The Prostate 67:1265 ^1276 (2007)
Prostatic Stromal Cells Derived From Benign Prostatic Hyperplasia Specimens Possess Stem Cell Like Property Victor K. Lin,1* Shih-Ya Wang,1 Dolores V. Vazquez,1 Chet C. Xu,2 Sheng Zhang,2 and Liping Tang2 1
Department of Urology,The University of Texas, Southwestern Medical Center, Dallas,Texas 2 Department of Bioengineering,The University of Texasin Arlignton, Arlington,Texas
INTRODUCTION. The hyper-proliferative activity of stromal smooth muscle (SM) cells is believed to be responsible for the pathogenesis of benign prostatic hyperplasia (BPH). We have observed that those stromal cells can differentiate into unrelated specialized cells. We thus hypothesize that stromal cells derived from adults prostate specimens may contain adult stem cells. To test this hypothesis, human prostate stromal primary cultures were established and used for characterization of their stem cell properties. METHODS. Immunoblotting, immunohistochemistry, RT-PCR, and tissue culture techniques were used to characterize the primary cultured human prostate-derived stromal cells for their stem cell and differentiation properties. The plasticity of these stromal cells was analyzed using cell culture and histology techniques. RESULTS. Primary cultured prostate stromal cells from BPH patient possess polygonal and elongated fibroblast/myofibroblast cellular morphology. They are positive in CD30, CD34, CD44, NSE, CD133, Flt-1, stem cell factor (SCF), and neuron-specific enolase (NSE), but negative in C-Kit, stem cell antigen (SCA), SH2, CD11b. Expression of SM myogenic markers in these cells may be induced by sodium butyrate (NaBu) treatment. Induction to osteogenic and adipogenic differentiation in these cells is also evident. CONCLUSIONS. Our study on primary stromal cells from BPH patients have yielded many interesting findings that these prostate stroma cells possess: (1) mesenchymal stem cell (MSC) markers; (2) strong proliferative potential; and (3) ability to differentiate or transdifferentiate to myogenic, adipogenic, and osteogenic lineages. These cell preparations may serve as a potential tool for studies in prostate adult stem cell research and the regulation of benign prostatic hyperplasia. Prostate 67: 1265–1276, 2007. # 2007 Wiley-Liss, Inc. KEY WORDS:
prostate stromal cells; adult stem cells; cell markers; differentiation; benign prostatic hyperplasia
INTRODUCTION Benign prostatic hyperplasia (BPH) is a common medical condition in the United States. It impacts over 80% of males aged 50 years and older with various degrees of bladder outlet obstruction associated with lower urinary tract symptoms (LUTS). In spite of the medical significance of BPH in aging males, the pathogenesis of this disorder is unclear, although theories such as aging, estrogen/androgen balance, embryonic reawakening, oxidoreductase removal of dihydrotestosterone, and inflammation/growth 2007 Wiley-Liss, Inc.
factors, have been postulated . It has been shown that, between the third and fifth decade of life, tissues in Grant sponsor: NIH; Grant numbers: RO1 GM074021, RO1DK52653; Grant sponsor: AHA Established Investigator Award. *Correspondence to: Victor K. Lin, PhD, Department of Urology, University of Texas, Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9110. E-mail: [email protected]
Received 2 January 2007; Accepted 12 March 2007 DOI 10.1002/pros.20599 Published online 27 June 2007 in Wiley InterScience (www.interscience.wiley.com).
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the prostate grow exponentially, with a doubling time of 4.5 years. However, a decreased hyper-plastic rate is also evident in the older age . There is a 9 and 37 times increase in the proliferative rate of the epithelium and stroma, respectively, in BPH compared to the normal prostate . Human prostates contain 45–60% stromal tissue, 30–34% acinar lumina, and 10–20% epithelial tissue . In studies using quantitative morphometry, the stroma-to-epithelium ratios in the normal and prostatic adenomas are 2– 1 and 5–1, respectively [4,5,6]. This age-related proliferative disorder of the prostate may be a process of trans-differentiation and activation of cellular proliferative potential. Given that stromal hyper-proliferation is a crucial characteristic in BPH pathogenesis, it is rational to speculate the existence of adult stem cell in prostate stromal compartment which could lead to the expansion of the stroma in response to hormonal, aging, and/or other stimuli during the pathogenesis of BPH. Adult type mesenchymal stem cells (MSC) have been characterized as a subset of precursor cells that are: (1) self-renewable; (2) with multilineage differentiation from a single cell; and (3) capable to in vivo functional reconstitution of the tissues to which they give rise . MSC have been identified and isolated form bone marrow [8,9], adipose tissue , and granulocyte colony stimulating factor mobilized peripheral blood . Those cells are shown to be multipotent and have proliferative potential. The biological characteristics and properties of MSC are limited. However, its applications in connective tissue engineering, cell transplantation, hematopoietic stem cell transplantation, and gene therapy have been well recognized . In an effort to uncover the processes and molecular mechanisms governing the hyper-plastic growth of prostate mass in BPH, we have studied the behavior and responses of stromal cells isolated from BPH patients. Rather surprisingly, we incidentally observed that cultured stromal cells exhibit phenotypic changes with the supplement of various differentiation cocktails. It is well established that stem cells are unique cell types with high plasticity . We thus assumed that adult stem cells are located in prostate stromal compartment. To test this hypothesis, we have established stromal cell primary cultures derived from BPH tissues and these stromal cells were systematically characterized for their stem cell-like markers and responses. The results from this study provide a fundamental piece of information about the potential role of stem cell-like stromal cells in BPH pathogenesis. A more comprehensive knowledge of these novel stem cells may permit the rational design of treatment to alleviate or control BPH symptoms. The Prostate DOI 10.1002/pros
MATERIALS AND METHODS Tissue Collection and Preparation BPH adenoma tissues were obtained from patients undergoing open prostatectomy or cystoprostatectomy surgery. Tissue specimens were all taken within an area 5mm lateral to the proximal urethra. The procured tissues were, either extensively washed thrice with ice-cold sterile PBS and then immediately processed for collagenase digestion for primary cell culture, or snapfrozen in liquid nitrogen and subsequently stored at 808C for further analysis. Histopathological analysis of these specimens was performed, and diagnoses of BPH were confirmed by staff pathologists with expertise in genitourinary pathology. Human tissue usage in this study was approved by the Institutional Review Board at The University of Texas Southwestern Medical Center at Dallas. Human Prostate Stroma Primary Cultures Human BPH stromal cell (HPS) primary cultures were prepared from fresh harvested surgical BPH tissues by collagenase digestion and cultured in Eagle modified minimum essential medium (MEM, Mediatech, Herndon, VA) with 10% fetal bovine serum (FBS, Gemini, West Sacramento, CA) and antibiotics. The cultures were maintained at 378C in 5% CO2 atmosphere with medium change every other day. Human dermal fibroblast primary culture (HDF) was kindly provided by Dr. Fred Grinnell, Department of Cell Biology and Anatomy, UT Southwestern Medical Center. HDF was grown in MEM with 10% FBS. For HPS cell clonal analyses, cells at passage 3 were seeded at clonal density (5 cells/cm2) with MEM with 10% FBS in 100 mm culture dishes and incubated for 3 weeks. The cells were then subjected to H&E or immunocytochemical staining. The results were examined under a Nikon Diaphot inverted microscope. Digitized images were then captured and analyses including colony scanning, sizing, counting were conducted using a MetaMorph software (Version 4.5, Universal Image Corp., West Chester, PA). Protein SDS ^PAGE Analysis and Immuno-Blot 4–20% gradient SDS–PAGE gels (Daiichi, Tokyo, Japan) were used to fractionate protein preparations from treated culture specimens. The proteins were electrophoretically transferred and immobilized to NitroPure Supported Nitrocellulose Membrane (Osmonics). Immunoblot analysis was carried out using antibodies against smooth muscle (SM) myosin heavy chain, SM a actin (1A4, Dako), b-actin (AC-40, Sigma), h-caldesmin (332M, Biogenex). Signal detection
Human Prostate Stromal Stem Cells
was done using ECL Plus Western detection system (Amersham, Piscatway, NJ).
mRNA expression between sodium butyrate (NaBu) treated HPS cell and non-treated was compared.
Stem Cell Characterization
HPS cells grown at designated density in eight well chamber slides (Lab-Tek II, Nalge Nunc, Naperville, IL) and cell clones grown in 100 mm culture dishes were washed three times with PBS and fixed in freshly prepared 4% paraformaldehyde for 15 min at room temperature followed by three PBS washes. The endogenous peroxidase was blocked by 3% hydrogen peroxide in methanol. The dishes were then blocked with DAKO blocker (protein free, Dacocytomation, Carpinteria, CA) and primary antibody was applied. After washing the excess antibody away, the biotinated or FITC conjugated second antibody (Jackson Laboratories, Bar Harbor, ME) was applied depending on the experimental design. RT-PCR Total RNA from HPS cells was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). The quantity of RNA was measured by spectrophotometer at A260. The quality of RNA isolated was verified by denaturing RNA gel for 18S and 28S integrities. cDNA was synthesized in a 20 ml reaction containing 1 mg RNA, 50 mM Tris, pH 8.3, 75 mM KCl, 3 mM MgCl2, 1 mM each of dNTP, 0.25 mg each of oligo-dT12–18 and random hexamer (PN6, Pharmacia Biochemicals, Piscataway, NJ), 20 units RNasin, and 400 units reverse transcriptase (Superscript II, Invitrogen, Carlsbad, CA). The reaction was incubated at 428C for 90 min and then inactivated at 908C for 10 min. The cDNA was stored in 808C until the PCR amplification. Real-time PCR analysis with quantification standard for assessment of SM myosin heavy chain and myocardin expression in HPS cells was conducted in a Bio-Rad MyIQ system (Bio-Rad, Hercules, CA) according to the manufacture’s instruction. The amplification reaction consists of 2X SYBR green supermix (Bio-Rad) and 50 ng equivalent cDNA templates in a 25 ml volume. PCR primers used to amplify SM myosin heavy chain cDNA were 50 -CAACGCCAACCGCAGGAAGCTGCA-30 and 50 - CCATTGAAGTCTGCGTCTCGAGTG-30 . Primers for myocardin cDNA amplification were 50 -CTTCCACTGCAGAGAGGTCC-30 and 50 GCTTCTTCACCTTTGGCTTG-30 . All cDNA samples were amplified in duplicates and the results were expressed as mean of copy number. Cyclophilin A cDNA was amplified (50 -GGTCAACCCCACCGTGTTCTTCG-30 and 50 -GTGCTCTCCTGAGCTACAGAAGG-30 ) for all specimens as internal quantity control. After normalization to cyclophilin A expression, the difference in SM myosin heavy chain and myocardin The Prostate DOI 10.1002/pros
Cell differentiation protocols. To verify the multipotential differentiation of mesenchymal characteristics of prostate SM cells, cells were subjected to a differentiation condition known to induce adipogenic and osteogenic in human cells. The adipogenic properties of HPS cells were analyzed by growing confluent cells in the presence of bone morphogenic protein-2 (BMP-2) for 3 weeks as described earlier . Mineralization of the extracellular matrix in the cell cluster was visualized by staining of the cultures with 2% Alzarin Red S for 5 min at room temperature. Alkaline phosphatase activity was detected histologically . On the other hand, adipocytic differentiation of HPS cells was carried out following culture with an adipocyte differentiation cocktail (a-MEM supplemented with 10% FBS, 10% rabbit serum, 10 nM dexamethasone, 1 U/ml insulin, and 50 mm 5,8,11,14eicosatetraynoic acid) for 1 week as previously described . At the end of the incubation, cells were stained with Oil Red O according to Kasturi and Joshi . Briefly, cells were washed twice with PBS and fixed with 10% formalin in PBS for 1 hr; then they were washed an additional three times with water and dried. Cells were stained with Oil Red O [six parts of saturated Oil Red O dye (0.6%) in isopropanol and four parts of water] for 15 min. Excess stain was removed by washing with PBS, and then stained cells were washed with water. Immunocytochemistry of cell surface markers. The surface markers of prostate stroma SM cells were determined using immunocytochemical analyses. Cells were grown on glass slides prior to the experiments. These slides were incubated with anti-human primary antibodies: AA4.1 , CD11b , CD34 [18,19,20,21,22], SH2 , CD44 [22,23], Flt-1 , NF-M , SCF [25,26], CD133 [21,27,28], and neuron-specific enolase (NSE) . In each case, incubation with the primary antibody for 3 hr was followed by respective FITC labeled secondary antibodies for 2 hr. The staining process was performed as per product protocols. Non-immunized IgG were used as negative controls for comparison. RESULTS Characterization of Primary Cultured Human Prostate Stromal Cells (HPS) HPS cells were prepared from surgical specimens obtained from men undergoing prostatectomy for
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BPH. At lower density, stromal cells were spindle shape and the morphology similar to fibroblasts (Fig. 1A). Upon confluency, cells tended to orient in the same direction and form typical ‘‘hill and valley’’ configurations consistent with most SM cells in culture (Fig. 1B). In the first few passages during culture, occasional cells with morphologic characteristics of epithelial cells were observed. Using immunocytochemistry, these cells were immunoreactive with antipan cytokeratin antibodies and thereby represented contaminating epithelial cells (Fig. 2A). Greater than 90% of the HPS cells were immunostained with antibodies specific to vimentin thereby confirming the stromal cell identity of the overwhelming majority of cells (Fig. 2B). HPS cells were also enriched with other mesenchymal markers such as a2-macroglobulin and Types I and III collagens (data not shown). Although, HPS remained viable and maintained growth potential for up to 15 passages, immunocytology and RT-PCR results indicated that in the intermediate passages (between passage 5 and 9), most of the stromal cells in culture were SM a actin positive, but expressed low levels of SM myosin heavy chain isoforms, h-caldesmin, and other SM specific markers such as a1a adrenergic receptors (data not shown). The downregulation of SM specific biomarkers may indicate that cultured prostate stromal cells have been de-differentiated due to rapid proliferation. Alternatively, it may suggest a small population of hyperproliferative cells, such as adult stem cells, to expand and then to overwhelm the existing SM cells. Under this condition, SM myosin heavy chain, h-caldesmon, and other SM markers including a1 adrenergic receptors appeared to be drastically downregulated and even became undetectable. Clonal Analysis of HPS Cells HPS cell preparation appeared to be heterogeneous in nature, particularly in the earlier passages. Thus, analysis of cloned cell population is essential to characterize these non-homogeneous HPS cells for their cellular properties. The clones formed from single cell (passage 3) were examined at 3 weeks after the
seeding. Gross microscopic observation indicated that, although single cells with epithelial cell morphology were occasionally seen, most of the colonies were made up from cells with morphological resemblance to fibro-muscular cells (Fig. 3A). However, few colonies composed of cells with epithelial morphology were also cited (Fig. 3B). Results from scanning of 153 colonies indicated that 138 of them appeared to consist of fibro-muscular cells (90.2%) while the rest of the colonies were epithelial in morphology. Even among those colonies made up by fibro-muscular cells, cells in some of the colonies were observed to exhibit variation in morphology than the cells in other colonies. Some of them showed relatively short pseudopodia with less elongated cell shapes (Fig. 3A, left colony). The others were with extended cell edge and showing extra in stress edges (Fig. 3A, right colony). These results suggest the existence of divergence in prostate stromal cell populations. To analyze the nature of the cells in each colony, immunocytochemistry results by computer-assisted colony scanning analysis revealed that only a fraction of these colonies show immunoreactivity to markers related to SM phenotype such as SM myosin heavy chain and SM a actin. Furthermore, the number of SM a actin-positive colonies per 0.5 X objective field under microscopic examination was similar to the number of SM myosin heavy chain positive colonies (17.0 1.58 and 16.8 3.19, respectively) while cytokaretinpositive colonies per field is 7 2.54 (Fig. 3C). These results suggest that, at passage 3, the HPS cell preparation primarily consists of stromal/mesenchymal cells. However, it also indirectly indicates that not all the mesenchymal colonies are SM marker positive since there are nine-fold excess of mesenchymal/stromal colonies to epithelial ones by morphological observation while only 2.4-fold excess of SM markers positive colonies than cytokaretin positive ones (Fig. 3C). Furthermore, our results also indicate that the stromal cells in the preparation exhibit different morphology and phenotype. Of major interest, our results indicated that the expression of SM a actin in the HPS population may not be a clonal
Fig. 1. HPS cellsin culture atlow density (A) andhigh density (B). The Prostate DOI 10.1002/pros
Human Prostate Stromal Stem Cells
Fig. 2. Fluorescent immunohistochemical results indicated that HPS cells are cytokeratin (green fluorescent) negative illustrated in (Panel A). Blue is DAPI nuclear staining. HPS cells are vimentin (red fluorescent) positive (Panel B). [Color figure can be viewed in the online issue, whichis available at www.interscience.wiley.com.]
characteristic since cells within a colony, which were derived from a single cell with an anticipated identical phenotype, expressed different levels of SM a actin (Fig. 4) and other markers. Taken together, these cloned HPS cells may not possess an identical phenotype, which strongly suggests that HPS cells, even when in culture for at least 3 passages, not only possess clonal differences, but also show an instability in their phenotypic expression. Results from analyzing randomly picked 14 colonies indicated that 7 colonies contain varying numbers of SM a actin positive cells and the percentage of SM a actin cells within the colony ranged from 0.1 to 54 (Table I). There was no correlation seen between the number of SM a actin positive cells and the size of the colony. Interestingly, higher SM a
actin positive cell containing colonies appeared to be smaller in colony size suggesting that the cells in colonies expressing SM a actin may have a retarded proliferative potential compared to those did not express SM a actin. These rapid proliferative, SM a actin negative clones may be representative of adult MSC, with the properties of high proliferative and multilineage potential, in the prostate. Spontaneous and Specif|c Differentiation of HPS Cells Osteogenic induction. Following long-term culture (6–8 weeks), we often observed the dense growth of clonal-like cells with morphology similar to
Fig. 3. ClonesderivedfromHPSprimaryculturedcells.HPScells,atpassage 3,were seededat20cells/cm2 inMEMwith10%FBS.Threeweeks after seeding, the cells were photographed under phase contrast inverted microscope.Though most of the colonies were fibro-muscular celllikeinmorphology (Panel A,100), a fewcolonieswith epithelialmorphology were observed (Panel B,100).Note thatboth coloniesin each panel are slightly differentin their gross morphology.The number of SM a actin SMAA positive colonies per 0.5 objective fieldis approximate to thenumberofSM myosinheavychain SMHCpositive colonies (17.0 1.58 and16.8 3.19, respectively) while cytokaretinpositive coloniesper fieldis 7 2.54 (Panel C). The Prostate DOI 10.1002/pros
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Fig. 4. SM a actin immuno staining results indicated that cells derived from same parental cell may not possess an identical phenotype. Panel A: 40; (Panel B) 100; (Panel C) 200; and (Panel D) 400. Arrows indicate SM a actin positive cells. [Color figure canbe viewed in the onlineissue, whichis available at www.interscience.wiley.com.]
osteo-differentiated bone marrow stem cells. To investigate whether HPS cells possess osteogenic properties, cultured cells were stained for calcium deposition and alkaline phosphatase production. Alzarin red S staining results clearly indicate that a wide spread calcium deposition in the center of HPS clonal growth (Fig. 5B). In addition, the osteogenic reaction of HPS cells can be confirmed with alkaline phosphatase staining (Fig. 5C). This finding suggests that HPS cells may contain stemlike cell characteristics. To broadly test the plasticity of
TABLE I. Cell Counts in Randomly Picked Colonies
Total cell numbering the colony 49 139 822 375 121 463 98 226 557 28 1,840 256 389 160
The Prostate DOI 10.1002/pros
SM a actin positive cell number
% of SM a actin in the colony
17 0 0 0 0 0 8 0 0 15 3 8 4 48
35 0 0 0 0 0 8 0 0 54 0.1 3 1 30
the HPS cells, we have cultured the cells in various culture conditions known to prompt differentiation of cells into bone cells, adipocytes, and neural cells. As expected, after incubated with osteogenic cocktail, majority of HPS cells are positive for Alzain red S (Fig. 5B) and alkaline phosphatase stain (Fig. 5C). Interestingly, HPS cells could also differentiate into lipid-droplet containing adipocytes (stained positive with Oil Red O dye) (Fig. 5D). However, HPS cells failed to differentiate into neurofilament-N positive neural cells. This series of studies support that, at least partially, some of the HPS cells have osteogenic and adipogenic differentiation properties. SMMyogenic Specif|c Protein Induction in HPS Cells The multipotent property of HPS cells may also be responsible to the differentiation of SM cells and the pathogenesis of BPH. To study the myogenic properties of HPS cells, cells were incubated with the wellestablished myogenic inducer NaBu for different periods of time and the expression of SM biomarkers in cells were assessed. Real-time RT PCR results shown in Figure 6A demonstrated a 10-fold increase in SMHC mRNA level in 2 mM NaBu-treated HPS cells in a period of 4 days compared to non-treated counterpart. Immuno-blot results indicated that a 2.4-fold increase in SM myosin heavy chain peptides were detected in HPS cells with 2 mM NaBu treatment for 3 days compared to non-treated one (Fig. 6C). The
Human Prostate Stromal Stem Cells
Fig. 5. Osteocyte-like cells with positive mineralization by Alizarin Red S staining was observed in HPS cells under expansion for 4 weeks (Panel A). Aftergrowinginmedia containingdifferentiationcocktails (seemethods),HPS cells showedpositiveincalcifiedbonymineraldeposition by Alizarin Red S staining (Panel B) and positive in alkaline phosphatase staining (Panel C) suggesting its osteocyte-like cell properties. Adipocyte-like cellswithlipidaceousvacuoles stainedwith Oil RedO stain (Panel D) is also seenin HPS cultures. [Color figure canbeviewedin the onlineissue, whichis available at www.interscience.wiley.com.]
Fig. 6. A:Real-time RT-PCR assessmentof NaBuinduction of SMHCmRNAin HPS cells.B:Immuno blotresultsindicated that HPS cells are induced to express SMHC by NaBu treatment.C: Myocardin expression was detected in HPS cells treated with NaBu. [Color figure can be viewedin the onlineissue, whichis available at www.interscience.wiley.com.] The Prostate DOI 10.1002/pros
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increase was higher at Day 5. However, the treatment at 5mM of NaBu did not lead to any gain of SM myosin heavy chain expression. This induction of SM myosin heavy chain expression by NaBu appears to be dose and time dependent. Furthermore, the expression of myocardin [29,30], a regulatory factor for SM myogenesis was also upregulated in response to NaBu treatment and the increase of myocardin mRNA appeared to be preceding to SM myosin heavy chain expression (Fig. 6B). In addition, SM a actin, hcaldesmon, both are known to be specific markers for SM differentiation, were seen upregulated in response to NaBu treatment in HPS (Fig. 7). These results further support that HPS cells may contain adult stem cells and are capable to be induced for SM myogenesis.
Stem Cell Marker Analysis on HPS Cells To determine whether HPS cells may contain multipotent stem cells, we conducted a series of immunocytochemical studies to determine stem cell marker expression on HPS cells. The results are shown in Table II. Our results indicated that HPS cells immunostained positive for CD30, CD44, CD54, and NSE. CD34, vascular endothelial growth factor receptor 1 (Flt-1), and SCF are also found to be positive in HPS cells, though expressed at a lower level compared to CD30, CD44, CD54, and NSE. Furthermore, CD133 is positive in a fraction of HPS cells. On the other hand, HPS cells appear to be negative for CD11b, SCA-1, SH2, AA4.1, and C-kit. Overall, the results support that HPS cells possess many common stem cell makers. Although the origin of the HPS cells is yet to be determined, the profile of surface markers suggests that HPS cells are in a lineage closely related to MSC.
Fig. 7. Immunoblot results indicate that the expression of h-caldesmin (upper panel) and SM a actin (middle panel) are concomitant with SMHC expression in responding to NaBu induction of SM myogenesis.Bottompanelshows the stainingofb actinexpressionin sameimmuno blot asinternalloading standard. The Prostate DOI 10.1002/pros
During the progression of BPH, one of the most common features is prostate enlargement. Morphometric analysis studies indicate that there is a 5:1 ratio of stromal to epithelial compartment in BPH patients compared to young non-hyperplastic normal prostate in which a 2:1 ratio of stromal to epithelial is observed [4,5,6]. These results strongly suggest that prostate enlargement in BPH is primarily caused by the unproportional volume increase in stromal compartment. Histology results also revealed that the volume increase of stromal compartment is the result of hyperproliferation of stromal cells. We thus assume that the abnormal proliferation of stromal cells is responsible to the BPH pathogenesis. To study the factors regulating stromal cell growth, we established a number of primary human stromal cell cultures from symptomatic BPH surgical specimens. This approach allows us to systematically study the characteristics and responses of BPH stromal cells in vitro. HPS cells, the primary cultured BPH stromal cells, have many unique characteristics. First, these cultured cells eventually are negative in expressing cytokeratins, which are considered markers of epithelial cells . This finding indirectly excludes the possibility of HPS being of epithelial origin. Second, these cultured stromal cells have low-expression of SM myosin heavy chain, h-caldesmin, and a1a AR which are the common markers for non-pathogenic SM cells . These results suggest that cultured BPH cells are intrinsically related to the lineage of SM cells. However, the causes of downregulation of SM myosin heavy chain, hcaldesmin, and a1a AR are yet to be determined. SM
Human Prostate Stromal Stem Cells
TABLE II. HPS Cells Expressed Markers Commonly Used to Identify Stem Cells and to Characterize Differentiated Cell Types Antibodies CD11b Cluster designation 30 (CD30) CD34 CD44 CD54 CD133 Neuron-specific enolase (NSE) Vascular endothelial growth factor receptor 1 (Flt-1) Stem cell antigen -1 (SCA-1) Stem cell factor (SCF) SH2 AA4.1 C-kit
Cell type Inflammatory cells Embryonic stem cells (ESC) and embryonic carcinoma cells Hematopoietic stem cells (HSC) Mesenchymal stem cells (MSC) Stem cells HSC Neural stem cell marker HSC and endothelial precursor cells
Negative Positive Positivelow Positive Positive Positive (5% cells) Positive Positivelow
HSC and MSC ESC, HSC and MSC Undifferentiated MHC Fetal liver stem cells HSC and MSC
Negative Positivelow Negative Negative Negative
has been known to switch its phenotype from contractive to proliferative in response to extrinsic and/or intrinsic stimuli, also known as SM phenotypic modulation . Currently, there is no specific marker(s) for SM de-differentiation other than the observed downregulation of SM markers. Another rational explanation for observing the decreased expression of SM markers in HPS cell population would be that a subpopulation of latent precursor cells in the prostate stromal compartment, with high proliferative potential and multilineage capacity reminiscent to adult stem cells, have been activated and preferentially propagated over the preexisting SM cells in vivo during BPH pathogenesis and in vitro cultivation. It has been documented that many mesenchymal tissues contain committed lineagedirected mesenchymal precursor cells capable of giving rise to same or different type of tissues . Our immunocytochemistry results from cell-cloning experiment showing that not all the stromal/mesenchymal colonies express SM markers (i.e., SM a actin and/ or SM myosin heavy chain) appear to support the notion that our stromal cells preparation contains adult mesenchymal precursor cells. As described in a study analyzing the homogeneity of colonies obtained from bone marrow stromal stem cell population, although full mesenchymal differentiation potential is evident, others show restricted lineage potential . Our results that the expression of SM a actin in the HPS population may not be a clonal characteristic (Fig. 4) and only a fraction of mesenchymal clones are SM marker positive further support the idea of existence of stromal/MSC in the prostate. Interestingly, following long-term in vitro culture, we have observed that cultured HPS cells underwent The Prostate DOI 10.1002/pros
HPS surface marker
spontaneous osteogenic changes similar to adult MSC . This phenomenon is also observed in five additional primary cultures prepared from different BPH tissues (data not shown). This finding suggests that cultured primary HPS cells may contain adult stem cells. Indeed, following established protocols for osteogenic and adipogenic differentiation, HPS cells did turn into calcium-depositing osteoblasts and lipid-droplet containing adipocytes. However, HPS cells fail to differentiate into neural cells. It is well established that bone marrow-derived MSC are capable of differentiating into adipocyte, chondrocytes, osteocytes, skeletal muscle, and neuronal/glial cells [35,36]. The limited plasticity in these cells suggests that HPS cells may be derived from stem cells and retain limited stem cell plasticity. The relationship between HPS cells and adult stem cells has been determined based on surface marker analyses. Indeed, HPS cells possess many common stem cell markers. Specifically, HPS stem cells are positive for CD30, CD34, CD44, CD54, CD133, NSE, Flt1, SCF, and negative on CD11b, CD133, SCA-1 SH2, AA4.1, and C-Kit. CD44, CD54, NSE, and SCF are commonly found on MSC and adipose tissue-derived stromal cells, but not hematopoeitic cells and endothelial cells [24,37,38,39,40,41]. CD30 antigen is expressed on a wide variety of hyper-proliferative cells, including tumor cells of Hodgkin’s disease, of anaplastic large cell lymphoma and T and B activated lymphocytes, decidual stroma, cultivated macrophages, lipoblasts, myoepithelial cells, reactive and neoplastic vascular lesions, mesotheliomas, embryonal carcinoma, and seminoma cells . CD30, a member of the tumor necrosis factor receptor superfamily, was recently reported to be an important biomarker which protects
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transformed stem cells from apoptosis . Flt-1, also called as vascular endothelial growth factor receptor-1 (VEGFR-1), has been shown to be present on MSC and to be downregulated during differentiation . Rather interestingly, HPS cells have low expression of CD34 which is a common marker for bone marrow cells . SCA-1 presents on the surfaces of a prostate cell subpopulation that possesses multiple stem cell properties. SCA-1 is also expressed by both basal and luminal cells in the proximal region of the adult prostate, but is not expressed by either lineage in more distal region . Remarkably, HPS shares many surface characteristics with a unique population of bone marrow derived cells-fibrocytes. Specifically, both fibrocytes and HPS cells share a set of unique markers, CD34, vimentin, collagen type I, and SM a actin [47,48,49,50]. The role of stromal stem cells in normal prostate tissue and in pathogenesis of BPH is yet to be established. It should also be noted that many studies have suggested that transit-amplifying cells (TAC) may serve as the link between prostate stromal cells and BPH [51,52,53]. TAC has been found in the skin, cornea, respiratory tract, teeth, gastrointestinal tract, liver, pancreas, salivary glands, kidney, breast, prostate, endometrium, mesenchyma, and bone . Furthermore, the observation of low expression of CD34 in HPS cells implies that these hyper-prolifeative cells are possibly derived from bone marrow cells. In addition, HPS cells possess vimentin, but not pan cytokeratin, clearly indicate that these cells belong to the stromal mesenchymal cells lineage. Our investigation has uncovered a line of multipotent stem cells coexisting in human prostate stromal tissue. These cells are hyper-proliferative and capable of differentiation into muscle cells, osteoblasts, and adipocytes. In addition, these cells have surface markers similar to bone marrow MSC. The presence of these hyper-proliferative and plastic stem cells in the stromal tissue of BPH patients provides a possible link between two unrelated responses—stem cell tissue regeneration and prostate stromal tissue hyperplasia. We believe that the existence of adult stem cells in prostate stromal compartment provide a rationale for the hyper-plastic expansion of prostate gland during the nature history of BPH. It has been reported that marrow stem cells are present in peripheral blood after mobilization from the bone marrow by administration of granulocyte-monocyte colony stimulating factor (GM-CSF) and may engraft within multiple tissues of mesenchymal origin in adult organism; However, the frequency is extremely low in normal peripheral blood without a GMCSF mobilization . Whether these cells originated from prostate or originally derived from marrow stem The Prostate DOI 10.1002/pros
cells, mobilized to, and colonized in the prostate before and/or during the pathogenesis of BPH remains to be investigated. Furthermore, it would be of great interest to learn the molecular mechanism of prostatic attraction of mobilized marrow stem cells, since the prostate carcinoma cells primarily metastasize to bone. It was demonstrated that adherent stromal cells can be transduced, with efficient and long-term expression [55,56]. Here, we demonstrate that prostate may be a potential source for MSC and the ability of these mesenchymal stromal cells to self-renew at a high proliferative rate. These results indicate that these HPS cells maybe a potential transplantable tool for gene therapy in cancer and tissue engineering. Additional investigations, such as whether these cells of the altered lineages have functional characteristics, assessment of clonal origin of these cells, and systemically evaluate the expression patterns of the lineage markers in cloned populations, may provide insight into the pathogenesis of BPH and may help to lay the foundation for the development of therapeutic approaches for controlling the hyper-plastic growth during the natural history of BPH. ACKNOWLEDGMENTS Part of this work was supported by NIH grants RO1 GM074021 (LT): RO1-DK52653 (VKL), and an AHA Established Investigator Award (LT). The authors wish to thank Dr. Jose Karam for his critical reading of this manuscript. REFERENCES 1. Bostwick DG. Pathology of benign prostatic hyperplasia. In: Kirby R, McConnell JD, Fitzpatrick JM, Roehrborn CG, Boyle P, editors. Textbook of benign prostatic hyperplasia. Oxford, UK: ISIS Medical Media; 1996. 2. Berry SJ, Coffey DS, Walsh PC, Ewing LL. The development of human benign prostatic hyperplasia with age. J Urol 1984;132: 474–479. 3. Claus S, Wrenger M, Senge T, Schulze H. Immunohistochemical determination of age related proliferation rates in normal and benign hyperplastic human prostates. Urol Res 1993;21:305–308. 4. Bartsch G, Muller HR, Oberholzer M, Rohr HP. Light microscopic stereological analysis of the normal human prostate and of benign prostatic hyperplasia. J Urol 1979;122:487–491. 5. Lin VK, Benaim EA, McConnell JD. Alpha-blockade downregulates myosin heavy chain gene expression in human benign prostatic hyperplasia. Urology 2001;57:170–175. 6. Shapiro E, Hartanto V, Lepor H. Quantifying the smooth muscle content of the prostate using double-immunoenzymatic staining and color assisted image analysis. J Urol 1992;147:1167–1170. 7. Verfaillie CM. Adult stem cells: Assessing the case for pluripotency. Trends Cell Biol 2002;12:502–508. 8. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie
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