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A putative mesenchymal stem cells population isolated from adult human testes


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Gonzalez et al 2009

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A putative mesenchymal stem cells population isolated from adult human testes

  1. 1. A putative mesenchymal stem cells population isolated from adult human testes R. Gonzalez a , L. Griparic a , V. Vargas a , K. Burgee a , P. SantaCruz c , R. Anderson d , M. Schiewe d , F. Silva a,*, A. Patel b a DaVinci Biosciences LLC, 1239 Victoria Street, Costa Mesa, CA 92627, USA b Cardiovascular Center, University of Utah, Salt Lake City, UT, USA c Department of Regenerative Medicine, Omni Hospital, Guayaquil, Ecuador, USA d Southern California Center for Reproductive Medicine, Newport Beach, CA, USA a r t i c l e i n f o Article history: Received 13 May 2009 Available online 29 May 2009 Keywords: Adult stem cells Human testis Pluripotent markers MSCs Multilineage differentiation Cell-based therapy a b s t r a c t Mesenchymal stem cells (MSCs) isolated from several adult human tissues are reported to be a promising tool for regenerative medicine. In order to broaden the array of tools for therapeutic application, we iso- lated a new population of cells from adult human testis termed gonadal stem cells (GSCs). GSCs express CD105, CD166, CD73, CD90, STRO-1 and lack hematopoietic markers CD34, CD45, and HLA-DR which are characteristic identifiers of MSCs. In addition, GSCs express pluripotent markers Oct4, Nanog, and SSEA-4. GSCs propagated for at least 64 population doublings and exhibited clonogenic capability. GSCs have a broad plasticity and the potential to differentiate into adipogenic, osteogenic, and chondrogenic cells. These studies demonstrate that GSCs are easily obtainable stem cells, have growth kinetics and marker expression similar to MSCs, and differentiate into mesodermal lineage cells. Therefore, GSCs may be a valuable tool for therapeutic applications. Ó 2009 Elsevier Inc. All rights reserved. Introduction The search for an ideal stem cell population for therapeutic pur- poses has been a challenge for years and remains elusive. Recent data on cell transplantation into animal models of degenerative diseases and injuries illustrated the feasibility of the use of adult stem cells for regenerative medicine [1–3]. Mesenchymal stem cells (MSCs) are one of the most investigated adult stem cells. Suc- cess in transplantation of these cells stimulated the search for other mesenchymal cell populations from different tissues. It has been illustrated that cells isolated from umbilical cord blood [4], placental cord blood [5], adipose tissue [6], and dental pulp [7] have similar properties to MSCs, yet also possess unique characteristics. Several groups have reported that following transplantation of adult MSCs, patients’ symptoms improved significantly in various disease states [8–10]. Despite the uncertainty around the mecha- nism of adult stem cells action upon transplantation into the in- jured site, these cells are presently the most promising tool for cell-based therapies. Studies have demonstrated that MSCs may be supportive to tissue recovery [11], stimulate the synthesis of cytokines and matrix molecules [3], be angiogenic [2], have immu- nomodulatory effects [10], and stimulate endogenous tissue pro- genitors [3]. Nevertheless, due to the heterogeneity of disease it is reasonable to assume that each disease condition will require different properties from transplanted cells in order to improve the disorder to which the cells are being applied. Thus, it is imper- ative to investigate the use of several different cell types for ther- apeutic applications to address the specific disease condition in the most appropriate way. Recently, it was demonstrated that pluripotent cells may be iso- lated from germ-line stem cells within the human testis [12]. How- ever, similar to embryonic stem cells, these cells generate teratomas when transplanted into immunodeficient mice bringing into question their potential clinical application. In order to broad- en the array of tools for cell-based autologous therapies, we iso- lated a novel renewable stem cell population from the adult testes that has characteristics of MSCs, termed gonadal stem cells (GSCs). Here we demonstrate that GSCs are easily isolated, have similar growth kinetics, expansion rates, clonogenic capacity and differentiation potential as MSCs. Materials and methods Testicular biopsies were obtained from DV Biologics LLC (Costa Mesa, CA). Biopsies were isolated from six 22–47 years old donors after informed consent as approved by an institutional regulatory board (IRB) (DV Biologics). Cell culture. Tissue was digested with 0.1% Collagenase type II solution for 10–20 min at 37 °C. After filtration through 40 lm 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.05.103 * Corresponding author. Fax: +1 949 515 2929. E-mail address: (F. Silva). Biochemical and Biophysical Research Communications 385 (2009) 570–575 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage:
  2. 2. strainer and centrifugation at 1500g for 10 min 4 °C, cells were counted and plated on 10 cm dishes coated with Fibronectin 10 lg/ml (Sigma). Cells were cultured in DMEM high glucose (Gib- co) supplemented with 10% FBS (Hyclone), FGF2 (10 ng/ml), GDNF (10 ng/ml) (Invitrogen), and penicillin/streptomycin (Gibco). After 3 days, non adhering cells were discarded and attached cells were cultured and expanded. At 70–80% confluency cells were detached using Trypzen (Sigma) and re-plated on flasks without coating at density 1000 cells/cm2 . Colony-forming units (CFU) assay and cell cloning. For colonies, cells were plated at a density of 150 cells/10 cm dish. After 17 days, cells were fixed and stained in a 9% Crystal Violet Methanol solu- tion for 1 min. Cloning efficiency was estimated as percentage of cells which generated clones from total cell number/dish. For cell cloning, 100 cells/10 cm dish were seeded. Selected clones were isolated using cloning rings (Sigma Aldrich) and each clone was re-plated in one well of a 6-well plate. After reaching 70% conflu- ency cells were seeded in 75 cm2 flasks for further expansion. Growth kinetics. For growth curve, cells were plated onto 24- well plates at a density of 4000 cells/well and counted in triplicates from day 3 to 8. Exponential intervals of the growth curve were used to calculate doubling time as previously described [13]. For population doublings (PD), cells were cultured on 25 cm2 flasks, harvested, counted and re-plated when 70–80% confluency. Cell culture was terminated when cell population failed to double after 2 weeks of culture. Population doubling was calculated using the formula PD = [log 10(N1) À log 10(N0)/log 10(2) as previously de- scribed [14]. Immunocytochemistry. Cells were fixed in 4% paraformaldehyde (PFA) and stored at 4 °C. After permeabilization in 0.1% of Triton X- 100 (Promega) and blocking in 2% BSA (Sigma), primary antibody diluted in blocking buffer was applied overnight at 4 °C. Staining for SSEA-4 was performed without using 0.1% Triton X-100 solu- tion. Cells were incubated with secondary antibody in blocking buffer for 1 h at RT. Cells were counterstained with DAPI (Molecu- lar Probes) and mounted with Fluoromount-G (Southern Biotech). Primary antibodies used were: Oct3/4 clone H-134 (Santa Cruz Bio- tech), Nanog (ReproCell), SSEA-4 (Millipore), vimentin (Dako), LHR (Milliepore), and 3b HSD (Santa Cruz Biotech). Secondary antibod- ies Alexa 488 and Alexa 594 (Molecular Probes) were used. For negative controls incubation without primary antibody and with corresponding specific non-immune immunoglobulins (Santa Cruz Biotech) were used. Staining was analyzed using an Olympus IX81 inverted microscope and SlideBook software. Flow Cytometry. Isolated cells were pelleted, resuspended in MEM + HEPES (Gibco) with 2% BSA and counted. Directly conju- gated antibodies were CD105, CD166, CD90, CD44, CD45, CD34, CD11b, CD19, HLA-ABC, HLA-DP DQ DR (Serotec), CD133 (Miltenyi Biotech) LIN, and CD73 (BD Pharmingen). For anti-SSEA-4 and anti- STRO-1 staining (Millipore) secondary antibody goat anti-mouse IgG + IgM-APC (Jackson Immunoresearch) was used. After staining, cells were fixed with 4% paraformaldehyde and analyzed using 0 20,000 40,000 60,000 80,000 100,000 0 2 4 6 8 10 0 10 20 30 40 50 60 70 0 2 4 6 8 10 12 14 16 18 days in culture passage number cumulativepopulationdoubling numberofcells/well GSCs GSC-cs GSCs GSC-cs GSCs GSC-cs Fig. 1. Characteristics of GSCs whole population and GSCs clone (GSC-cs). Phase contract images exhibit differences in morphology of cells maintained under the same culture conditions (10Â) (A). Comparison between growth curves and cumulative population doublings for GSCs and GSC-cs (B,C). Normal karyotype of GSC-cs after seven passages (D). R. Gonzalez et al. / Biochemical and Biophysical Research Communications 385 (2009) 570–575 571
  3. 3. CyAn ADP Analyzer 9 color (Beckman Coulter). Histograms were generated by using Flowjo software (Treestar Inc.). PCR analysis. To analyze gene expression profile, cells were col- lected in RLT buffer (Qiagen) and stored at À80 °C. NT2 cells used as a control for pluripotency genes were purchased from ATCC (Manassas, VA). Total RNA was isolated with the RNeasy Plus kit (Qiagen). 200–300 ng RNA was reverse transcribed using Thermo- Script (Invitrogen). Table 2 in Supplemental Materials shows gene specific primers that were used in both end point and real time PCRs. Real-time PCR was performed with a CFX96TM Real Time Sys- tem and iQTM SybrGreen Supermix (Bio-Rad Laboratories) to assess the expression of osteocalcin. GAPDH mRNA was used as a control. Each sample was measured in triplicate. End point PCR was con- ducted in a C1000TM Thermal Cycler (Bio-Rad) using GoTaqÒ Hot Start Polymerase (Promega) and 1 ll of cDNA product for the anal- yses of all other genes. ‘‘No RT” and ‘‘no template” controls were included in each experiment. Student t-test was done in order to establish statistical differences in induced samples as compared to controls. Cell differentiation Adipogenic differentiation. Cells were plated in 12-well plates. At 90–100% confluency cells were switched to adipogenic induction medium according to manufacturer’s protocol (Lonza). After 3 days, medium was changed to adipogenic maintenance medium and kept for 1 day. Cycles of 3 days induction + 1 day maintenance medium were repeated for 12–19 days. Control cells were kept in regular culture medium. At 12 and 19 days cells were fixed with 4% PFA and stored at 4 °C until staining. Staining was performed using 0.3% Oil Red O solution (Sigma–Aldrich). For quantitative as- say, Oil Red O bound to lipid droplets was extracted with 100% Eth- anol solution and absorbance was measured at 550 nm with reference wavelength 650 nm. Absorbance measurements of Oil Red O release were compared to standard titration curve of corre- sponding dye. Obtained quantity of dye accumulation/well was normalized to cell number determined by Hoechst 33342 (Molec- ular Probes) staining of nuclei. Osteogenic differentiation. Cells plated on 12-well dishes were switched to osteogenic differentiation medium (HyClone) accord- ing to manufacturer’s protocol. After 12 and 19 days of induction cells were fixed in 4% PFA and stained with 2% Alizarin Red S (Sig- ma–Aldrich). To detect calcium deposit accumulation, Ca-bound Alizarin Red S was extracted in 10% of cetylpyridinium (Sigma) in phosphate buffer (8 mM Na2HPO4 + 1.5 mM KH2PO4, Sigma–Al- drich). Alizarin Red S release was measured at 550 nm with refer- ence wavelength 650 nm. Absorbance measurements of Alizarin Red S release were compared to standard titration curve of corre- sponding dye. Obtained quantity of dye accumulation/well was normalized to cell number determined by Hoechst 33342 staining of nuclei. Chondrogenic differentiation. Cells were placed in either control media or chondrogenic differentiation medium according to man- ufactures’ protocol (Lonza). Briefly, 300,000 cells/15 ml tube were pelleted and control or chondrogenic differentiation medium was added. After 28 days, pellets were fixed with 4% PFA. Pellets were placed in 25% sucrose solution and frozen embedded 48 h after in OCT compound (Sakura Finetek). Pellets were sectioned at 10 lm and stained with 1% Alcian Blue (Sigma) and counter stained with nuclear fast red (Sigma) using standard protocols. Karyotyping. Karyotype analysis was performed by Cell Line Genetics using standard cytogenetic protocols. Briefly, G-banding technique was applied to karyograms produced from at least 20 metaphases. Fig. 2. Flow cytometry of GSCs isolated from testis at passage 3. GSCs express markers indicative of MSCs. Blue histograms—antibody staining, open histograms indicate appropriate isotype controls. Percent of positive cells is indicated for each antigen studied. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.) 572 R. Gonzalez et al. / Biochemical and Biophysical Research Communications 385 (2009) 570–575
  4. 4. Results Expansion and growth kinetics of GSCs In order to isolate a novel stem cell population we dissociated cells isolated from a single small biopsy of adult human testes. Supernatant containing spermatogonial cells and dying cells were discarded. Attached cells were fed every 3–4 days. Under these conditions, cells reached 70% confluency after 7–10 days and exhibited MSC-like morphology (Fig. 1A). Clonogenic efficiency of whole cell population was 35 + 1.8% (n = 5). In total, 7 clones (GSC-cs) were collected and every clone was successfully ex- panded. GSC-sc exhibited statistically significant (p < 0.001) de- crease in clonogenic efficiency (7 + 0.6%, n = 5) in comparison with starting population. Doubling times were similar for both populations (33.8 ± 6.5 h GSC and 32.4 + 4.4 h GSC-cs) (Fig. 1B). However, the proliferative capacity of GSC-cs was markedly re- duced in comparison to that of GSCs (Fig. 1C). GSCs propagated for 17 passages with at least 64 population doublings (Fig. 1C) and were easily expanded to therapeutically necessary amounts by passage 3 (>2.0 Â 108 ). GSC-cs exhibit diploid cells without chromosomal aberrations as determined by karyotype analysis (Fig. 1D). Characterization of GSCs In order to characterize GSCs, we performed flow cytometry, immunocytochemistry, and RT-PCR. GSCs exhibit characteristics of MSCs. Flow cytometry analysis revealed that GSCs have charac- teristics typical of MSCs isolated from bone marrow in accordance with the International Society for Cellular Therapy minimum crite- ria for defining MSCs [15]. GSCs were positive for CD105, CD73, CD166 and negative for CD34, CD45, HLA-DR, CD11b and CD19 (Fig. 2). In addition, GSCs expressed high levels of CD44, CD90 and STRO-1 which are expressed on MSCs [16,17]. Interestingly, a small percentage of GSCs express the pluripotent stem cell mar- ker stage-specific embryonic antigen 4 (SSEA-4) (Fig. 2). Based on morphology similar to the several distinct cell types described in MSCs, GSCs are also a heterogeneous population. When comparing GSCs with GSC-cs, their morphology (Fig. 1A) and antigen expres- sion (Supplementary Table 1) were different. GSCs had a morphol- ogy similar to MSCs while GSC-cs were much smaller and with less processes. Specifically, GSC-cs were mostly CD90 (Thy-1) negative and demonstrated increased expression of SSEA-4 and CD34. Immunocytochemistry analysis demonstrates that GSCs express the pluripotent markers Oct 4, Nanog, and SSEA-4 (Fig. 3A). RT-PCR experiments confirms that GSCs express Oct 4 and Nanog but are negative for Sox 2 (Fig. 3B). GSCs also express vimentin which is a major subunit protein of the intermediate filaments of mesen- chymal cells (Fig. 3A). There are possibilities that GSCs are derived from other cell lineages present in testes, namely germ or Leydig. RT-PCR for Vasa and Dazl confirms that GSCs are not of the germ cell lineage (Fig. 3B). Additionally, the GCS are not precursors or adult Leydig cells, based on the negative immunocytochemistry staining for luteinizing hormone (LH) receptor and 3b-hydroxy- steroid dehydrogenase [18] (data not shown). Vimentin SSEA-4 Oct4 Oct4 Merged Nanog Merged MergedDAPI Oct4 Nanog Sox2 Vasa Dazl GAPDH no RT 21 3 4 5 6 A B Fig. 3. Pluripotent marker expression of GSCs. Immunocytochemistry analysis revealed expression of pluripotent stem cell markers Oct4, Nanog and SSEA-4 in GSCs population. GSCs also express the intermediate filament marker vimentin. DAPI nuclear staining is in blue (A). PCR analysis confirmed staining results. Germ cell specific genes Vasa and Dazl were not expressed in GSCs indicating other than germ cell origin of isolated population. Lane 1, whole testes; lane 2, GSCs passage 1; lane 3, GSCs passage 4; lane 4, GSCs passage 9; lane 5, GSC-cs passage 4; and lane 6, NT2 control cells (B). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.) R. Gonzalez et al. / Biochemical and Biophysical Research Communications 385 (2009) 570–575 573
  5. 5. GSCs differentiate into mesodermal lineage The hallmark of MSCs is its ability to differentiate into mesoder- mal lineage. We therefore undertook studies differentiating GCS into adipogenic, osteogenic and chondrogenic lineage. Both GSCs and GSC-cs were induced to the adipogenic, osteogenic, and chon- drogenic lineages using standard MSC differentiation protocols. Specifically, GSCs and GSC-cs induced to adipogenic lineage dis- played lipid vacuoles (Fig. 4A and C) and increased expression of lipoprotein lipase and PPARyIso2 (Fig. 4B) as compared to non-in- duced controls. When subjected to osteogenic differentiation, GSCs and GSC-cs displayed calcium deposits typical of bone (Fig. 4D and F) and increased expression of osteocalcin and DLX5 (Fig. 4E) as compared to non-induced controls. The chondrogenic potential of GSCs and GSC-cs was confirmed by sulfated proteoglycans staining (Fig. 4G) and increased expression of aggrecan and link protein (Fig. 4H) as compared to non-induced controls after 28 days in cul- ture. All together, these data clearly demonstrate that GSCs are eas- ily differentiated into mesodermal lineage and have MSC properties. Discussion Presently, hematopoietic stem cells (HSCs) and MSCs are the most widely investigated stem cells for therapeutic applications because they are easily obtained, autologous, expandable to therapeutic amounts, and most importantly, have been shown to improve symptoms in several disease states [19–21]. To expand the array of cell lines for therapeutic applications, we isolated a new population (GCS) from adult human testis. With a small biopsy, GSCs are easily expandable to therapeutic suitable amounts making this cell an attractive tool for regenerative medicine. GSCs described in this study possess fundamental stem cell properties such as clonogeneity, multipotentiality and self-renewal. Further- more, Isolated GSCs were negative for germ cell specific markers [12], thus representing a new cell population different from germ cells. Interestingly, GSCs express the pluripotent markers Oct 4 and Nanog typical of embryonic stem cells (ESCs) [22,23]. GSCs are characteristically similar to MSCs isolated from bone marrow, based on their morphology, antigen expression pattern and differentiation potential [4,17]. GSCs exhibit substantially ex- panded life span (>60 population doublings) when compared to adult MSCs derived from bone marrow which normally produce approximately 35 population doublings [4,17,24]. This may be due to the expression of Oct-4 and Nanog in GSCs. It has been shown that MSCs can also display marked heterogeneity in mor- phology, growth kinetics, differentiation potential and gene expression profiles [4,17,25]. Our flow cytometry data demon- strates differences between GSCs population as compared to GSC-cs in regards to surface antigen expression. GSCs were mostly positive for CD90 and negative for CD34, while GSC-cs were mostly negative for CD90 and had a greater percentage of SSEA-4 and CD34 positive cells (Supplementary Table 1). Interestingly, both SSEA-4 and CD34 are stem cell markers that are associated with AdipogenesisChondrogenesis 0 10 20 30 40 50 60 70 80 1 2 3 4 5 6 7C CI I GSCs GSC cs relativeugofARS/well GSCs GSC cs Osteogenesis control control induction induction 1 10 100 1000 C CI I Aggrecan Link relativeexpression 0 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7C CI I GSCs GSC cs relativeugof ORO/well 0 1 2 3 4 5 6 7 8 9 10 day 12 day 19 day 12 day 19 lipoprotein lipase PPAR iso 2 relativeexpression Control Induced** ** * 0 2 4 6 8 10 12 14 16 osteocalcin DLX5 relativeexpression Control Induced *** ** day 12 day 19 day 12 day 19 Fig. 4. GSCs and GSC-c9 have multilineage differentiation potential. Staining of lipid droplets with Oil Red O—adipo differentiation for both GSCs and GSC-cs (small inserts are controls) (A). Gene expression of lipoprotein liapase and PPAR iso 2 in cells undergoing adipogenic differentiation demonstrated upregulation in induced GSCs as compared to controls (B). Quantification of dye accumulation/well for Oil Red O (ORO) demonstrated increased oil red O accumulation in induced cells as compared to controls for both GSCs and GSC-cs (C). Calcium deposits with Alizarin Red S—osteo differentiation for both GSCs and GSC-cs (small inserts are controls) (D). Gene expression of osteocalcin and DLX5 in cell subjected to osteogenic differentiation demonstrated upregulation in induced GSCs as compared to controls (E). Quantification of dye accumulation/well for Alizarin Red S (ARS) staining demonstrated increased dye accumulation in induced cells as compared to controls for both GSCs and GSC-cs. (G) Staining with Alcian blue for sulfated proteoglycans—representative cells undergoing chondro differentiation are shown for both GSCs and GSC-cs (F). Controls are top panel and induced are lower panels. Gene expression of aggrecan and link in samples subjected to chondrogenic differentiation. Induced GSCs demonstrate an upregulation of studied genes as compared to controls (H). Pictures A, D, G 40Â; inserts in G are 4Â low magnification. Data are means + SEM of triplicate samples. The ratio was calculated against the values in control that was set to 1. * p < 0.05; ** p < 0.01; *** p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.) 574 R. Gonzalez et al. / Biochemical and Biophysical Research Communications 385 (2009) 570–575
  6. 6. growth; yet GSC-cs underwent replicative arrest much earlier than GSCs (Fig. 1B). Moreover, we found that lack of CD90 expression and increase of SSEA-4 and CD34 expression correlates with enhancement of osteogenic differentiation potential for GSC-cs as compared to GSCs (Fig. 4D and F). Recently, it was shown that the expression of another marker, CD106/VCAM correlates with preferential adipogenic versus osteogenic differentiation [26]. Thus, our study demonstrates that CD90À/SSEA-4+/CD34+ expres- sion on cells may correlate with increased osteogenic differentia- tion potential. Novel cell types may be more effective in the treatment of degenerative disorders. Although the unique properties of GSCs re- main to be determined, we hypothesize that these cells may be therapeutically advantageous over MSCs for a specific pathological state, since GSCs have a greater life span and are easily differenti- ated to mesodermal lineage cells. These cells may be used for pa- tients with disorders like atrophic nonunion [27] for which MSCs may not be a good candidate. Future studies involving transplant- ing GSCs into diseased animal models will help elucidate the use of GSCs for therapeutic application. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2009.05.103. References [1] M. Chopp, X.H. Zhang, Y. Li, L. Wang, J. Chen, D. Lu, M. Lu, M. Rosenblum, Spinal cord injury in rat: treatment with bone marrow stromal cell transplantation, Neuroreport 11 (2000) 3001–3005. [2] T. Onda, O. Honmou, K. Harada, K. Houkin, H. Hamada, J.D. Kocsis, Therapeutic benefits by human mesenchymal stem cells (hMSCs) and Ang-1 gene-modified hMSCs after cerebral ischemia, J. Cerebral Blood Flow Metabol. (2007) 1–12. [3] D.J. Prockop, ‘‘Stemness” does not explain the repair of many tissues by mesenchymal stem/multipotent stromal cells (MSCs), Clin. Pharmacol. 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