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First published online July 19, 2007
Stem Cells Vol. 25 No. 11 November 2007, pp. 2760 -2769
doi:10.1634/stemcells.2007-0355; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Self-Renewal and Multilineage Differentiation In Vitro from Murine Prostate Stem Cells

Li Xina, Rita U. Lukacsa, Devon A. Lawsona, Donghui Chengb, Owen N. Wittea,b,c

Departments of aMicrobiology, Immunology, and Molecular Genetics and
cMolecular and Medical Pharmacology, David Geffen School of Medicine,
bHoward Hughes Medical Institute, University of California Los Angeles, Los Angeles, California, USA

Key Words. Prostate sphere assay • P63 • Androgen receptor • Integrin {alpha}6 • Prostate stem cell antigen

Correspondence: Owen N. Witte, M.D., Howard Hughes Medical Institute, University of California Los Angeles, 675 Charles E. Young Drive South, 5-748 MRL, Los Angeles, California 90095-1662, USA. Telephone: 310-206-0386; Fax: 310-206-8822; e-mail: owenw{at}microbio.ucla.edu

Received on May 9, 2007; accepted for publication on July 9, 2007.

First published online in STEM CELLS EXPRESS  July 19, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Murine prostate stem cells express integrin {alpha}6, which modulates survival, proliferation, and differentiation signaling through its interaction with the extracellular protein laminin. When plated in vitro in laminin containing Matrigel medium, 1 of 500–1,000 murine prostate cells can grow and form clonogenic spheroid structures that we term prostate spheres. Prostate spheres can be serially passaged individually or in bulk to generate daughter spheres with similar composition, demonstrating that sphere-forming cells are capable of self-renewal. Spheres spontaneously undergo lineage specification for basal and transit-amplifying cell types. P63-expressing cells localized to the outer layers of prostate spheres possess higher self-renewal capacity, whereas cells toward the center display a more differentiated transit-amplifying phenotype, as demonstrated by the expression of the prostate stem cell antigen. When dihydrotestosterone is added to the medium, the androgen receptor is stabilized, is imported to the nucleus, and drives differentiation to a luminal cell-like phenotype. A fraction of sphere cells returned to an in vivo environment can undergo differentiation and morphogenesis to form prostate tubular structures with defined basal and luminal layers accompanied by prostatic secretions. This study demonstrates self-renewal and multilineage differentiation from single adult prostate stem/progenitor cells in a specific in vitro microenvironment.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
The self-renewal property of stem cells varies in different tissue types in vivo. Stem cells in skin, gastrointestinal tract, and the hematopoietic system display extensive and continuous self-renewal capacity [1–3], whereas those in the nervous system show massive self-renewal during early development but become more quiescent in adulthood [4]. In certain organs, such as the mammary gland and prostate, the self-renewal activity of stem cells is hormonally regulated [5, 6].

The serial transplantation of stem cells in vivo has been used to study their self-renewal capacity. This includes hematopoietic reconstitution by bone marrow transplantation methods and the cleared mammary gland fat pad assay [3, 7, 8]. In vitro assays for the self-renewal activity of certain stem cell types have also been developed. When grown in serum-free medium supplemented with peptide growth factors, neural stem cells and mammary stem cells can proliferate and form floating spherical structures termed neurospheres and mammospheres, respectively. These spheres can be serially passaged in bulk or individually, demonstrating their ability to maintain and expand a self-renewing population of cells [9–11]. Such techniques have been instrumental in defining signaling transduction pathways that control self-renewal [12–15].

Adult prostate epithelium is composed of three major cell types: secretory luminal cells, basal cells aligned along the basement membrane, and rare neuroendocrine cells [16]. Adult prostate epithelial cells turn over infrequently. However, adult murine prostate tissues atrophy dramatically upon hormonal depletion, largely because of the apoptosis of luminal cells, but can regenerate after androgen is added back. This cycle can be repeated many times, defining a unique property of prostate stem cells [6]. This and related observations have lead to a hypothesis suggesting that prostate stem cells exist in the androgen-independent basal cell layer, where they self-renew and give rise to intermediate transit-amplifying cells that undergo further differentiation [17, 18].

The role of androgen in prostate growth control is partly through the expression of andromedins, which stimulate the regeneration of prostatic epithelia in a tissue fragment recombination assay [19]. This assay has been further modified by us for use with dissociated cell populations [20]. Single-cell suspensions of prostate epithelial cells were engrafted under the kidney capsules of immunodeficient host mice with embryonic urogenital sinus mesenchymal (UGSM) cells and regenerated tissues resembling adult murine prostate tubules. The surface antigens Sca-1 and CD49f highly enrich for the murine prostate stem cell activity in this assay [21, 22]. Although the multilineage differentiation capacity of prostate stem cells was demonstrated by the prostate regeneration assay, their self-renewal capacity has not yet been demonstrated.

Immortalized human prostate epithelial cell lines have been established that display the lineage marker characteristics of immature cells [23], but a robust in vitro assay to measure the self-renewal capacity of prostate stem cells is still lacking. Normal human and rodent prostate cells have been cultured in vitro to form colonies or three-dimensional outgrowths, but serial passage has not been demonstrated [24–27]. In one study, murine prostate cells grown on feeder cells formed colonies, but only a fraction generated daughter colonies for one or two passages [28]. Because of these limitations, most work to define genetic components and mechanisms of self-renewal for the murine prostate has been performed using in vivo models [29, 30].

We demonstrated the extensive in vitro self-renewal capacity of murine prostate stem/progenitor cells. Murine prostate cells can grow in Matrigel and form clonal spheroids termed prostate spheres. Sphere-forming cells can self-renew and be serially passaged. Sphere-forming cells are also capable of multilineage differentiation. Prostate spheres contain cells at various lineage stages that could undergo further differentiation when induced by adhesion to substrata and stimulation with dihydrotestosterone. A fraction of the sphere-forming cells retain the capacity to regenerate globular structures in vivo, resembling murine prostate tissues. In contrast to floating neurospheres that are composed of an unorganized mass of cells, the prostate spheres grown in Matrigel spontaneously develop an organized structure displaying a lineage hierarchy. Cells in the outer layers of prostate spheres express P63 and possess higher capacity to self-renew, whereas more differentiated prostate stem cell antigen (PSCA)-expressing cells appear within inner layers. These combined results demonstrate that the essential features of stem cell self-renewal and response to microenvironmental cues to drive differentiation can be modeled in a defined in vitro system.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mouse Strains
The wild-type C57BL/6, β-actin green fluorescent protein (GFP) (C57BL/6-Tg[ACTbEGFP]1Osb), β-actin DsRed (C57BL/6-Tg[ACTB-DsRed.MST]1Nagy/J), and CB17Scid/Scid mouse strains were purchased from the Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). The PSCA-GFP transgenic mouse strain was generated in our laboratory previously [31]. Mice were housed and bred under the regulation of the Division of Laboratory Animal Medicine at the University of California Los Angeles.

Prostate Sphere Assay
The conditions to culture and passage prostate spheres were adapted from previously published protocols [9, 10, 32]. Dissociated prostate epithelial cells were prepared from 6- to 10-week-old adult mice as described previously [20]. Dissociated cells were run through a BD FACSVantage (BD Biosciences, Bedford, MA, http://www.bdbiosciences.com) to exclude cell doublets and small clusters. Prostate cell preps were counted by hemocytometer and suspended in 1:1 Matrigel (BD Biosciences)/prostate epithelial growth medium (PrEGM) (Lonza, Walkersville, MD, http://www.lonza.com) in a total volume of 100 µl. Samples were plated around the rims of wells in a 12-well plate and allowed to solidify at 37°C for 10 minutes, before 1 ml of PrEGM was added. Medium was replenished every 3 days. Ten days after plating, spheres with a diameter over 40 µm were counted.

To passage spheres, medium was aspirated off, and Matrigel was digested by incubation in 500 µl of dispase solution (Invitrogen, Carlsbad, CA, http://www.invitrogen.com; 1 mg/ml, dissolved in PrEGM medium) for 30 minutes at 37°C. Digested cultures were collected, pelleted, resuspended, and incubated in 1 ml of type I collagenase solution (190 units/ml; Invitrogen) for 45 minutes at 37°C. Cells were then pelleted, resuspended, and incubated in 0.05% Trypsin/EDTA (Invitrogen) for 10 minutes at room temperature, passed through a 27-gauge syringe 5–10 times, and passed through a 40-µm filter. Cells were counted by hemocytometer and replated.

Histology, Immunohistochemistry, and Western Blot Analyses
Western blot, histological, and immunohistochemical (IHC) analyses were performed as described previously [20]. Slides were made from formalin-fixed and paraffin-embedded cells or tissues. Antibodies used are listed in detail in the supplemental online data.

Qtracker Cell Labeling
Prostate sphere cultures were treated with dispase to release the spheres from Matrigel. Spheres were pelleted and washed twice with 500 µl of PrEGM medium. Prostate spheres were labeled with a Qtracker 525 cell labeling kit (Invitrogen) according to the manufacturer's instructions.

RNA Isolation and Regular and Quantitative Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from cells using the Versagene RNA Kit from Gentra Systems (Minneapolis, MN, http://www.gentra.com). Reverse transcription was performed using Superscript III first-strand synthesis system (Invitrogen). Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed using a Quantitect SYBR Green RT-PCR kit (Qiagen, Valencia, CA, http://www1.qiagen.com) on an Applied Biosystems 7700 Real-Time PCR system (Foster City, CA, http://www.appliedbiosystems.com). Primer sequences are listed in the supplemental online data.

In Vivo Regeneration of Prostate Tissue Using Prostate Sphere Cells
Urogenital sinus mesenchyme cells were prepared as described previously [20]. Dissociated prostate sphere cells were mixed with UGSM cells and pelleted before resuspending in 40 µl of Matrigel. Samples were kept on ice and then injected subcutaneously into CB17Scid/Scid host mice. Grafts were harvested 6–8 weeks later. Serial tissue sections were stained with H&E to visualize and quantify the glandular structures.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
A Small Fraction of Primary Murine Prostate Epithelial Cells Can Form Spheroid Structures in a Semisolid Matrigel Matrix
Stem cells from the mammary gland, brain, and skin have been successfully maintained and propagated in vitro as floating spheroids [9, 10, 33]. Prostate stem cells reside within the basal cell layer and express high levels of the integrin {alpha}6 [17, 22]. Interactions of integrins with their ligands in the extracellular matrix (ECM) have been shown to regulate stem cell function and tissue homeostasis [34, 35]. To simulate this environment, cells were cultured in Matrigel, which contains high levels of the integrin {alpha}161 ligand laminin. Culturing the cells in Matrigel also immobilizes them and allows the enumeration of clonal outgrowths from single cells [36].

As detailed in Materials and Methods, dissociated prostate cells were prepared from adult C57BL/6 mice and passed through a fluorescence-activated cell sorter (FACS) to produce single-cell suspensions. Cells were serially diluted and plated in triplicate in Matrigel/PrEGM at final densities of 10,000, 5,000, 2,500, and 1,250 cells per well in a 12-well plate.

Small, solid spheroid structures became visible after 5 days in culture. Ten to 13 days after plating, the diameter of the prostate spheres reached 100–200 µm, and the spheres began to canalize and displayed a distinct double-layered appearance (Fig. 1A, insets). Some smaller and more lucent spheroids with a diameter of less than 40 µm were also observed in the culture. These small spheroids stopped growing after day 5 and were not counted as prostate spheres. The number of spheres was plotted against the number of seeded cells, and the frequency of sphere-forming cells was calculated to be approximately 1 out of 500–1,000 by linear regression analysis (Fig. 1A). The sphere-forming capacity is exclusively found in the integrin {alpha}6-expressing prostate stem cell fraction [22].


Figure 1
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  		Figure 1. A small fraction of murine prostate epithelial cells form clonally derived prostate spheres in Matrigel. (A): Graph shows the number of prostate spheres formed at each dilution plotted against the input cell numbers. Insets show representative images of a solid and a double-layered prostate sphere. Scale bars = 100 µm. (B): TI and fluorescence images of the prostate spheres derived from a mixture of prostate cells from β-actin -GFP and -DsRed transgenic mice. Scale bars = 200 µm. (C): Bar graph shows the number of prostate spheres grown from each mixture of GFP+ and DsRed+ cells. The table indicates the number of cells seeded in each condition. Abbreviations: GFP, green fluorescent protein; RFP, red fluorescent protein; TI, transillumination.

 
Prostate Spheres Are Clonally Derived
Dissociated prostate cells were independently prepared from β-actin GFP mice or DsRed mice and sent through a FACS to collect single cells. The green and red cells were seeded separately as controls or mixed in various ratios (1:1, 5:1, and 25:1) and seeded in triplicate as described in Materials and Methods. As shown in Figure 1B, individual spheres were exclusively monochromatic, indicating that spheres were clonally derived. The ratios of different color spheres also matched the ratios of the seeded cells (Fig. 1C).

Sphere-Forming Cells Are Capable of Self-Renewal
Prostate spheres were dissociated into single cells as described in Materials and Methods. Dissociated single cells were reseeded to form secondary spheres (Fig. 2A). As summarized in the table in Figure 2A, prostate spheres continued to form at each generation, and the percentage of the sphere-forming cells varied between 1% and 3% during the first three generations. We repeatedly dissociated and passaged spheres for up to 12 cycles. Interestingly, the majority of the spheres in later passages appeared solid instead of double-layered.


Figure 2
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  		Figure 2. Sphere-forming cells are capable of self-renewal. (A): Schematic illustration of the bulk passaging of prostate spheres. Table shows data from a representative experiment of prostate spheres passaged in bulk. SFU was calculated by dividing the number of input cells by the number of spheres formed. (B): Schematic illustration of the serial passaging of individual prostate spheres. The table summarizes the number of the spheres that can replate out of 24 spheres picked in the experiment and the number of daughter spheres formed from each individual parental sphere. Abbreviations: G, generation; SFU, sphere-forming unit.

 
To truly show self-renewal, individual spheres must be able to give rise to daughter spheres that retain that same capacity (Fig. 2B). Twenty-four generation 1 (G1) spheres derived from DsRed prostate epithelial cells were picked at day 10. To minimize cell loss during the procedures, each sphere was mixed with approximately 20 spheres generated from wild-type epithelial cells, and the mix was digested. The resulting single-cell mixture was seeded into 12-well plates, and the number of cells derived from each red sphere was counted under a fluorescent microscope. Daughter spheres were counted at day 10, and individual secondary red spheres from several representative wells were picked again to repeat the process.

As shown in the table in Figure 2B, on average, there were approximately 200 cells in each G1 sphere, and all 24 G1 spheres were capable of generating various numbers of daughter spheres (generation 2 [G2]) ranging from 1 to 115 (average, 38). In the second round, G2 spheres consisted of an average of 170 cells, and 19 of 24 G2 spheres formed tertiary spheres. The number of generation 3 spheres formed from each G2 sphere ranged from 1 to 85 (average, 25). These data show that prostate spheres can be serially passaged in bulk or individually and demonstrate that sphere-forming cells are capable of self-renewal. In addition, one sphere can give rise to multiple new spheres, indicating that prostate stem/progenitor cells not only self-renew but may also expand in this assay.

Prostate Spheres Contain Organized Structures with Lineage Hierarchy
Sphere cells uniformly expressed CD24, CD49f, and Sca-1 or lacked CD90, CD133, and CD44 surface markers (supplemental online Fig. 1). Tissue section analysis showed that a G1 prostate sphere is composed of a central cavity filled with secretions that is surrounded by a compact outer shell made of four or five layers of cells (Fig. 3A). The centers of the more solid spheres from secondary and tertiary generations consist of a mixture of secretions and sparsely distributed cells that are also surrounded by a few compact layers of cells (Fig. 3B).


Figure 3
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  		Figure 3. Prostate spheres spontaneously evolve organized structures with a lineage hierarchy. (A, B): H&E staining of double-layered and solid prostate spheres. (C, E, G, J) and (D, F, H, K) show the immunohistochemical (IHC) analyses of cytokeratin 5, cytokeratin 8, AR, and P63 on double-layered and solid spheres, respectively. (I): Western blot analysis of AR expression in prostate spheres. (L): IHC analysis of Ki67 expression. (M): TUNEL assay analysis of prostate spheres. (N): TI and fluorescence images of the prostate spheres formed from cells derived from PSCA-GFP transgenic mice. (O): Diagram modeling the cell lineage hierarchy in prostate spheres based on the protein expression profiles of the different layers. Scale bars = 100 µm. Abbreviations: {alpha}-, anti-; AR, androgen receptor; GFP, green fluorescent protein; PSCA, prostate stem cell antigen; TI, transillumination; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

 
We performed IHC analysis using antibodies against several epithelial markers. Prostate sphere cells strongly expressed the basal cell marker K5 (Fig. 3C, 3D) but expressed a very low level of the luminal cell markers K8 (Fig. 3E, 3F). The expression of androgen receptor (AR) was undetectable by IHC analysis (Fig. 3G, 3H). However, Western blot analysis showed that prostate sphere cells expressed AR, but the majority of the protein was partially degraded and displayed an apparent molecular mass of approximately 87 kDa (Fig. 3I) [37]. The expression of the neuroendocrine cell marker synaptophysin was not detected by IHC analysis. In summary, prostate sphere cells are of epithelial origin and uniformly express a high level of the basal cell marker K5 and low levels of luminal cell markers K8 and AR.

P63, a transcription factor belonging to the P53 superfamily, is expressed in prostatic basal cells and plays a critical role in prostate development [38–40]. IHC analysis showed that P63 is only present in the cells of the outer layers of prostate spheres (Fig. 3J, 3K). Most P63-expressing cells were actively proliferating as demonstrated by Ki-67 staining, whereas apoptotic cells were mainly observed in the cores of the spheres by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay (Fig. 3L, 3M). These data demonstrate that prostate spheres maintain a distinct cellular hierarchy and structural organization.

P63 is essential for prostate development and is only expressed by basal cells where stem cells are suspected to reside. To explore whether cells in the inner layers of prostate spheres are comparably more differentiated, we took advantage of a PSCA-GFP transgenic mouse model that harbors a GFP transgene driven by the human PSCA promoter [31]. PSCA is a member of the Ly-6/Thy-1 family of GPI-anchored glycoprotein and has been shown to be a marker for the transit-amplifying cell population in the prostate [31, 41].

Dissociated prostate cells from PSCA-GFP mice were fractionated into GFP-positive and -negative populations using a FACS and seeded in the sphere assay separately. Prostate spheres grew only from the GFP-negative cells, indicating that PSCA-expressing transit-amplifying cells are not the sphere-forming cells. GFP fluorescence was examined daily on the spheres. Prostate spheres did not express GFP at first (Fig. 3N). However, cells at the center of the spheres turned GFP-positive after 7 days in culture, indicating that they started to differentiate and express PSCA. From the rim to the center of the spheres, cells displayed a phenotype from early stem/progenitor cells to more differentiated transit-amplifying cells (Fig. 3O).

P63-Expressing Cells Possess Higher Self-Renewal Activity
Since P63 is critical for prostate development, we evaluated whether the p63-expressing cells in the spheres possessed higher self-renewal capacity. At least six p63 isoforms are transcribed through alternative promoter use and splicing [38]. The major p63 isoform expressed in normal prostate epithelia is {Delta}Np63{alpha} [39]. RT-PCR showed that the {Delta}Np63 isoforms were also dominant in prostate spheres (Fig. 4A). Western blot confirmed that {Delta}Np63{alpha} protein was the major isoform detected (Fig. 4B).


Figure 4
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  		Figure 4. P63-expressing prostate sphere cells possess higher self-renewal activity. (A): RT-PCR analysis of p63 isoforms expressed by prostate spheres. (B): Western blot analysis of the P63 isoform expressed by prostate spheres. (C): Fluorescence-activated cell sorter analysis of dissociated cells from Q+ prostate spheres. (D): Quantitative analysis of the expression of p63 in Q+ and Q– prostate sphere cells. (E): Western blot analysis of P63 expression in Q+ and Q– prostate sphere cells. (F): Bar graph shows the percentage of sphere-forming cells in Q+, Q–, and unsorted prostate sphere cells. Abbreviations: FACS, fluorescence-activated cell sorter; M, marker; PE, phycoerythrin; Q+, Qtracker-labeled; Q–, Qtracker-unlabeled; Qdot, Qtracker; qPCR, quantitative polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction.

 
A Qtracker 525 cell labeling kit was used to enrich the p63-expressing cells. This kit delivers fluorescent nanocrystals into the cytoplasm of live cells. Intact spheres were incubated with the nanocrystals so that the p63-expressing cells in the outer layer of the spheres were fluorescently marked and could be separated from the unlabeled inner-layer cells using a FACS. Approximately 30% of sphere cells were labeled by the fluorescent nanocrystals (Fig. 4C). RNA and protein lysates were made from both fractions. Quantitative RT-PCR and Western blot analysis confirmed that there was a twofold enrichment of p63 expression in the Q dot-labeled sphere cells (Fig. 4D, 4E).

The sphere-forming activities of total sphere cells and FACS-sorted fluorescence-labeled and unlabeled cells were determined using the prostate sphere assay. Three independent experiments showed that the sphere-forming capacity of the Qtracker-labeled cells was three times that of the nonlabeled cells, demonstrating that the P63-expressing population displays a higher self-renewal activity (Fig. 4F).

Prostate Sphere Cells Undergo Further Differentiation upon Induction by 5{alpha}-Dihydrotestosterone Stimulation and Attachment to Substrata
Prostate spheres consistently express a high level of the basal cell marker K5 and low levels of the luminal cell markers K8 and AR. Androgen-mediated signals in normal prostatic epithelia induce differentiation [42]. Since prostate sphere cells express low levels of AR, we monitored the effects of the AR ligand 5{alpha}-dihydrotestosterone (DHT) on the differentiation of prostate sphere cells in Matrigel.

DHT was added to G2 prostate sphere cultures at a final concentration of 10–8 M. DHT was replenished every 3 days, and cells were cultured for 10 days. The spheres formed under these conditions were morphologically distinct from the relatively homogeneous solid spheres generated in the control culture treated with vehicle alone. Some spheres consisted of a single lucent cavity surrounded by a thin layer of cells (Fig. 5A1, 5A2), whereas other spheres developed multiple lucent cavities in their centers, causing a lobulated appearance (Fig. 5A, 5A4). H&E staining confirmed this morphology (Fig. 5A5) and showed that these lumens were free of protein secretions.


Figure 5
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  		Figure 5. Prostate sphere cells undergo further differentiation upon stimulation with DHT and substrata adhesion. (A): Effects of DHT treatment on morphology and degree of differentiation in prostate spheres. Panels 1–4, representative images of lucent hollow spheres formed in the presence of DHT; panel 5, H&E staining; panels 6–9, immunohistochemical analysis of K5, P63, K8, and AR; panel 10, Western blot analysis of AR. Scale bars = 100 µm. (B): Quantitative RT-PCR analysis of changes of the expression levels of the lineage markers K5, p63, K8, and Ar. (C): Effects of DHT treatment and attachment to substrata on the differentiation of prostate sphere cells. Images show the immunocytochemical analysis of basal cell marker k5 and luminal cell markers K8 and AR. Abbreviations: {alpha}-, anti-; AR, androgen receptor; DHT, 5{alpha}-dihydrotestosterone; Kd, kilodaltons.

 
IHC analyses showed that K5 was still expressed by all sphere cells, and P63 continued to be expressed by the cells in the outer layer of the spheres (Fig. 5A, 5A7). However, expression of K8 was increased in the inner layer of the prostate spheres, and nuclear staining of AR was also detected (Fig. 5A, 5A9). Crucially, Western blot analyses showed that most of the AR in DHT-treated prostate spheres had a molecular mass of 113–120 kDa in contrast to 87 kDa in the vehicle-treated group (Fig. 5A). This is consistent with the previous report that ligand binding stabilizes the AR protein [37]. We also observed degraded AR fragments with molecular masses less than 50 kDa in the vehicle-treated prostate sphere samples. Quantitative RT-PCR was performed to evaluate changes in the expression levels of different prostate lineage markers (Fig. 5B). Whereas the expression of K5 decreased only slightly, p63 was reduced significantly, and K8 expression increased approximately fourfold with the addition of DHT. Consistent with the Western blot analysis, AR expression did not change dramatically, suggesting that protein stabilization and nuclear import lead to the ability to detect AR by IHC analysis. Nkx3.1, a homeobox gene expressed specifically by differentiated prostatic luminal cells [43], was not detectable by RT-PCR in the vehicle or DHT-treated spheres (data not shown).

Human mammary sphere cells can terminally differentiate when plated on collagen [10]. We evaluated the potential of the prostate sphere cells to undergo further differentiation in vitro upon simultaneous adhesion to substrata and hormonal stimulation. Dissociated prostate sphere cells were cultured in PrEGM medium on irradiated NIH3T3 cells. Epithelial colonies became visible after 5 days. DHT was added at day 8 to a final concentration of 10–8 M and incubated for another 6 days. IHC analyses were performed to evaluate the expression of the lineage markers (K5, K8, and AR) in day 8 (d8) colonies and DHT-treated or vehicle-treated d14 colonies. Cells in d8 colonies strongly expressed both K5 and K8 (Fig. 5C, left panels). K5 expression was reduced or lost in a few cells in d14 colony cells (Fig. 5C, middle panels), suggesting that these cells underwent further differentiation. AR remained undetectable at these stages. However, when treated with DHT, most colony cells became K5lowK8high, and AR expression became detectable via IHC analysis (Fig. 5C, right panels). These data demonstrate that attachment to substrata in combination with hormonal stimulation promotes prostate sphere cells to further differentiate.

Prostate Sphere Cells Can Form Glandular Structures Resembling Murine Prostate Tissue In Vivo
We tested whether sphere-forming cells retain differentiation capacity in vivo. G1 sphere cells (5 x 104) were mixed with 1 x 105 UGSM cells in Matrigel and injected subcutaneously into CB17Scid/Scid mice. Grafts were collected 7 weeks later, fixed with formalin, and embedded in paraffin to prepare tissue sections. H&E staining revealed that sphere cells regenerated glandular structures consisting of a single layer of epithelial cells surrounding lumens that were occasionally filled with secretions (Fig. 6A). IHC analyses showed that these glandular structures were reminiscent of adult murine prostate tissues and were composed of K8- and AR-positive luminal cells and K5- and P63-positive basal cells (Fig. 6B–6E). Protein secretions that accumulated within some lumens were immunoreactive to an antiserum prepared against murine dorsolateral prostate secretions [44] (Fig. 6F).


Figure 6
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  		Figure 6. Some prostate sphere cells retain the capacity to regenerate globular structures resembling adult murine prostate glands. (A): H&E staining of a regenerated glandular structure. (B–F): Immunohistochemical analyses of the expression of K5, K8, P63, AR, and mDLP secretions. Insets show details of higher magnification. Scale bars = 100 µm. Abbreviations: AR, androgen receptor; mDLP, murine dorsolateral prostate.

 
Although these data demonstrate that prostate sphere-forming cells retain the capacity of multilineage differentiation in vivo, we noticed that the efficiency of in vivo glandular structure formation was low. If each sphere-forming cell can generate a glandular structure in vivo, we would expect approximately 500 regenerated glands from 5 x 104 prostate sphere cells. However, we observed fewer than 20 glandular structures. The dissociated G2 prostate sphere cells were also tested in the in vivo assay; very few globular structures were observed, and none of them contained secretions inside the lumen. These results may indicate that not all sphere-forming cells in vitro are capable of forming glandular structures in vivo. Alternatively, the conditions for in vivo regeneration may not be optimal for prostate stem cells to function efficiently.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
Self-renewal and multilineage differentiation are two key features of stem cells. The differentiation capacity of prospectively isolated human and murine prostate stem cells has been demonstrated using prostate regeneration methods [20, 22, 45]. The self-renewal capacity of prostate stem cells has been implicated by castration and androgen cycling experiments performed in rodents [6]. We and others have also pursued the self-renewal activity of prostate stem cells using the prostate regeneration system. Regenerated murine prostate tissues can be serially transplanted for up to 2–4 passages (L. Xin and O.N. Witte, unpublished data; [46]). However, the self-renewal capacity of prostate stem cells has not previously been demonstrated at a clonal level.

In Vitro Maintenance and Characterization of Prostate Stem Cells
We show here that approximately 0.1% of the epithelial cells in murine prostate possess prostate stem/progenitor cell activity as demonstrated by their growth in Matrigel. These spheres can be serially passaged in bulk or individually, definitively demonstrating the self-renewal capacity of prostate stem cells in vitro. Interestingly, although only 1%–3% of prostate sphere cells in the first three generations could form new spheres, the percentage of the sphere-forming cells increased in later passages. Potential mechanisms underlying this change are under investigation.

Previous studies have shown that inhibition of Sonic Hedgehog (Shh) and Notch signaling interferes with normal prostate development and morphogenesis, indicating that these pathways play important roles in prostate stem cell maintenance [29, 30]. Disrupting these signaling pathways in the prostate sphere assay using small molecule inhibitors such as cyclopamine (Shh inhibitor) [47] and DAPT (LY-374973; a {gamma}-secretase inhibitor) [48] significantly decreased the cells' sphere-forming capacity and self-renewal activity (R.U. Lukacs and O.N. Witte, unpublished data).

Not all self-renewing prostate sphere cells can generate globular structures in vivo. This was also observed in the mammary system. Stingl et al. showed that the CD24lowCD49fhigh and CD24medCD49fhigh mammary epithelial cells display myoepithelial lineage identities [7]. Both populations can form solid spheres in a Matrigel assay; however, only CD24lowCD49fhigh cells could reconstitute mammary glands in vivo.

Role of Microenvironment in the Maintenance and Differentiation of Prostate Stem Cells
A unique feature of the prostate sphere assay is the immobilization of the cells in a semisolid matrix that contains high levels of laminin and other factors (Matrigel; product description by BD Biosciences). Interactions of cell surface receptors with ECM proteins in Matrigel have been shown to regulate breast epithelial cell polarity and morphology and mediate signals of survival, proliferation, and differentiation [49]. Human prostate cancer cell lines have been shown to form spheroids with glandular differentiation in Matrigel [50]. Bello-DeOcampo et al. further demonstrated that normal and malignant human prostate epithelial cells develop acinar morphogenesis in Matrigel through interaction of their surface receptor integrin {alpha}6 with the laminin in Matrigel [51]. Comparably, we found that murine prostate stem cells, which express high levels of integrin {alpha}6 [22], cannot generate prostate spheres when cultured in rat tail collagen alone. However, prostate spheres formed at low efficiency in collagen supplemented with laminin alone, suggesting that laminin is necessary but not sufficient for sphere formation (data not shown).

Differentiation Program of the Prostate Stem Cell
The differentiation program of the prostate stem cell remains unclear. Human prostate basal cells have been shown to differentiate into luminal cells in in vitro culture systems [41, 52]. These data suggest that prostate stem cells may differentiate in a linear fashion in which stem cells generate basal cells that in turn differentiate into luminal cells [18]. Alternatively, stem cells may differentiate via a branching pathway to generate progenitor cells for each lineage that separately undergo further maturation. This theory is supported by the observation that human and rodent embryonic prostatic epithelial progenitor cells express lineage markers of both basal and luminal cells [53, 54].

We showed that adhesion to substrata and stimulation by DHT downregulate the expression of basal cell markers but increase luminal cell marker expression in prostate sphere cells. These data support the linear model for prostate stem cell differentiation; however, a branching model cannot be ruled out. A branching model for mammary stem cell differentiation has been proposed recently; Asselin-Labat et al. defined mammary epithelial cells displaying a CD24lowCD29+CD61+ expression profile as luminal progenitor cells [55]. These cells can form luminal colonies in an in vitro assay [55]. The mammary and prostate epithelia share highly similar anatomic and phenotypic features. Prostate stem cells may use a similar branched differentiation program.

A key feature of the terminally differentiated prostatic luminal cells is their secretory function. However, some of the globular structures derived from prostate sphere cells in vivo lacked secretions (data not shown). In the prostate sphere assay, the K8+AR+ prostate sphere cells in the DHT-treated condition failed to produce secretions, and we were unable to detect the expression of the luminal cell marker Nkx3.1. It has been shown that prostatic secretory proteins were greatly reduced or eliminated in Nkx3.1-null mice [43]. These observations indicate a lack of terminally differentiated luminal cells in prostate spheres under the selected conditions we have used and support the branching model. Additional experiments using lineage tracing approaches are necessary to definitively elucidate the differentiation program of prostate stem cells [56].

P63 in Prostate Development and Homeostasis
P63 is a transcriptional factor belonging to the P53 superfamily whose deletion causes a failure to develop stratified epithelial and epithelial appendages in mice [38, 40]. These phenotypes have led to two alternative functions for p63: proliferative potential of the epithelial stem cells and the commitment to stratification [38, 40, 57, 58]. P63 is highly expressed in rabbit limbal stem cells and is a marker for keratinocyte stem cells maintained in vitro [59, 60]. In addition, P63 has been shown to regulate pathways mediating stem cell function, such as Shh and Notch [61, 62]. Studies on rodent skin development have demonstrated that the balance of signaling of p63 isoforms acts as a molecular switch for initiation of the epithelial stratification program [57]. {Delta}Np63 was shown to maintain the integrity of the basement membrane and regulate cell adhesion, tissue morphogenesis, and homeostasis [63]. P63 has also been found to affect breast cancer epithelial cell survival by regulating adhesion-associated genes [64].

Although prostate epithelium is not considered a stratified epithelium, P63 deletion causes prostate agenesis [38, 40]. It is not clear how P63 functions in prostate. Signoretti et al. complemented p63–/– blastocysts with p63+/+ β-galactosidase-positive embryonic stem cells and found that luminal cells of the prostate originated only from p63-positive donors, which suggests the defects of the p63-null stem cells in proliferation and self-maintenance [39]. Kurita et al. grafted p63–/– urogenital sinus into adult male nude mice and found that prostate tissues were generated, except that the ducts were less dilated, basal cells were absent, and atypical mucin-producing cells were produced [65]. This result highlights the defects in the differentiation potential of p63-null stem cells.

In our prostate sphere assay, the P63-expressing cells possess higher stem cell activity in forming secondary spheres. Interestingly, these cells are actively proliferating and located in the outer layer of the spheres, where they are directly in contact with ECM components. These observations suggest a link between the two functions of p63 in prostate development and homeostasis: P63 may regulate the expression of adhesion molecules such as the integrins to modulate signaling networks between stem cells and their niche that subsequently control the proliferation capacity and the differentiation program of the prostate stem cells.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Dr. Michael Teitell for helpful discussion, Dr. Gerald Cunha for the polyclonal antiserum reactive with mouse dorsolateral prostate secretions, Shirley Quan and Lakeisha Perkins for outstanding technical assistance, and Barbara Anderson for help with preparation of the manuscript. L.X. was supported by funds from the Prostate Cancer Foundation and the University of California Los Angeles Special Program of Research Excellence in Prostate Cancer (Jean deKernion, Principal Investigator) and a training grant from the California Institute for Regenerative Medicine. O.N.W. is an Investigator of the Howard Hughes Medical Institute. L.X. and R.U.L. contributed equally to this paper.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

  1. Potten CS, Loeffler M. Stem cells: Attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development 1990;110:1001–1020.[Abstract/Free Full Text]

  2. Tumbar T, Guasch G, Greco V et al. Defining the epithelial stem cell niche in skin. Science 2004;303:359–363.[Abstract/Free Full Text]

  3. Spangrude GJ, Heimfeld S, Weissman I. Purification and characterization of mouse hematopoietic stem cells. Science 1988;241:58–62.[Abstract/Free Full Text]

  4. Kornblum HI. Introduction to neural stem cells. Stroke 2007;38 (suppl):810–816.[Abstract/Free Full Text]

  5. Stingl J, Raouf A, Emerman JT et al. Epithelial progenitors in the normal human mammary gland. J Mammary Gland Biol Neoplasia 2005;10:49–59.[CrossRef][Medline]

  6. Isaacs JT. Control of cell proliferation and death in the normal and neoplastic prostate: A stem cell model. In: Rodgers CH, Coffey DS, Cunha G, eds. Benign Prostatic Hyperplasia.Bethesda, MD: Department of Health and Human Services, National Institutes of Health,1987;85–94 Report no. NIH 87-2881.

  7. Stingl J, Eirew P, Ricketson I et al. Purification and unique properties of mammary epithelial stem cells. Nature 2006;439:993–997.[Medline]

  8. Shackleton M, Vaillant F, Simpson KJ et al. Generation of a functional mammary gland from a single stem cell. Nature 2006;439:84–88.[CrossRef][Medline]

  9. Reynolds BA, Weiss S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 1996;175:1–13.[CrossRef][Medline]

  10. Dontu G, Abdallah WM, Foley JM et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 2003;17:1253–1270.[Abstract/Free Full Text]

  11. Reynolds BA, Rietze RL. Neural stem cells and neurospheres—Re-evaluating the relationship. Nat Methods 2005;2:333–336.[CrossRef][Medline]

  12. Dontu G, Jackson KW, McNicholas E et al. Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells. Breast Cancer Res 2004;6:R605–15.[CrossRef][Medline]

  13. Hitoshi S, Alexson T, Tropepe V et al. Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells. Genes Dev 2002;16:846–858.[Abstract/Free Full Text]

  14. Molofsky AV, He S, Bydon M et al. Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by repressing the p16Ink4a and p19Arf senescence pathways. Genes Dev 2005;19:1432–1437.[Abstract/Free Full Text]

  15. Park IK, Qian D, Kiel M et al. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 2003;423:302–305.[CrossRef][Medline]

  16. Abate-Shen C, Shen MM. Molecular genetics of prostate cancer. Genes Dev 2000;14:2410–2434.[Free Full Text]

  17. Uzgare AR, Xu Y, Isaacs JT. In vitro culturing and characteristics of transit amplifying epithelial cells from human prostate tissue. J Cell Biochem 2004;91:196–205.[CrossRef][Medline]

  18. Litvinov IV, Vander Griend DJ, Xu Y et al. Low-calcium serum-free defined medium selects for growth of normal prostatic epithelial stem cells. Cancer Res 2006;66:8598–8607.[Abstract/Free Full Text]

  19. Cunha GR, Lung B. The possible influence of temporal factors in androgenic responsiveness of urogenital tissue recombinants from wild-type and androgen-insensitive (Tfm) mice. J Exp Zool 1978;205:181–193.[CrossRef][Medline]

  20. Xin L, Ide H, Kim Y et al. In vivo regeneration of murine prostate from dissociated cell populations of postnatal epithelia and urogenital sinus mesenchyme. Proc Natl Acad Sci U S A 2003;100 (suppl):11896–11903.[Abstract/Free Full Text]

  21. Xin L, Lawson DA, Witte ON. The Sca-1 cell surface marker enriches for a prostate-regenerating cell subpopulation that can initiate prostate tumorigenesis. Proc Natl Acad Sci U S A 2005;102:6942–6947.[Abstract/Free Full Text]

  22. Lawson DA, Xin L, Lukacs RU et al. Isolation and functional characterization of murine prostate stem cells. Proc Natl Acad Sci U S A 2007;104:181–186.[Abstract/Free Full Text]

  23. Tokar EJ, Ancrile BB, Cunha GR et al. Stem/progenitor and intermediate cell types and the origin of human prostate cancer. Differentiation 2005;73:463–473.[CrossRef][Medline]

  24. Hudson DL, O'Hare M, Watt FM et al. Proliferative heterogeneity in the human prostate: Evidence for epithelial stem cells. Lab Invest 2000;80:1243–1250.[Medline]

  25. Robinson EJ, Neal DE, Collins AT. Basal cells are progenitors of luminal cells in primary cultures of differentiating human prostatic epithelium. Prostate 1998;37:149–160.[CrossRef][Medline]

  26. Kinbara H, Cunha GR, Boutin E et al. Evidence of stem cells in the adult prostatic epithelium based upon responsiveness to mesenchymal inductors. Prostate 1996;29:107–116.[CrossRef][Medline]

  27. Garraway LA, Lin D, Signoretti S et al. Intermediate basal cells of the prostate: In vitro and in vivo characterization. Prostate 2003;55:206–218.[CrossRef][Medline]

  28. Sawicki JA, Rothman CJ. Evidence for stem cells in cultures of mouse prostate epithelial cells. Prostate 2002;50:46–53.[CrossRef][Medline]

  29. Karhadkar SS, Bova GS, Abdallah N et al. Hedgehog signalling in prostate regeneration, neoplasia and metastasis. Nature 2004;431:707–712.[CrossRef][Medline]

  30. Wang XD, Shou J, Wong P et al. Notch1-expressing cells are indispensable for prostatic branching morphogenesis during development and re-growth following castration and androgen replacement. J Biol Chem 2004;279:24733–24744.[Abstract/Free Full Text]

  31. Watabe T, Lin M, Ide H et al. Growth, regeneration, and tumorigenesis of the prostate activates the PSCA promoter. Proc Natl Acad Sci U S A 2002;99:401–406.[Abstract/Free Full Text]

  32. Petersen OW, Rønnov-Jessen L, Howlett AR et al. Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proc Natl Acad Sci U S A 1992;89:9064–9068.[Abstract/Free Full Text]

  33. Medina RJ, Kataoka K, Takaishi M et al. Isolation of epithelial stem cells from dermis by a three-dimensional culture system. J Cell Biochem 2006;98:174–184.[CrossRef][Medline]

  34. Qian H, Tryggvason K, Jacobsen SE et al. Contribution of alpha6 integrins to hematopoietic stem and progenitor cell homing to bone marrow and collaboration with alpha4 integrins. Blood 2006;107:3503–3510.[Abstract/Free Full Text]

  35. Jones RG, Li X, Gray PD et al. Conditional deletion of beta1 integrins in the intestinal epithelium causes a loss of Hedgehog expression, intestinal hyperplasia, and early postnatal lethality. J Cell Biol 2006;175:505–514.[Abstract/Free Full Text]

  36. Singec I, Knoth R, Meyer RP et al. Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology. Nat Methods 2006;3:801–806.[CrossRef][Medline]

  37. Gregory CW, He B, Wilson EM. The putative androgen receptor-A form results from in vitro proteolysis. J Mol Endocrinol 2001;27:309–319.[Abstract]

  38. Yang A, Schweitzer R, Sun D et al. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 1999;398:714–718.[CrossRef][Medline]

  39. Signoretti S, Waltregny D, Dilks J et al. p63 is a prostate basal cell marker and is required for prostate development. Am J Pathol 2000;157:1769–1775.[Abstract/Free Full Text]

  40. Mills AA, Zheng B, Wang XJ et al. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 1999;398:708–713.[CrossRef][Medline]

  41. Tran CP, Lin C, Yamashiro J et al. Prostate stem cell antigen is a marker of late intermediate prostate epithelial cells. Mol Cancer Res 2002;1:113–121.[Abstract/Free Full Text]

  42. Isaacs JT, Isaacs WB. Androgen receptor outwits prostate cancer drugs. Nat Med 2004;10:26–27.[CrossRef][Medline]

  43. Bhatia-Gaur R, Donjacour AA, Sciavolino PJ et al. Roles for Nkx3.1 in prostate development and cancer. Genes Dev 1999;13:966–977.[Abstract/Free Full Text]

  44. Donjacour AA et al. Characterization of antibodies to androgen-dependent secretory proteins of the mouse dorsolateral prostate. Endocrinology 1990;126:1343–1354.[Abstract/Free Full Text]

  45. Richardson GD, Robson CN, Lang SH et al. CD133, a novel marker for human prostatic epithelial stem cells. J Cell Sci 2004;117:3539–3545.[Abstract/Free Full Text]

  46. Goto K, Salm SN, Coetzee S et al. Proximal prostatic stem cells are programmed to regenerate a proximal-distal ductal axis. STEM CELLS 2006;24:1859–1868.[Abstract/Free Full Text]

  47. Cooper MK, Porter JA, Young KE et al. Teratogen-mediated inhibition of target tissue response to Shh signaling. Science 1998;280:1603–1607.[Abstract/Free Full Text]

  48. Dovey HF, John V, Anderson JP et al. Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain. J Neurochem 2001;76:173–181.[CrossRef][Medline]

  49. Bissell MJ, Radisky DC, Rizki A et al. The organizing principle: Microenvironmental influences in the normal and malignant breast. Differentiation 2002;70:537–546.[CrossRef][Medline]

  50. Lang SH, Sharrard RM, Stark M et al. Prostate epithelial cell lines form spheroids with evidence of glandular differentiation in three-dimensional Matrigel cultures. Br J Cancer 2001;85:590–599.[CrossRef][Medline]

  51. Bello-DeOcampo D, Kleinman HK, Deocampo ND et al. Laminin-1 and alpha6beta1 integrin regulate acinar morphogenesis of normal and malignant human prostate epithelial cells. Prostate 2001;46:142–153.[CrossRef][Medline]

  52. Liu AY, True LD, LaTray L et al. Cell-cell interaction in prostate gene regulation and cytodifferentiation. Proc Natl Acad Sci U S A 1997;94:10705–10710.[Abstract/Free Full Text]

  53. Wang Y, Hayward S, Cao M et al. Cell differentiation lineage in the prostate. Differentiation 2001;68:270–279.[CrossRef][Medline]

  54. Xue Y, Smedts F, Debruyne FM et al. Identification of intermediate cell types by keratin expression in the developing human prostate. Prostate 1998;34:292–301.[CrossRef][Medline]

  55. Asselin-Labat ML, Sutherland KD, Barker H et al. Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation. Nat Cell Biol 2007;9:201–209.[CrossRef][Medline]

  56. Dor Y, Brown J, Martinez OI et al. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 2004;429:41–46.[CrossRef][Medline]

  57. Koster MI, Kim S, Mills AA et al. p63 is the molecular switch for initiation of an epithelial stratification program. Genes Dev 2004;18:126–131.[Abstract/Free Full Text]

  58. Senoo M, Pinto F, Crum CP et al. p63 is essential for the proliferative potential of stem cells in stratified epithelia. Cell 2007;129:523–536.[CrossRef][Medline]

  59. Pellegrini G, Dellambra E, Golisano O et al. p63 identifies keratinocyte stem cells. Proc Natl Acad Sci U S A 2001;98:3156–3161.[Abstract/Free Full Text]

  60. Epstein SP, Wolosin JM, Asbell PA. p63 expression levels in side population and low light scattering ocular surface epithelial cells. Trans Am Ophthalmol Soc 2005;103:187–199 discussion 199.[Medline]

  61. Caserta TM, Kommagani R, Yuan Z et al. p63 overexpression induces the expression of Sonic Hedgehog. Mol Cancer Res 2006;4:759–768.[Abstract/Free Full Text]

  62. Nguyen BC, Lefort K, Mandinova A et al. Cross-regulation between Notch and p63 in keratinocyte commitment to differentiation. Genes Dev 2006;20:1028–1042.[Abstract/Free Full Text]

  63. Koster MI, Dai D, Marinari B et al. p63 induces key target genes required for epidermal morphogenesis. Proc Natl Acad Sci U S A 2007;104:3255–3260.[Abstract/Free Full Text]

  64. Stern CD, Fraser SE. Tracing the lineage of tracing cell lineages. Nat Cell Biol 2001;3:E216–8.[CrossRef][Medline]

  65. Kurita T, Medina RT, Mills AA et al. Role of p63 and basal cells in the prostate. Development 2004;131:4955–4964.[Abstract/Free Full Text]




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