanced Salt Solution) with 100 U/mL penicillin, 100 mg mL streptomycin, 20 U/mL DNase I, and 0.147 mg/mL Tosyl-lysine-chloromethyl ketone at pH 7.4. This mixture was incubated in a water bath at 37 °C for 60 min with agitation. The mixture was then centrifuged at 600 g for five minutes at 4 °C. The pellet was resuspended in 10 mL 25% BSA (bovine serum albumin) in HBSS and centrifuged at 1000 g for 15 min at 4 °C. The pellet was then resuspended in 1 mL of 0.1% collagenase/Dispase solution as previously outlined. After 10 min of incubation at 37 °C with sporadic agitation, it was again centrifuged at 1000 g for 15 min at 4 °C, yielding a pellet that contained a fraction abundant with retinal capillaries.
For seeding the fraction abundant with capillaries, tissue culture dishes (Becton Dickinson, Bedford, MA, USA) with a coating of rat tail type I collagen were used. Cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) with a composition of 20 mm of sodium bicarbonate, 15 ng/mL of ECGF (endothelial cell growth factor), 10 U/mL of heparin, 100 U/mL of penicillin, 100 mg/mL of streptomycin, 2.5 mg/mL of amphotericin B, and 20% FBS (fetal bovine serum). Culture conditions were: temperature of 37 °C, humidified atmosphere, and 5% carbon dioxide/air. A variety of colony types were distinguished after 14 days. The colony with a spindle-fiber morphology was enveloped with a stainless-steel penicillin cup and subjected to selective trypsinization. Meanwhile, colonies of pericytes, fibroblasts and other types of cells were eliminated via aspiration. After they underwent passage two to three times, cells underwent cloning from one cell via colony formation and double separation from other cells with a penicillin cup.
Once migration of circulating endothelial progenitor cell (CEPC), explants were eliminated. Colonies were monitored to keep track of contamination of smooth muscle cells. CEPC proliferation was permitted. A cell sorter (FACS Vantage, Becton Dickinson, Bedford, MA, USA) with fluorescence activation to classify cells with PE-conjugated anti-CD31 antibody (1:50; Becton Dickinson, Bedford, MA, USA), yielding more than 95% CD31-positive cells. Cells were validated to have an endothelial source via assimilation of Alexa-488-acetylated-LDL (Molecular Probes, Eugene, OR, USA), immunofluorescence for CD-31 (Becton Dickinson, Bedford, MA, USA), Vascular Endothelial Growth Factors (VEGF), von-Willebrand factor (vWF) (Abcam, Cambridge, UK), and vascular endothelial growth factor receptor2 (KDR) (Sigma Co., St Louis, MO, USA).
Immortalized human endothelial progenitor cells
Transduction of telomerase into CEPC was performed. Previously characterized primary dissociated telencephalic cells were subjected to replication-incompetence amphotropic infection with a retroviral vector underpinned by a Maloney murine leukemia viral (MMLV) backbone. A tetracycline (Tet)-response regulator vector was initially inserted into the culture. Resistance to G418 (300 μg/mL) was the basis for infectant selection. Subsequently, a telomerase-encoding retroviral vector was introduced into chosen clones. Infectant selection was based on hygromycin resistance of 200 μg/mL. A significant proportion of cells died during antibiotic-based selection (resistance to G418 and to hygromycin). This led to the emergence of colonies of phase-dark bipolar or polygonal cells displaying short processes [5]
Cell culture
Tissue culture dishes with a coating of 2% gelatin type B (Sigma Co., St Louis, MO, USA) were used for culturing hTERT immortalized HEN6. The culture environment was M199 (medium 199) (GIBCO, Waltham, MA, USA), to which 20% FBS, EGM-2MV (Clonetics), L-glutamine, 10,000 /mL penicillin, 10,000 /mL streptomycin, and 25 g/mL Fungizone were added. Growth took place in a humidified incubator at a temperature of 37 °C and 5% carbon dioxide, with a change of medium at three-day intervals [5]
Cytogenetics
Cytogenetic analysis of hTERT-hEPCs were performed to confirm the normal human karyotype of chromosomes.
Confirmation of HEN6
Cell lines were isolated by limiting dilution. Their clonal identity was confirmed. reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm the presence of telomerase messenger ribonucleic acid (mRNA) [5]
In vitro angiogenesis assay
We used 96-well tissue culture plates with a coating of 30 μL Matrigel (BD Biosciences, Franklin, NJ, USA) for seeding ECs, each well containing 5000–20,000 cells. After one day, we used an inverted microscope with 40 × magnification to view cells and to ascertain whether tubules resembling capillaries had formed.
Generation of cavernous nerve injured ED model and transplantation of HEN6 into the penile cavernosum
The Institutional Animal Care and Use Committee of the researcher’s hospital granted approval for all procedures, which we performed in line with the guidelines of the National Institute of Health Guide for the Care and Use of Laboratory Animals (2001). A total of 54 Sprague–Dawley eight-week-old male rats were equally divided into three groups at 2, 4 and 12 weeks: group 1, sham operation; group 2, bilateral CNI; and group 3, treated with HEN6 (1 × 106 cells) following CNI.
A solution of 30 mg/kg of 1% ketamine and 4 mg/kg xylazine hydrochloride was used to anesthetize rats. Cavernous nerve (CN) and major pelvic ganglion (MPG) posterolaterally on each side of the prostate were observed. No additional procedures in group 1. For rats in group 2 and 3, a hemostat was employed to isolate and crush the CN for two minutes on each side at a distance of 5 mm from its MPG origin. Next, a penile skin incision was made to dissect and palpate the penile cavernosum. For rats in group 3, HEN6 (1 × 106 cells) was transplanted into the cavernosa [9]. To prevent infection, Flomoxef (cephalosporin; Ildong, Seoul, Korea) was injected intraperitoneally at 10 mg/kg daily. The penile cavernosum was harvested at 2, 4, and 12 weeks after cell transplantation.
Measurement of erectile function after transplantation
After hTERT-hEPCs were transplanted, erectile responses in groups 1 to 3 were assessed at two, four, and 12 weeks after transplantation. A solution of 30 mg/kg 1% ketamine and 4 mg/kg xylazine hydrochloride was administered through an intraperitoneal injection to anesthetize rats. A midline laparotomy was subsequently performed for bilateral exposure of the main pelvic ganglia and cavernous nerve. Exposure of the penis and mobilization of the corpus spongiosum was then performed so that a 25-gauge needle could be inserted into the corpus cavernosum. A polyethylene-50 tube was used to attach a needle to a pressure transducer containing heparinized saline. After that, a bipolar electrode made of platinum was positioned around the cavernous nerve so that penile erections could be induced through electric stimulation. For electric stimulation, 1 and 5 V were used at 12-Hz frequency with a 1-ms square-wave interval for 60 s. The highest intracavernous pressure (ICP) was then calculated [10].
Immunohistochemical study
For immunohistochemistry, he cells were placed onto glass slides and incubated with 5% goat normal serum with 1% BSA (Jackson ImmunoResearch, West Grove, PA, USA) for blocking purposes. Slides were then incubated with primary antibodies of SMA (smooth muscle actin) (1:100; Sigma Co., St Louis, MO, USA) and vWF (1:100; Sigma Co., St Louis, MO, USA) at 22 °C for half an hour without light exposure. After washing out, slides were incubated Alexa-488, and Alexa-594 conjugated goat anti-mouse or anti-rabbit secondary antibodies (1:800; Molecular Probes, Eugene, OR, USA) diluted in blocking buffer at 22 °C for half an hour without. Slides were subsequently washed and incubated with DAPI (4’,6-diamidino-2-phenylindole) (1:30,000; Molecular Probes, Eugene, OR, USA) for five minutes to achieve cell nucleus staining [10]. Cell morphology and fluorescence were observed at 488- and 594-nm excitation wave-lengths using a microscope (Olympus, Tokyo, Japan). Quantitative analysis of histologic examinations was processed with an image analyzer system (National Institutes of Health [NIH] Image J 1.34, https://imagej.nih.gov/ij/).
Statistical analysis
Transplantation of stem cells was investigated using via two-way ANOVA (Analysis of variance) and post-hoc Tukey test. Data are expressed as mean ± SE (standard error), with statistical significance indicated by a P-value of less than 0.05.
