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 mesenchymal stem cell transplanted in corpus cavernosum toward endothelial or 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 and harvested 2 weeks later. The expression of CD31, von Willebrand factor (vWF), smooth muscle cell (SMA), 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 ) 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- 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 2% paraformaldehyde for 5 min and then was made and the penile cavernosum was dissected analyzed with flow cytometer (FACS Vantage, and palpated. B10 human MSCs (1 Â 106 cells) were Becton Dickinson, Franklin Lakes, NJ, USA). transplanted into the rat cavernosum using 500 ml syringe with 26G needle and the incised penile skin was sutured. To prevent rejection or infection, Differentiation culture conditions 10 mg/kg of intraperitoneal Cypol (cyclosporine; Adipogenic differentiation of B10 cells was induced Chong Kun Dang, Seoul, Korea) with 10 mg/kg by growing the cells in a 6-well plate in Dulbecco’s injection of Flomoxef (cephalosporin; Ildong, Seoul, modified Eagle’s medium (DMEM) containing 10% Korea) was administered intraperitoneal daily. The FBS, 5 mg/ml insulin, 1 mM dexamethasone, 100 nM penile cavernosum was harvested 2 weeks after the indomethacin and 0.5 mM methylisobutylxanthine cell transplantation. (Sigma) for 48 h, then cells were incubated in the same medium without dexamethasone. One week after the induction, cells were stained with oil-red. Immunofluorescence microscopy To induce osteogenic differentiation, B10 cells were Each animal was anesthetized and perfused through cultured in DMEM containing 10 mM b-glyceropho- the heart with 100 ml cold saline and 100 ml of 4% sphate, 0.2 mM ascorbate-2-phosphate and 10 nM paraformaldehyde in PBS. After 24 h of fixation in dexamethasone. After 14 days, osteogenic differen- 4% paraformaldehyde, the penis was cryoprotected tiation was evaluated by alkaline with 30% sucrose for 24 h and cut into 30 mm staining. For chondrogenic differentiation, pellets sections in a cryostat (Leica CM 1900, Houston, of B10 cell were cultured for 3 weeks in the presence TX, USA). Adjacent serial coronal sections were of transforming growth factor-b1 (TGFb-1) in DM4 processed for human nuclear immunofluorescent serum-free medium (DMEM containing 10 mg/ml staining with human antinuclear antibody (1:400, human insulin, 10 mg/ml human transferrin, 3 nM Chemicon & Abcam, Cambridge, MA, USA) to sodium selenite, 5 nM hydrocortisone and 100 pM 13 identify the human nuclear-positive, transplanted triiodothyronine). This medium was replaced cells. Monoclonal antibodies specific for CD31 every 3–4 days for 21 days. Development of (1:200, R&D System, Mineapolis, MN, USA) and chondrogenic differentiation was determined by von Willebrand factor (1:100, R&D) were used for staining the pellet with Alcian blue. cell-type identification of ECs, whereas antibodies specific for smooth muscle a-actin (1:1000; Sigma), desmin (1:10 000, Chemicon, Temecula, CA, USA) Immunocytochemistry and calponin (1:500; Sigma) for cell-type identifica- Immunochemical determination of cell type-specific tion of SMCs. Penis sections were incubated in markers in B10 cells was performed as follows: B10 primary antibodies overnight at 41C as free-floating cells were grown on poly-D-lysine-coated Aclar sections, and followed by Alexa Fluor 488-conju-

International Journal of Impotence Research Potential differentiation of human MSC in corpus cavernosum YS Song et al 381 gated anti-mouse immunoglobulin G (IgG) and determined by staining the cell pellets with Alcian Alexa Fluor 594-conjugated anti-mouse IgG (1:400, blue, which positively identifies proteoglycan ex- Molecular Probe, Invitrogen, Carlsbad, CA, USA) for tracellular matrix, specific components of cartilage 1 h at room temperature. Negative control sections tissues (Figure 2c). As in primary human MSCs, B10 from each animal were prepared for immunohisto- MSCs expressed markers of adipocytes, of osteo- chemical staining in an identical manner except the blasts and of chondrocytes following culture under use of normal goat serum as a secondary antibody. the appropriate differentiation-inducing condi- The following steps were performed at room tions.6 temperature and all washing steps were performed twice with phosphate buffer saline Tween-20 (PBST) for 5 min. Nonspecific binding sites were blocked by Properties of human MSCs incubating with normal goat serum (10%, n/v in In B10 human MSCs, B10 cells expressing immu- PBS) for 40 min. These procedures were repeated six noreactivities against CD31 or vWF were not found times. Stained sections were then examined under indicating that the B10 cells do not differentiate into an Olympus laser confocal fluorescence microscope. ECs (Figure 3). In B10 human MSCs, B10 cells expressing immunoreactivities against desmin, ca- lonin or smooth muscle actin protein were not Results found indicating that the B10 cells do not differ- entiate into SMCs (Figure 3). Phenotype of human MSCs One of immortalized human mesenchymal stem cell lines, B10 were isolated. Phase contrast microscopy Differentiation of human MSCs toward ECs of primary human MSC culture and B10 human In penile sections transplanted earlier with B10 mesenchymal stem cell line showed spindle shape human MSCs, B10 cells expressing CD31 or vWF (Figure 1b). were found indicating that the B10 cells differen- Karyotype of B10 is 46, XX that is normal human tiated into ECs (Figure 4). The merged cells expres- karyotype (data not shown). B10 cells express cell sing yellow fluorescence were CD31 þ or vWF þ type-specific markers for MSC markers CD29, CD44 cells that derived from B10 human MSCs (Figure 2). and CD166 (Figure 1c), but do not express cell type markers for hematopoietic stem cells CD24, CD34 or CD45 (data not shown). Differentiation of human MSCs toward SMCs In penile sections transplanted earlier with B10 human MSCs, B10 cells expressing immunoreactiv- Differentiation of human MSCs ities against calponin, smooth muscle actin or After 7 days of culture in adipogenic culture desmin protein were found indicating that the B10 medium, more than 80% of B10 MSCs differentiated cells differentiated into SMCs. A large proportion of into lipid-laden cells that stained with oil-red the transplanted HuNu-positive cells (15–20%) was (Figure 2a). Total cell numbers were counted by calponin-positive and took the morphology of hematoxylin staining, and then the percentage of smooth muscle. Many of the HuNu/calponin dou- oil-red positive cells was calculated. After 14 days of ble-positive cells were found in the smooth muscle culture in osteogenic medium, B10 MSCs differen- of corpus cavernosum. A smaller proportion (3–5%) tiated into osteoblasts and this was confirmed by of transplanted B10 MSCs differentiated into SMA- strong alkaline phosphatase staining (Figure 2c).14,15 or Desmin-positive SMCs in the corpus cavernosum Differentiation of B10 MSCs into chondroblasts was (Figure 5).

Figure 2 Multipotency of B10. (a) Adipogenic differentiation, oil red O staining. Original magnification  100. (b) Osteogenic differentiation, alkaline phosphatase staining. Original magnification  100. (c) Chondrogenic differentiation, toluidine blue staining. Original magnification  100.

International Journal of Impotence Research Potential differentiation of human MSC in corpus cavernosum YS Song et al 382 a c e g i

hPECAM-I vWF Desmin Calponin SMA

b d f h j

merge merge merge merge merge

Figure 3 Expression of cell type markers for ECs or SMCs in B10 human mesenchymal stem cell line. (a and c) The cells expressing red fluorescence are CD31 or vWF-positive cells were not found. (e, g and i) The cells expressing red fluorescence are desmin-, calponin- or smooth muscle actin-positive cells were not found. (b, d, f, h and j) The cells expressing blue are DAPI-positive human cell nuclear cells derived from B10 cells. Scale bar ¼ 50 mm. (For colour figure see online version.)

a bc

hNu CD31 merge

d ef

hNu vWF merge

Figure 4 Expression of cell type markers for ECs CD31 and vWF protein in B10 human mesenchymal stem cell line after the cell transplantation into the rat cavernosum. (a) The cells expressing green fluorescence are human nuclear antibody-positive cells derived from B10 cells. (b) The cells expressing red fluorescence are CD31-positive cells. (c) The merged cell expressing yellow fluorescence are CD31-positive cells derived from B10. (d) The cells expressing green fluorescence are human nuclear antibody-positive cells derived from B10. (e) The cells expressing red fluorescence are vWF-positive cells. (f) The merged cell expressing yellow fluorescence are vWF- positive cells derived from B10. Scale bar ¼ 50 mm. (For colour figure see online version.)

Discussion (EPCs) may contribute to re-endothelization after endothelial injury.12,19 However, one major criticism The integrity of cavernous ECs and SMCs is critical of studies describing plasticity of bone marrow cells in maintaining and regulating erectile function. is the heterogeneity of the cell population. EPCs Accelerating intact ECs and SMCs repair is a key show some phenotypic overlaps with hematopoietic method for treating ED. Endothelial regeneration cells and controversy still exists with respect to the depends on the proliferation and migration of the identification and origin of EPCs.20 adjacent uninjured ECs toward injury site and the MSCs are self-renewing and multipotential stem recruitment of bone marrow-derived progenitor cells in the bone marrow. The human MSCs, with cells.16–18 Several investigators have suggested that their attributes of (1) ease of isolation, (2) high ex vivo expanded endothelial progenitor cells expansion potential, (3) genetic stability, (4) repro-

International Journal of Impotence Research Potential differentiation of human MSC in corpus cavernosum YS Song et al 383 abc

hNu calponin merge

def

hNu SMA merge

ghi

hNu desmin merge

Figure 5 Expression of cell type markers for SMCs calponin, smooth muscle actin (SMA) and desmin in B10 human mesenchymal stem cell line. (a) The cells expressing green fluorescence are human nuclear antibody-positive cells derived from B10. (b) The cells expressing red fluorescence are calponin-positive cells. (c) The merged cell expressing yellow fluorescence are calponin-positive cells derived from B10. (d) The cells expressing green fluorescence are human nuclear antibody-positive cells derived from B10. (e) The cells expressing red fluorescence are SMA-positive cells. (f) The merged cell expressing yellow fluorescence are SMA-positive cells derived from B10. (g) The cells expressing green fluorescence are human nuclear antibody-positive cells derived from B10s. (h) The cells expressing red fluorescence are desmin-positive cells. (i) The merged cell expressing yellow fluorescence are desmin-positive cells derived from B10. Scale bar ¼ 50 mm. (For colour figure see online version.)

ducible attributes from isolate to isolate, (5) repro- types that are similar to the cells in the local ducible characteristics in widely dispersed labora- microenvironment.27 Recent report suggested that tories, (6) compatibility with tissue engineering cell fusion might explain some of the observed principles and (7) potential to enhance repair in plasticity of adult stem cells.28,29 Our results many vital tissues, may be the current preferred support the possibility of the transplantability of stem cell model for cellular therapeutic develop- MSCs and their potential utility in the treatment of ment.21 One might expect allogenic MSCs would ED. We examined the phenotype of human MSCs stimulate T-cell proliferation and that donor MSCs after in vivo transplantation to the penile caverno- would be recognized by responder T cells and sum, and the grafted MSCs expressed EC antigens rejected by a recipient host. However, examination (CD31 and vWF) as well as SMC antigens (SMA, of the interaction of allogenic MSCs with cells of the calponin or desmin)30 after transplantation into the immune system indicates little rejection by T penile cavernosum. Therefore, these results show cells.22–24 Recent studies have shown that MSCs that MSCs have the milieu-dependent differentia- can acquire several features of mature ECs when tion potential along endothelial and smooth muscle cultured under optimal condition.25 MSCs may lineage when transplanted into the penile caverno- therefore be an alternative source for EPCs.26 sum. Animal experiments have shown that MSCs can Our study shows for the first time that human prevent deleterious remodeling and improve recov- bone marrow-derived MSCs (CD29 þ CD44 þ ery after myocardial infarction.21 Thus, it may be CD166 þ ) are capable of differentiating into ECs hypothesized that MSCs also contribute to replace- and SMCs in vivo, which make them attractive ment of SMCs after carvernous SMC loss. candidates for cell therapy in patients suffering from Adult stem cells from bone marrow undergo ED. Although we found that the transplanted MSCs milieu-dependent differentiation to express pheno- to penile cavernosum had the ability to differentiate

International Journal of Impotence Research Potential differentiation of human MSC in corpus cavernosum YS Song et al 384 toward ECs or SMCs in the local microenvironment, 2 Sainz I, Amaya J, Garcia M. Erectile dysfunction in heart it is not clear if these cells respond same way toward disease patients. Int J Impot Res 2004; 16: S13–S17. injury in the penile cavernosum. 3 Liechty KW, MacKenzie TC, Shaaban AF, Radu A, Mosley AM, Deans R et al. Human mesenchymal stem cells engraft and The source of the cells that repair tissues after demonstrate site-specific differentiation after in utero trans- injury remains poorly defined and controversial. plantation in sheep. Nat Med 2000; 6: 1282–1286. One possible source is stem cell-like progenitors 4 Deng J, Petersen B, Steindler DA, Jorgensen ML, Latwell ED. that are endogenous to injured tissues. 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Science 1999; 284: 143–147. to the tissue.35,36 It is not known if the in vivo- 7 Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, transplanted MSCs act completely as a mature ECs Kearney M et al. Transplantation of ex vivo expanded or SMCs in the early phase following transplanta- endothelial progenitor cells for therapeutic neovasculariza- tion. Proc Natl Acad Sci USA 2000; 97: 3422–3427. tion. However, it is possible that certain injuries on 8 Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B mature ECs or SMCs play the important role in the et al. Bone marrow cells regenerate infarcted myocardium. induction of MSC differentiation.25 Nature 2001; 410: 701–705. Our study has some limitations. First, at present, 9 Bang OY, Lee JS, Lee PH, Lee G. Autologous mesenchymal we do not know whether cellular therapy may help stem cell transplantation in stroke patients. Ann Neurol 2005; 57: 874–882. or hurt penile erectile function. 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