Research Article 3539 CD133, a novel marker for human prostatic epithelial stem cells

Gavin D. Richardson1, Craig N. Robson1, Shona H. Lang2, David E. Neal3, Norman J. Maitland2 and Anne T. Collins2,* 1Prostate Research Group, Surgical Oncology, Medical School, Framlington Place, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, UK 2YCR Cancer Research Unit (Area 13), Department of Biology, University of York, PO BOX 373, York, YO10 5DD, UK 3Department of Oncology, Addenbrooke’s Hospital, University of Cambridge, Hills Road, Cambridge, CB2 2QQ, UK *Author for correspondence (e-mail: [email protected])

Accepted 15 March 2004 Journal of Cell Science 117, 3539-3545 Published by The Company of Biologists 2004 doi:10.1242/jcs.01222

Summary Stem cells are clonogenic cells with self-renewal and cells express the cell surface marker CD133 and are hi differentiation properties, which may represent a major restricted to the α2β1 population, previously shown to be target for genetic damage leading to prostate cancer and a marker of stem cells in prostate epithelia (Collins, A. T., benign prostatic hyperplasia. Stem cells remain poorly Habib, F. K., Maitland, N. J. and Neal, D. E. (2001). J. Cell hi + characterised because of the absence of specific molecular Sci. 114, 3865-3872). α2β1 /CD133 cells exhibit two markers that permit us to distinguish them from their important attributes of epithelial stem cells: they possess a progeny, the transit amplifying cells, which have a more high in vitro proliferative potential and can reconstitute restricted proliferative potential. Human CD133 antigen, prostatic-like acini in immunocompromised male nude also known as AC133, was recently identified as a mice. haematopoietic stem cell marker. Here we show that a small population (approximately 1%) of human prostate basal Key words: Stem cells, CD133, Integrins, Prostate

Introduction characterisation of human prostate epithelial stem cells (hPE- Considerable research efforts have been directed towards the SC). In the haematopoietic system, multilineage stem and identification of markers associated with the initiation and progenitor cells are isolated using monoclonal antibodies progression of prostate cancer, yet there is little consensus against cell surface markers for enriching rare subpopulations about the target cell within prostate epithelium that is that are clonal self-renewing and multipotent (Morrison and susceptible to malignant transformation. Stem cells may Weissman, 1994; Spangrude et al., 1988). This strategy was represent a major target for mutations leading to cancer as their used to identify and isolate a candidate hPE-SC. The longevity assures continued presence during the long latency monoclonal antibody CD133 recognises the CD133/1 antigen: between exposure and cancer development (Pierce and a five-transmembrane glycoprotein, localised to membrane Wallace, 1971; Reya et al., 2001). protrusions or microvilli. Antibodies to CD133/1 have been The existence of stem cells in the prostate is probably best used to enrich for human haematopoietic stem cells (Yin et al., illustrated by animal studies investigating the effect of 1997), endothelial cells (Peichev et al., 2000), neurons and androgen on the prostate. Castration leads to rapid involution glial cells (Uchida et al., 2000). CD133 is also expressed by of the gland, but once androgen levels are restored, the the intestine-derived epithelial cell line Caco-2 where it is gland completely regenerates. As this cycle of involution- down-regulated upon differentiation (Corbeil et al., 2000). regeneration can be repeated many times, a population of long- CD133 is believed to be the human orthologue of mouse lived, prostatic epithelial stem cells must exist (Isaacs et al., Prominin, a protein expressed on the apical surface of 1987). In human prostate, stem cells can be distinguished from neuroepithelial cells as well as several other embryonic transit amplifying cells (daughter cells that have a more limited epithelia, and on brush border membranes of adult kidney proliferative capacity), by two- to threefold higher surface proximal tubules (Weigmann et al., 1997). + levels of integrin α2β1 (Collins et al., 2001). In addition, a third Here we show that CD133 selected cells significantly population of basal cells that are destined to differentiate after expand in culture and form acini with evidence of prostatic- a few rounds of cell division can also be identified (Collins et specific differentiation when grafted subcutaneously into the al., 2001; Hudson et al., 2001). α2β1 integrin expression can flanks of male, athymic nude mice: properties consistent with be used to enrich for stem cells directly from prostate tissue a stem cell origin. using differential adhesion to type I collagen. Depending upon the methodology used, between 1-15% of the basal cells exhibit high levels of α2β1 integrin expression (Collins et al., Materials and Methods 2001). Clearly, the availability of further cell surface markers Tissue collection and isolation of hPE-SC for prostate basal cells would greatly facilitate the isolation and Human prostatic tissue was obtained, with patient consent, from 38 3540 Journal of Cell Science 117 (16) patients (age range 53 to 99) undergoing transurethral and retropubic isotype control was added for 1 hour. Cells were washed three times prostatectomy for benign prostatic hyperplasia and cytoprostatectomy in TBS and incubated with goat anti-mouse Ig conjugated to for bladder cancer. The condition of benign prostatic hyperplasia was tetramethylisothiocyanate (TRITC) for 45 minutes. After blocking confirmed by histological examination of representative fragments. free Ig-binding sites with normal mouse serum (1:5 dilution for 30 The tissue was prepared as described previously (Collins et al., minutes), the second primary antibody, directly labelled with PE or 2001). Briefly, collagenase digestion released epithelial structures fluorescein (FITC), was applied and the cells incubated for 1 hour. (organoids; glands and ducts) which where subsequently separated After washing, cells were mounted in the anti-photobleaching from the stromal fraction by repeated unit gravity centrifugation. medium Vectashield containing diamino-2-phenylindole DAPI Organoids were dis-aggregated into a single cell suspension by (Vector Laboratories, Peterborough, England). incubation with trypsin/EDTA (InVitrogen, Paisley, Scotland) for The antibodies used for confocal analysis of cell fractions were 30 minutes, at 37°C. Luminal cells were depleted from the sample 34βE12, which identifies keratins 1, 5, 10 and 14 of the basal cell by linking anti-CD57 antibody to magnetic beads (Dynal Biotech, compartment in prostate (Dako Ltd), RCK103 (BD PharMingen) and UK). LL002 (Serotec Ltd), which react with keratins 5 and 14 respectively, Basal cells were further fractionated on the basis of adhesion to CY-90 designated K18, which reacts with keratin 18 and identifies type I collagen, as described previously (Collins et al., 2001). Cells differentiating epithelium in prostate (Sigma) (Robinson et al., 1998) that had adhered after 5 minutes constituted the stem cell-enriched and RCK 108 (designated K19) which binds to basal and luminal fraction (approximately 3% of the basal population). The non- epithelial cells and recognises keratin 19. In all cases a Leica adherent (transit amplifying and post-mitotic basal cells) were also Microscope, running spot advanced software, was used for image recovered during washing (Collins et al., 2001). Anti-human epithelial capture. antigen, linked to MACS microbeads (Miltenyi Biotec Ltd, Surrey, Basal cells were processed for single (CD133 (CD133-1)- PE; UK) was further used to purify the non-adherent population. CD133 Miltenyi Biotec Ltd) or double (CD133 (CD133-1)- allophycocyanin cells were selected from the above fractions using MACS microbeads (APC; Miltenyi Biotec Ltd) and Ki-67 (MIB-1) – FITC; linked to anti-human CD133, according to the manufacturer’s DakoCytomation) staining along with appropriate negative controls instruction. CD133-positive (CD133+) cells were not detected within and single colour positive controls. Double staining was performed on the CD133-negative (CD133–) fraction and the purity of the MACS cells fixed and permeablised in 1% saponin (Sigma). Cells were system was calculated as 98%. analysed using Beckton Dickinson FACScan software. At least 10,000 events where acquired for each sample. Colony forming and long-term serial culture assays Selected basal cells were counted and plated on collagen-coated Transplantation of CD133+ sorted cells into athymic nude mice (BD-BiocoatTM) plates (BD Biosciences, Oxford, UK) for the To determine the ability of selected CD133+ cells to induce prostatic determination of colony forming efficiency (CFE) and long-term morphogenesis, growth and functional cytodifferentiation in vivo, hi + proliferative potential. After plating, CFE was determined by marking α2β1 /CD133 cells were combined with cultured stromal cells single cells and examining at intervals up to 28 days when they were (passage 1-4) and injected subcutaneously into 6- to 8-week old male, subsequently fixed and colonies scored under a phase contrast nude mice (strain ICRF-nu) at a ratio of 1:10 epithelial (1×104 to 4 hi – microscope. Colonies were scored if they contained >32 cells. As the 5×10 ) to stromal cells. α2β1 /CD133 cells (with stroma) were used number of cells selected was small, irradiated (60 Grays) STO (mouse as a control. After 8 weeks, grafts were removed, fixed in phosphate- embryonic fibroblasts) cells were added as feeders. For the long-term buffered formalin, embedded in paraffin, and cut into 4 µm thick serial culture assays, used to determine the proliferative potential of sections. Serial sections 35 µm apart were stained with Mayer’s various basal subpopulations, cells were passaged continuously until Haematoxylin and counterstained with Eosin. The capacity of grafted their growth capacity had been exhausted. At every passage, the cells to differentiate in vivo to the secretory phenotype was taken as number of cells generated by each fraction was determined by cell evidence of an epithelial stem cell population. counts. The cumulative total cell output from the initial 5000 cells was calculated at the end of the experiment, assuming that all the cells from each passage had been replated. The results obtained were Immunocytochemical analysis of transplanted CD133 cells subjected to a paired t-test to determine statistical significance. The Paraffin embedded serial sections (4 µm) taken from xenografts were duration of each experiment was dependent on the individual culture, incubated with primary antibody. A biotinylated secondary antibody but was generally between 55-75 days. was applied to the specimens followed by incubation with avidin- biotin complex reagents (Dako Ltd., Bucks, UK). The staining was developed with diaminobenzidine tetrahydrochloride (DAB; Sigma). Immunofluorescent staining of tissue sections Primary antibodies used were 34βE12, K18, anti-PAP (clone Prostate cells expressing CD133 antigen were identified by direct PASE/4LJ: a marker of secretory luminal cells; Dako Ltd) and anti- immunofluorescent staining using anti-CD133 mouse monoclonal androgen receptor (clone AR27) antibody (Novocastra Laboratories antibody (clone 293C3, Miltenyi Biotec Ltd) directly conjugated with Ltd, Newcastle upon Tyne, UK). phycoerythrin (PE). A series of 1 µm optical sections through the entire thickness of the tissue was obtained using a 60× objective of the confocal microscope, and a Z series was constructed from these Results sections. CD133+ cells are located within the basal layer of prostate epithelium Confocal microscopy and FACS analysis of Isolated basal cells We initially determined the location of CD133 cell within prostatic epithelium using anti-CD133 directly conjugated to Cells were processed for triple colour staining for confocal microscopy by fixing in ice-cold methanol (20 minutes) and PE (Fig. 1). Two hundred cross sections of acini, from five permeabilising in 0.4% Triton X-100 (Sigma) containing 0.3% patients samples, were examined for CD133 expression. Only normal goat serum (NGS; Sigma) for 10 minutes. After blocking five percent (10/200) of sections examined for CD133 [1:5 dilution of NGS in Tris-buffered saline (TBS) for 10 minutes] expression were found to contain positive cells; which were primary antibody (diluted in blocking serum) or a non-specific always located in the basal layer. Between 0 and 5 CD133+ Prostate epithelial stem cells 3541 cells were present in each cross section of an acinus and were Clonogenic cells can be enriched by CD133 selection hi either found alone, or when clustered together, were often from the α2β1 population situated at the base of a budding region or branching point (Fig. To determine the proliferative potential of putative hPE-SC, hi + 1). There was no significant difference in the number of CD133 α2β1 basal cells were separated into two populations (CD133 cells and the size of acini (P<0.05), i.e. CD133 cells were not and CD133–) using MACS immunomagnetic beads, and plated confined to tips of acini, for example, but were randomly to determine CFE (Fig. 3A). CD133+ cells displayed a located throughout each acinus. 10.6±0.45-fold greater CFE than the non-selected basal population and CD133– cells had a 4.5±1.2-fold greater CFE than non-selected basal cells. The other important difference Candidate hPE-SC-expressing CD133 are restricted to between the CD133+ and CD133– founded colonies was the α β hi the 2 1 population and represent a quiescent time taken by the cells founded by the CD133+ cells to begin subpopulation of the basal layer a phase growth in vitro, perhaps reflecting the slow cell cycle hi + Basal cells were selected at 5 minutes (α2β1 ) and 20 minutes time of stem cells in vivo. For example, colonies founded by low (cells that had not adhered within 20 minutes; α2β1 ) on type CD133+ selected cells first appeared 5±1.5 days after colonies I collagen-coated plates and were subsequently incubated with founded by the CD133– population. 87.5% of CD133+ founded anti-CD133 and analysed by confocal microscopy. Cells were colonies contained >32 cells, whereas only 36% of the colonies also stained for expression of 34βE12 to confirm that we had founded by CD133–-selected cells were >32 cells in size. isolated basal epithelium. All cells, from both populations, We compared the long-term proliferative capacity of the stained positively for 34βE12, whilst CD133 expression was CD133+ and CD133– populations by assaying total cell output hi restricted to the α2β1 population (Fig. 2A). Expression of following serial passage until all growth potential was CD133 was punctate, similar to that described by Corbeil and exhausted (typically 55-75 days). The total cell output from co-workers (Corbeil et al., 2000) in developing epithelia. selected cells demonstrated clearly that basal cells with the Expression of CD133, in the absence of 34βE12 expression greatest, long-term proliferative capacity reside within the was not observed. Both populations were also analysed by flow CD133+ population: the CD133+ population generated more cytometry (Fig. 2B,C). Similarly, CD133 expression was only than twofold more cells than the CD133– population (Fig. 3B; hi observed within the α2β1 population (25% of cells within the P<0.05). α β hi hi + 2 1 population expressed CD133; 0.75% of the total basal The phenotype of α2β1 /CD133 cells was examined and population). the results are shown in Fig. 4. All CD133+ cells examined In vivo cell kinetic studies have established that prostate expressed the basal cell specific marker, 34βE12 (Fig. 4A). stem cells in the mouse are largely quiescent and do not Within this population, 75±8.2% of cells expressed K14 with proliferate at high rates, whereas transit amplifying cells are K5 (Fig. 4B). A minority of cells (11±2.6%) expressed K18 or actively cycling (Tsujimura et al., 2002). To determine whether K19 (Fig. 4C), but never with K14 (Fig. 4D,E). Markers of this is true for human prostate, basal cells were labelled with prostate-specific differentiation, such as prostatic acid Ki-67 and CD133 and analysed by flow cytometry (Fig. 2D). phosphatase (PAP), prostate-specific antigen (PSA) and the The results obtained from three separate experiments shown in androgen receptor were not expressed within the CD133+ Fig. 2D demonstrate that the majority of actively cycling basal population (results not shown). cells (i.e. cells expressing Ki-67) reside in the CD133– fraction, whereas the CD133+ population contain significantly more quiescent cells. CD133+ selected cells form fully differentiated acini in immunocompromised mice One of the properties attributed to stem cells is the ability to regenerate the different cell types that constitute the tissue in which they exist (Morrison et al., 1997). We therefore determined whether the candidate hPE-SC, isolated directly from the basal layer, had the potential to self-renew and form fully differentiated glands when grafted into athymic male hi mice. Basal cells were selected for α2β1 /CD133 expression and were immediately grafted, together with human stromal cells, into the flanks of mice. After 8 weeks, a fully formed epithelium was retrieved from 2/10 (20%) mice grafted with CD133 selected cells. From 4/10 (40%) mice, epithelial nests were recovered. In the remaining mice epithelium was not found, but a dense inflammatory infiltrate was observed. In mice grafted with CD133– cells, epithelium was not observed (0/10), but a fibromuscular stromal matrix was apparent in 7/10 of these grafts. In the four grafts containing epithelium, Fig. 1. A rare sub-set of basal cells express CD133+. A paraffin variable gland formation was observed, with morphologic and section of prostatic acini labelled with the nuclear stain DAPI (blue) immunohistochemical evidence of both secretory and and anti-CD133 directly conjugated to PE (red). 200 cross sections squamous cell differentiation (Fig. 5). The xenografts often of acini were studied by confocal microscopy for the presence and consisted of multiple acini within connective tissue. Lumina location of CD133+ cells. Scale bar: 40 µm. were either well defined (Fig. 5A) or when less apparent, 3542 Journal of Cell Science 117 (16)

Fig. 2. CD133+ cells are restricted to the hi α2β1 population and are largely quiescent. hi Basal cells were selected for α2β1 and low hi α2β1 expression. (A) α2β1 population labelled with antibodies to CD133 (red) and basal cell marker 34βE12 (green). Co- localization (yellow/orange; yellow arrow). hi Scale bar: 5 µm. (B,C) Graph of α2β1 low population (B) and α2β1 population (C) labelled with anti-CD133 antibody (shaded area) and analysed by flow cytometry. Isotype control is depicted as a solid black line. (D) Dot-plot showing flow cytometric analysis of basal cells double-labelled with anti-CD133 (APC) and Ki-67 (FITC) from a representative experiment (n=3).

containing large, squamous-like cells with secreted material (Fig. 5D). In the well-defined acini, flattened or cuboidal cells lined the periphery and stained intensely with the basal cell- specific marker 34βE12 (Fig. 5B) and lacked expression of the differentiation marker K18 (Fig. 5C). The cells facing the lumen or adjacent to the basal layer were either morphologically squamous or columnar and stained with antibodies against K18 (Fig. 5C), PAP (Fig. 5D), a marker of functional cytodifferentiation, while lacking expression of the basal cell marker 34βE12 (Fig. 5B). As the secretory luminal hi cells are dependent upon androgen for survival, we expected Fig. 3. α2β1 /CD133 population have a high proliferative potential hi + that androgen receptor expression would also be present in in vitro. (A) Basal cells with the phenotypes α2β1 /CD133 and hi – these grafts; indeed this was the case (Fig. 5E). Expression was α2β1 /CD133 were plated onto type I collagen plates and the confined to the nucleus of a proportion of basal cells as well colony forming efficiency (CFE) determined. Cells were counted as the columnar or squamous-like epithelium. It is also worth before the addition of irradiated feeders and were cultured for up to noting that AR was also focally expressed within the nucleus 28 days. Controls (basal cells) were plated with no pre-selection. of stromal cells surrounding the epithelium (Fig. 5E). Colonies containing 32 or more cells were scored. CFE was calculated as the number of colonies formed per number of selected cells ×100. CFEs are expressed as the ratio of the selected population Discussion to control (unselected basal cell) population. Results show means ± s.e.m. of four experiments. (B) Long-term proliferative potential of hi + hi – 3 In the present study we have shown that a subpopulation of α2β1 /CD133 and α2β1 /CD133 cells. 5×10 cells were seeded hi α2β1 basal cells express the CD133 antigen and that this onto type I collagen plates, in triplicate, and the total cell output was expression correlates with a high proliferative potential and determined at the end of the serial culture when their growth capacity ability to regenerate a fully differentiated prostatic epithelium was exhausted. Results show means ± s.e.m. of four experiments. with expression of prostatic secretory products in vivo. P<0.05. Prostate epithelial stem cells 3543

Fig. 4. Phenotype of hi + α2β1 /CD133 basal cells. hi + α2β1 /CD133 basal cells were isolated, plated on to collagen I-coated culture slides and triple labelled with anti-keratin antibodies and the nuclear stain, DAPI. (A) 34βE12 (green). (B) K5 (green) and K14 (red). (C) K18 (green) and K19 (red). (D) K14 (red) and K19 (green). (E) K14 (red) and K18 (green). Approximately 100 cells were isolated for each experiment (n=10). Scale bar: 10 µm.

a model whereby proximally located stem cells give rise to a population of transit-amplifying cells that migrate distally. However, this model does not take into account the nature of the cells in the intermediate regions and the label-retaining cells that are also present in the distal regions of the duct. hi + Unlike α2β1 cells, CD133 cells were often found in clusters, most frequently at the base of a budding region or branching point. This discrepancy may reflect the strict criteria hi that were used to identify α2β1 cells (those cells with a fluorescence intensity across cell borders which was at least twice the average of the other cells in the transect). Using these hi In the past decade, haematopoietic stem and progenitor cells criteria, only 1% of basal cells were regarded as α2β1 (Collins + hi have been identified using monoclonal antibodies against cell et al., 2001). CD133 cells were restricted to the α2β1 surface markers to enrich for rare subpopulations that are population and had a greater colony-forming ability and clonally self-renewing and multipotent (Spangrude et al., 1988; proliferative potential in vitro than CD133– cells within the hi Yin et al., 1997). One such antigen, CD133, has not only been α2β1 population. The enrichment of colony forming cells seen used to isolate haematopoietic stem cells (Yin et al., 1997), but with CD133 was similar to that observed by Yin et al. (Yin et it also has an expression restricted to the stem cell population al., 1997) for haematopoietic stem cells. Although the total cell within the central nervous system (Uchida et al., 2000). CD133, output was relatively low compared to rapidly renewing although it has no known function, is expressed by developing tissues, such as the epidermis and bone marrow, this may epithelial cells, and is rapidly down regulated upon reflect the age of the patients in this study (average age 71 differentiation (Corbeil et al., 2000). In this study, we examined years). It is well documented that the proliferative capacity of the position and number of CD133-expressing cells within stem cells decreases with age (Marley et al., 1999). Moreover, prostatic acini. CD133 expression was restricted to a small compared with tissues that undergo rapid cell turnover, the population of cells within the basal layer, but was randomly adult prostate is slow growing [epithelial cells within the adult located throughout acini. High surface expression of integrin gland have a doubling time of approximately 200 days (Isaacs α2β1 has been shown to be associated with stem cells in the and Coffey, 1989)], thus the proliferative capacity would be prostate (Collins et al., 2001), as well as other tissues systems expected to be lower. CD133+ cells also founded colonies at a such as the epidermis (Jones et al., 1995). We have previously slower rate; possibly reflecting the slow cycling times of stem hi shown that α2β1 cells (which make up 3% of the total basal cells in vivo; resembling the type I colonies previously population) are also randomly located throughout acini (Collins described by our group (Collins et al., 2001). Indeed, the et al., 2001). In contrast, Tsujimura and co-workers (Tsujimura majority of CD133+ basal cells did not express the et al., 2002) found that stem cells (label-retaining cells) in the proliferation-associated marker, Ki-67. rodent prostate are concentrated in the proximal region of the In vivo, epithelial cells exhibit ordered pairs of keratins. Thus, prostatic ducts. However, they also detected label-retaining cells K5 and K14 are expressed by basal cells, whereas K8 and K18 in more distal locations, albeit in lower numbers. They proposed are predominantly expressed by the luminal cells in the prostate 3544 Journal of Cell Science 117 (16)

Fig. 5. CD133+ selected cells form fully differentiated acini in immunocompromised mice. Xenografts of prostate acini formed by transplantation of hi + α2β1 /CD133 basal cells stained with (A) Haematoxylin and Eosin, (B) 34βE12, (C) anti-K18, (D) anti-PAP (E) Anti-androgen receptor. Scale bar: 40 µm.

a fully differentiated epithelium, expressing markers associated with prostate-specific differentiation when grafted into nude hi – mice. In contrast, α2β1 /CD133 were incapable of forming prostate epithelium in vivo. Prostate development and maintenance of function is dependent upon androgen via interaction with the stroma (Cunha et al., 1983). Therefore, in our experiments epithelial cells were grafted together with stroma, into male mice, to induce epithelial morphogenesis and cytodifferentiation. Often within the same graft, varying degrees of organisation were (Sherwood et al., 1990; Sherwood et al., 1991). An intermediate observed. Some acini had a fully formed lumen and a clear population has also been identified within the basal layer that distinction between the basal and lumen compartment was expresses basal and luminal keratins (Hudson et al., 2001; van observed, yet in adjacent acini the distinction between these Leenders et al., 2000) suggesting the presence of committed compartments was not as clear and lumen were not present. It cells within the basal layer. All CD133+ cells directly isolated is possible that prostatic regeneration may have been more from prostate tissue reacted with antibody 34βE12, which effective in the presence of a powerful inducer of prostate identifies keratins 1, 5, 10 and 14. Whilst the majority of cells development; urogenital sinus mesenchyme (UGM) (Cunha expressed K5 and K14, a minority also expressed K18 or K19. et al., 1987). Tissue recombination and grafting experiments It is possible that K18 and K19 are not as robust indicators of have demonstrated that epithelial budding, branching differentiation as previously believed. For example K19 is a morphogenesis, and the functional differentiation of luminal marker of skin stem cells (Michel et al., 1996). Several secretory cells are dependent on mesenchymal androgen investigators have hypothesised that K5/14 stem cells give rise receptor (Donjacour and Cunha, 1993; Cunha and Lung, 1978; to a transit amplifying population that express K5 with K18 and Lang et al., 2001). In all cases, in our study, stroma surrounding K19 (van Leenders et al., 2000). As the K5/K14-expressing cells acini expressed the androgen receptor. However the signals that made up the largest proportion within the CD133 population our are involved in lumen formation are less well understood. results suggest that stem cells express this phenotype. CD133, Epithelial cells have default apoptotic machinery and require although enriching for the stem cell population may also detect survival signals to avoid engaging in a suicide program (Raff an early transit amplifying population. et al., 1993). There is now good evidence that survival signals One important property attributed to stem cells is their are provided by soluble factors, including hormones and ability to regenerate all of the cell types specific to the tissue cytokines, and more importantly through cell contacts with from which they are derived. Therefore, transplanted stem cells extracellular matrix (ECM) proteins and neighbouring cells should have the potential to self renew and to produce progeny (Frisch and Francis, 1994; Pullan et al., 1996) In skin, growth that differentiate into a fully functional epithelium. By and differentiation of the overlying epidermis is dependent hi + definition, only stem cells have this capacity. α2β1 /CD133 upon synthesis of an ECM by the supporting fibroblasts where able to fulfil these criteria, as they were able to produce (Demarchez et al., 1992). In the prostate, the differences Prostate epithelial stem cells 3545 observed in acini formation may represent the time taken for identification of intermediate phenotypes by keratin expression. J. Histochem. the prostatic fibroblasts to form a functional basement Cytochem. 49, 271-278. membrane. Isaacs, J. T. and Coffey, D. S. (1989). Etiology and disease process of benign prostatic hyperplasia. Prostate Suppl. 2, 33-50. It has been shown in Chinese hamster ovary three- Isaacs, J. T., Schulze, H. and Coffey, D. S. (1987). Development of androgen dimensional cultures, apoptosis is in part due to the loss of resistance in prostatic cancer. Prog. Clin. Biol. Res. 243, 21-31. integrin signalling (Zhang et al., 1995). Integrin-regulated Jones, J., Downer, C. S. and Speight, P. M. (1995). Changes in the expression apoptosis has also been suggested as a possible mechanism of of integrins and basement membrane proteins in benign mucous membrane pemphigoid. Oral Dis. 1, 159-165. cavitation. This is accomplished by apoptosis, within the Lang, S. H., Stark, M., Collins, A., Paul, A. B., Stower, M. J. and Maitland, region where a space forms, caused by a lack of integrin N. J. (2001). Experimental prostate epithelial morphogenesis in response to signalling. This has been shown to occur in embryonal stroma and three-dimensional matrigel culture. Cell Growth Differ. 12, 631-640. carcinoma cell lines that form hollow embryoid bodies Marley, S. B., Lewis, J. L., Davidson, R. J., Roberts, I. A., Dokal, I., Goldman, J. M. and Gordon, M. Y. (1999). Evidence for a continuous (Coucouvanis and Martin, 1995). What is evident however, is decline in haemopoietic cell function from birth, application to evaluating that even within acini with no clear lumen or cellular bone marrow failure in children. Br. J. Haematol. 106, 162-166. compartmentalisation, basal and luminal cells could still be Michel, M., Torok, N., Godbout, M. J., Lussier, M., Gaudreau, P., Royal, A. detected immunohistochemically. Similarly, we have recently and Germain, L. (1996). Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro, keratin 19 expressing cells are differentially localized reported successful tissue reconstructions from prostate cell in function of anatomic sites, and their number varies with donor age and lines that display a degree of polarisation and differentiation culture stage. J. Cell Sci. 109, 1017-1028. (Lang et al., 2001). Morrison, S. J. and Weissman, I. L. (1994). The long-term repopulating subset In conclusion, we have shown that cells expressing the of hematopoietic stem cells is deterministic and isolatable by phenotype. hi + Immunity 1, 661-673. α2β1 /CD133 phenotype have properties that indicate that Morrison, J., Shah, N. M. and Anderson, D. J. (1997). Regulatory mechanisms they are equivalent to the stem cells of the prostate, as they in stem cell biology. Cell 88, 287-298. have the potential to establish and maintain a prostate Peichev, M., Naiyer, A. J., Pereira, D., Zhu, Z., Lane, W. J., Williams, M., epithelium similar to that found in vivo, with associated Oz, M. C., Hicklin, D. J., Witte, L., Moore, M. A. et al. (2000). Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a secretory activity. The degree of enrichment of stem cells population of functional endothelial precursors. Blood 95, 952-958. attainable by the use of CD133+ will allow further analysis of Pierce, G. B. and Wallace, C. (1971). 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