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Potential Differentiation of Human Mesenchymal Stem Cell Transplanted in Rat Corpus Cavernosum Toward Endothelial Or Smooth Muscle Cells

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Potential Differentiation of Human Mesenchymal Stem Cell Transplanted in Rat Corpus Cavernosum Toward Endothelial Or Smooth Muscle Cells International Journal of Impotence Research (2007) 19, 378–385 & 2007 Nature Publishing Group All rights reserved 0955-9930/07 $30.00 www.nature.com/ijir ORIGINAL ARTICLE Potential differentiation of human mesenchymal stem cell transplanted in rat corpus cavernosum toward endothelial or smooth muscle cells YS Song1,6,HJLee2,6,IHPark2, WK Kim3,JHKu4, SU Kim3,5 1Department of Urology, Soonchunhyang School of Medicine, Seoul, Korea; 2Brain Disease Research Center, Ajou University School of Medicine, Suwon, Korea; 3Institute of Regeneration Medicine, Gacheon University Ghill Hospital, Inchon, Korea; 4Department of Urology, Seoul National University Hospital, Seoul, Korea and 5Division of Neurology, Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada One of the causes of erectile dysfunction (ED) is the damaged penile cavernous smooth muscle cells (SMCs) and sinus endothelial cells (ECs). To investigate the feasibility of applying immortalized human mesenchymal stem cells (MSCs) to penile cavernous ECs or SMCs repair in the treatment of ED, the in vivo potential differentiation of the immortalized human MSCs toward penile cavernous endothelial or smooth muscle was investigated. One clone of immortalized human bone marrow mesenchymal stem cell line B10 cells via retroviral vector encoding v-myc were transplanted into the cavernosum of the Sprague–Dawley rats and harvested 2 weeks later. The expression of CD31, von Willebrand factor (vWF), smooth muscle cell actin (SMA), calponin and desmin was determined immunohistochemically in rat penile cavernosum. Multipotency of B10 to adipogenic, osteogenic or chondrogenic differentiation was found. Expression of EC specific markers (CD31 or vWF protein) and expression of SMC specific markers (calponin, SMA or desmin protein) were demonstrated in grafted B10 cells. When human MSCs were transplanted into the penile cavernosum, they have the potential to differentiate toward ECs or SMCs. Human MSCs may be a good candidate in the treatment of penile cavernosum injury. International Journal of Impotence Research (2007) 19, 378–385; doi:10.1038/sj.ijir.3901539; published online 26 April 2007 Keywords: injury repair; human mesenchymal stem cell; endothelium; smooth muscle; penis; erectile dysfunction Introduction sclerosis or surgery,1,2 and accelerating repair of intact cavernous SMCs and sinus ECs repair can be a One of causes of erectile dysfunction (ED) is the novel way in the treatment of ED. damage in penile cavernous smooth muscle cells Bone marrow contains two types of stem cells: (SMCs) and sinus endothelial cells (ECs) by the hematopoietic stem cells and mesenchymal stem various metabolic conditions or mechanical manip- cells (MSCs). MSCs are a population of self-renew- ulations such as diabetes, hypertension, athero- ing stem cells with pluripotent capacity to differ- entiate into different cell types.3 Previous studies have demonstrated in experimental animals that the MSCs transplanted into the brain, heart and other Correspondence: Dr JH Ku, Department of Urology, Seoul organs differentiate into cell types of host-grafted National University Hospital, 28, Yongon Dong, Jongno site.3–6 In a canine chronic ischemia model, MSCs Ku, Seoul 110-744, Korea. differentiated into SMCs and ECs, resulting in E-mail: [email protected] or Dr SU Kim, Institute of increased vascularity and improved cardiac func- Regeneration Medicine, Gacheon University Ghill Hospi- tion.5 Transplantation studies in mouse ischemia tal, 534-2, Yonsu-dong, Inchon 406-799, Korea. E-mail: [email protected] models also showed an engraftment at ischemic 6 hind limb or myocardium lesions induced func- These authors contributed equally to this work. 7,8 Received 8 September 2006; revised 18 December 2006; tional recovery. accepted 21 December 2006; published online 26 April More recent studies in human patients have 2007 reported that the transplantation of autologous Potential differentiation of human MSC in corpus cavernosum YS Song et al 379 MSCs provided improvement in clinical outcome.9,10 gestational age). The permission to use the fetal Therefore, MSCs fulfill all criteria of true stem cells, tissues was granted by the Clinical Screening that is, self-renewal, multilineage differentiation and Committee for Research involving human subjects in vivo reconstitution of tissue.11 The major advantage of the University of British Columbia. Bone marrow of MSCs is the vast number of cells that can be cells were grown in modified Eagle medium -a harvested from one bone marrow aspirate. The (MEM-a) supplemented with 10% fetal bovine relative ease of isolating MSCs from bone marrow serum (FBS) and 25 mg/ml gentamicin. After the and the great plasticity of the cells make them ideal cultures reached confluency, the cells were lifted tools for an autologous or allogeneic cell therapy. with phosphate-buffered saline (PBS) containing Although previous studies have shown that MSCs 0.1% trypsin and 1 mM ethylenediamine tetraacetic transplantation can regenerate damaged vascular acid (EDTA) at 371C for 3 min and passaged into new endothelium and myocardium,12 it remains un- dishes at 1:3 dilution. known whether MSCs can be used to repair penile cavernous smooth muscle and sinus endothelium. Because primary MSCs can be provided for only limited time before they undergo senescence, we Immortlized human MSC cell line have generated an immortalized human MSC cell An amphotropic replication-incompetent retroviral line via retroviral vector encoding v-myc and vector encoding v-myc oncogene (transcribed from utilized for the study to investigate if immortalized mouse leukemia virus LTR plus neomycin-resistant human MSCs could have the potential to differenti- gene transcribed from a SV40 early promotor) was ate toward ECs or SMCs. used to infect human fetal bone marrow MSCs inducing propagation of immortalized human bone marrow MSC cell lines (Figure 1a). This ampho- Patients and methods tropic vector, LMmyc, was generated in our labora- tory using the ecotropic retroviral vector encoding Primary cell culture v-myc (ATCC, Manhasset, VA, USA) to infect PA317 Primary cultures of human bone marrow cells were amphotropic packaging cell line. MSCs were sub- obtained from fetal spinal vertebrae (14–18 weeks jected to retrovirus-mediated transduction of v-myc Figure 1 Human MSC cell line (B10 cells). (a) B10 was generated by a retroviral vector encoding v-myc. (b) Phase-contrast microscopy of primary human MSC culture (left) and B10 human mesenchymal stem cell line (right). BM3 is one of primary human fetal bone marrow colonies and B10 was established from BM3. (c) Cell markers of B10 cells. B10 cells express MSC markers CD29, CD44 and CD166. Surface phenotype was analyzed by FACS. The x axis of the histogram displays the fluorescence intensity, which is usually measured on a log scale. The y axis displays the number of cells found within each parameter. Fl1H represents fluorescein isothiocyanate (FITC) labeled-conjugated antibody and FL2H represents phycoerythrin (PE) labeled-conjugated antibody, respectively. B10 cells were labeled with FITC-coupled antibodies specific for CD29, CD44 and CD166 or immunoglobulin isotype control antibodies. Open histograms are for control immunoglobulins and colored histograms are for specific antibodies. International Journal of Impotence Research Potential differentiation of human MSC in corpus cavernosum YS Song et al 380 by LMmyc construct and subsequent cloning. Infec- plastic coverslips (9 mm in diameter) for 3–14 days, tion of human MSCs in 6-well plates was performed fixed in cold methanol for 10 min at À201C, air-dried twice by the established procedures. Briefly, 2 ml of and incubated with antibodies specific for each supernatant (4 Â 105 CFUs) from the packaging cell antigen marker. Cultures incubated with primary line and 8 mg/ml polybrene (Sigma, St Louis, MO, antibodies were followed by biotinylated secondary USA) were added to target cells in six-well plates antibodies and avidin–biotin complex (ABC, Vector, and incubated for 4 h at 371C; the medium was then Burlingame, CA, USA) and visualized with 3-amino- replaced with fresh growth medium; infection was 9-ethyl carbazole (Sigma) chromogen development. repeated 24 h later. Seventy-two hours after the Cell type-specific markers used were CD31 and vWF second infection, infected cells were selected with for ECs, desmin, calponin and smooth muscle actin G418 (250 mg/ml; Sigma) for 7–14 days and large for SMCs. Cells were counterstained with 40,6- clusters of clonally derived cells were individually diamino-2-phenylindole (DAPI, Sigma) to identify isolated and grown in six-well plates. Individual cellular nuclei. Following immunostaining, cells clones were generated by limited dilution and were mounted on glass slides using gelvatol and propagated further. At this phase of isolation, viewed under an Olympus laser-scanning confocal individual clones were designated as human MSC microscope (Tokyo, Japan). cell lines. One of these clones, B10 was subjected to further study. Transplantation of human MSCs into the rat penile cavernosum Fluorescence-activated cell sorting Ten week-old male Sprague–Dawley rats (300–320 g, For fluorescence-activated cell sorter (FACS), B10 n ¼ 20) were used in this study. Rats were anesthe- cells were detached and stained sequentially with tized with 1% ketamine (30 mg/kg) and xylazine immunofluorescence conjugated antibodies, fixed hydrochloride (4 mg/kg). The penile skin incision with
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