A Role for AngiotensinConverting Enzyme in the ... - Wiley Online Library

TISSUE-SPECIFIC STEM CELLS A Role for Angiotensin-Converting Enzyme in the Characterization, Enrichment, and Proliferation Potential of Adult Murine Pituitary Colony-Forming Cells DIANA A. LEPORE,a VANTA J. JOKUBAITIS,b PAUL J. SIMMONS,b KELLY N. ROESZLER,a RALPH ROSSI,c KARL BAUER,d PAUL Q. THOMASe a

Pituitary Research Unit, Murdoch Childrens Research Institute, Royal Childrens Hospital, Parkville, Victoria, Australia; bStem Cell Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; cFlow Cytometry Unit, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; dMax-Planck-Institut fur Experimentelle, Endokrinologie, Hannover, Germany; eSchool of Molecular & Biomedical Science, The University of Adelaide, Adelaide, South Australia, Australia Key Words. Angiotensin-converting enzyme • Pituitary • Tissue stem cells • 7-Amino-4-methylcoumarin-3-acetic acid

ABSTRACT Recently, we described a rare cell type within the adult murine pituitary gland with progenitor cell hallmarks (PCFCs). PCFCs are contained exclusively within a subpopulation of cells that import fluorescent ␤-Ala-Lys-N␧AMCA (7-amino-4-methylcoumarin-3-acetic acid). Herein, we investigate the utility of cell surface molecules angiotensin-converting enzyme (ACE) and stem cell antigen-1 (Sca-1) to further enrich for PCFCs. ACE and Sca-1 were expressed on 61% and 55% of AMCAⴙCD45ⴚCD31ⴚ cells, respectively, and coexpressed on 38%. ACEⴙSca-1ⴙAMCAⴙ cells enriched for PCFCs by 195-fold over unselected cells. ACEⴙAMCAⴙ cells enriched for PCFCs by 170-fold, and

colonies were twofold larger than for AMCAⴙ selection alone. Conversely, ACEⴚ-selected cells reduced both colony-forming activity and size. Notably, colonies generated from AMCAⴙ cells obtained from ACEnull mice were 2.7-fold smaller than for wild-type mice. These data identify ACE as a previously unrecognized marker of PCFCs and suggest that ACE is functionally important for PCFC proliferation. Anatomically, the cells that imported AMCA and expressed ACE were situated in the marginal epithelial cell layer of the pituitary cleft and in the adjacent subluminal zone, thus supporting previous proposals that the luminal zone is a source of precursor cells in the adult pituitary. STEM CELLS 2006;24:2382–2390

INTRODUCTION

itary with the properties of progenitor cells and have termed these cells pituitary colony-forming cells (PCFCs) [5]. PCFCs comprise approximately 0.2% of the anterior pituitary population and exhibit robust colony-forming activity (CFA) in vitro. PCFC colonies derived from single cells contain a low frequency of GH and prolactin immunopositive cells, suggesting that at least some PCFCs possess differentiation potential. We have also shown that PCFCs can be enriched 62-fold by means of their ability to import the fluorophore-tagged dipeptide ␤-Ala-Lys-N␧-AMCA (7-amino-4methylcoumarin-3-acetic acid) via the PepT2 transporter [5]. However, many of the key features of PCFCs are unclear, including their precise location within the pituitary gland and their repertoire of expression of cell surface molecules. We reasoned that identifying cell surface molecules whose expression was restricted to PCFC would

Growth hormone (GH) deficiency is a significant clinical problem [1] and in many cases is caused by a paucity of functional GH-secreting cells (somatotropes) in the anterior pituitary. Somatotrope function is controlled principally through the hypothalamic peptide GH releasing factor, which stimulates cell division and the synthesis/release of GH [2, 3]. An alternative mechanism by which somatotropes may be generated is via differentiation of a progenitor or stem cell located in the anterior pituitary. Direct supportive evidence for a GH progenitor cell was first published by Borrelli et al., who demonstrated that somatotropes of the pituitary gland are capable of complete repopulation after their selective depletion by pharmacological ablation [4]. Recently, we reported for the first time the existence of a subpopulation of cells within the adult murine pitu-

Correspondence: Diana Lepore, Ph.D., Pituitary Research Unit, Murdoch Childrens Research Institute, Royal Childrens Hospital, Parkville, Victoria, 3052, Australia. Telephone: 61-3-8341-6286; Fax: 61-3-8341-6366; e-mail: [email protected] Received February 11, 2006; accepted for publication June 22, 2006; first published online in STEM CELLS EXPRESS July 20, 2006. © AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2006-0085

STEM CELLS 2006;24:2382–2390 www.StemCells.com

Lepore, Jokubaitis, Simmons et al. provide an improved means to prospectively isolate these progenitors and thereby facilitate the study of their properties in vitro and their anatomical location in vivo in the adult pituitary gland. To this end, we examined the expression of two cell surface glycoproteins, stem cell-associated antigen-1 (Sca-1) and angiotensin-converting enzyme (ACE), on PCFCs. Initially defined as a marker of murine hemopoietic stem cells [6], Sca-1 has since been identified on putative stem cells in a range of nonhemopoietic tissues, including skeletal and cardiac muscle, mammary tissue, lung, and mesenchymal stem cells [7, 8] (I. Bertoncello et al., manuscript in preparation; M. Short et al., manuscript in preparation). Angiotensin I-converting enzyme (kininase II, EC 3.4.15.1, CD 143) is a zinc-dependent carboxydipeptidase that, as a component of the renin-angiotensin system (RAS), is best known for its key role in the regulation of blood pressure and the development of vascular pathology and remodeling. Constitutively expressed by a range of cell types, including endothelial cells, epithelial and neuroepithelial cells, and dendritic cells of the immune system, ACE is also involved in neuropeptide metabolism and reproductive and immune functions [9, 10]. Although relatively few studies have examined a role for ACE in the context of stem cell regulation, recent observations by several of the current authors demonstrate that ACE is also constitutively expressed by both human hemopoietic stem cells (V.J. Jokubaitis et al., manuscript in preparation) and mesenchymal stem cells [11, 12]. In recent years, it has become clear that ACE is also a regulator of hemopoietic stem cell proliferation. This was first indicated by the observation that occasional hypertensive patients treated with specific inhibitors of ACE activity developed granulocytopenia, aplastic anemia, and pancytopenia [13–18]. In vitro and in vivo studies in mice have since shown that inhibition of ACE activity inhibits the proliferation of primitive hematopoietic cells [11, 19 –24]. ACE exerts its proliferative effect by hydrolyzing of N-acetyl-Ser-Asp-Lys-Pro (AcSDKP), a natural inhibitor of hemopoietic stem cell proliferation, which prevents entry into S-phase of the cell cycle [25, 26]. Given that stem cells from diverse origins have considerable overlap in their transcriptional profiles [27, 28], ACE may also control proliferation of other stem/progenitor cells. To date, this possibility has not been explored. In the current study, we investigated the potential of ACE and of Sca-1 to enrich for PCFCs in the pituitary gland. Here, we show that 60% of AMCA⫹ cells express ACE and that this marker, in combination with positive selection for AMCA uptake, allows PCFCs to be enriched 170-fold. Further, we show that PCFC proliferation is increased in ACE-selected cultures and markedly reduced in cells isolated from ACEnull mice. Dual fluorescence analysis of adult pituitaries revealed that ACE⫹AMCA⫹ cells are located in the marginal epithelial cell (MEC) layer and in the subluminal region. Together, the data indicate that ACE may be an important mediator of PCFC proliferation and provide novel insight into the topographical location of PCFCs within the pituitary gland.

MATERIALS

AND

METHODS

Isolation of Pituitary Single-Cell Suspensions and Uptake of ␤-Ala-Lys-N␧-AMCA The pituitary gland was removed from HS DOLA female mice aged 5– 6 weeks, digested into a single-cell suspension, and www.StemCells.com

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incubated in the presence of the dipeptide-fluorophore ␤-AlaLys-N␧-AMCA [29] as described previously [5]. ␤-Ala-LysN␧-AMCA was synthesized by K. Bauer as described previously [30].

Antibody Labeling and Fluorescence-Activated Cell Sorting Single-cell pituitary suspensions were prepared and loaded with AMCA as described above. Cells were then incubated with a panel of rat anti-mouse antibodies to cell membrane-bound Sca-1, ACE, CD45 (leukocytes), and CD31 (endothelium). Briefly, cells were incubated with rat anti-mouse ACE (10 ␮g/ml, clone 230214; R&D Systems Inc., Minneapolis, http:// www.rndsystems.com) or immunoglobulin (Ig)G2a nonbinding isotype control antibody (10 ␮g/ml; BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen) for 30 minutes on ice, followed by two washes in wash buffer (phosphatebuffered saline [PBS] containing 2% [vol/vol] newborn bovine serum [NBS]). Secondary antibody, goat anti-rat IgG-R-phycoerythrin (PE) (1:500; Biosource International, Camarillo, CA, http://www.biosource.com), was then added for 20 minutes on ice and washed twice. To block unbound secondary antibody, rat serum (5% [vol/vol]) was added to cells for 5 minutes. To this were added rat-anti-mouse CD45-PE-cyanine (Cy7) (0.4 ␮g/ml), Sca-1-fluorescein isothiocyanate (FITC) (2.5 ␮g/ml), and CD31-allophycocyanin (APC) (0.5 ␮g/ml) or relevant nonbinding isotype control antibodies (all from BD Pharmingen) for a further 20 minutes on ice. Flow cytometric analysis and sorting were performed on a FACS Vantage SE with DIVA option (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) using Fluoro Gold (1:10,000 methanosulfonate; Molecular Probes Inc., Eugene, OR, http://probes. invitrogen.com) to exclude nonviable cells. Live, single-nucleated pituitary cells were sorted by size and their exclusion of Fluoro Gold. Cell doublets were excluded by a combination of high forward scatter height and area characteristics [31]. AMCA⫹ cells were excited using an I90 laser (Coherent Scientific Pty. Ltd., Inc., Hilton, South Australia, Australia, http://www.coherent.com.au) tuned to 350 nm and run at 50 mW; the emission was then collected with a 450/ 30-nM filter. Relevant nonbinding isotype control-labeled cells were used to set gates for Sca-1-FITC⫹ and ACE-PE⫹ cells. Individual, viable AMCA⫹ CD45⫺ CD31⫺ cells were sorted on the basis of Sca-1 and ACE positivity into 96-well culture plates (NUNC A/S, Roskilde, Denmark, http://www.nuncbrand.com). Cells were cultured in medium containing Ham’s F-12 medium/ Dulbecco’s modified Eagle’s medium (50%:50% vol/vol) supplemented with 10% fetal calf serum (FCS) at 36°C.

Single-Cell Cloning, Clonogenic Titration Single cells selected by fluorescence-activated cell sorting (FACS) and seeded into 96-well plates were scored for CFA after 10 –15 days in culture. The criterion used for defining a colony was one cluster of closely packed cells per culture of greater than or equal to approximately 25 cells in number. Single-cell colonies were stained with nuclear stain 4⬘,6-diamidino-2-phenylindole, dilactate (DAPI) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) at a final concentration of 2.5 ␮g/ml. Colonies were photographed using a Leica DM IRB inverted microscope and Leica DFC 480 camera (Leica, Heer-

ACE and Pituitary Colony-Forming Cells

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Figure 1. Flow cytometric analysis of Sca-1 and ACE expression in AMCA-loaded pituitary cell preparations. Pituitary single-cell suspensions incubated in the presence of AMCA were labeled with antibodies to Sca-1 and ACE or their respective nonbinding isotype control antibodies. (A): Single cells were selected by excluding doublets and debris (gate 1). (B): Live cells were selected by their exclusion of the viability dye (gate 2). (C): Leukocytes and endothelial cells were excluded by selecting cells that were CD45⫺ and CD31⫺ (gate 3). (D): AMCA⫹ cells were selected (gate 4). (E): Isotype control antibodies IgG2a-FITC and IgG2a-PE. (F): Flow cytometric analysis showing Sca-1 and ACE expression on AMCA-loaded CD45⫺CD31⫺ pituitary cell preparations. AMCA⫹CD45⫺CD31⫺ cells that were ACE⫹Sca-1⫺, ACE⫹Sca-1⫹, Sca-1⫺ACE⫺, or Sca-1⫹ACE⫺ were selected for single-cell cloning and for limiting dilution assays. Abbreviations: ACE, angiotensin-converting enzyme; AMCA, 7-amino-4-methylcoumarin-3-acetic acid; FITC, fluorescein isothiocyanate; Sca-1, stem cell antigen-1.

brugg, Switzerland, http://www.leica.com). The size of the colonies was determined by counting the number of DAPI⫹ nuclei using the semiautomated cell-counting program Image J 1.36B (NIH, Bethesda, MD, http://www.rsb.info.nih.gov/ij). Clonogenic titer curves were performed by seeding cells (1, 3, 5, 10, or 20 cells per well, n ⫽ 24 cultures per cell concentration) into 96-well plates. The clonogenicity was determined as previously described [5] and as originally described [32]. In two experiments, ACEnull [33] or wild-type mice (n ⫽ 5 each), a generous gift from Dr. Michael McKinley (Howard Florey Institute, Melbourne, Australia), were used to assess the clonogenic activity of cells isolated from pituitaries in an ACEnull background.

Statistical Analysis Multiple group comparisons of the single-cell cloning data or clonogenic activity were performed using the one-way analysis of variance (ANOVA) and Tukey’s post hoc test. Comparison of two groups such as the colony size was carried out using the Student’s t-test for unequal variances.

Immunofluorescence Staining of Pituitary Sections

For in situ analysis of the ␤-Ala-Lys-N␧-AMCA⫹ cell population, the whole pituitary was placed in HEPES-buffered saline (HBS) containing 20 ␮M AMCA at 36°C for 3 hours. The AMCA-labeled pituitary was rinsed in HBS and post-fixed in 4% wt/vol paraformaldehyde in PBS for 15 minutes in the dark, embedded in Optimal Cutting Temperature medium (Tissue Tek, Torrance, CA, http://www.sakura-americas.com), and then cryosectioned into 5-␮M slices. Sections were analyzed using ultraviolet fluorescence microscopy.

To detect ACE⫹ cells in pituitary sections, we used an ACE rabbit polyclonal antibody raised against the cell membranebound amino terminus of ACE (amino acids 1–170; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com) at a final concentration of 4 ␮g/ml. Briefly, pituitary sections were treated with 0.1% vol/vol Triton X in PBS for 10 minutes. Sections were then blocked for 15 minutes using 1% wt/vol bovine serum albumin and 10% vol/vol FCS in PBS and incubated in the presence of the primary antibody diluted in the blocking agent overnight at 4°C. Excess primary antibody was rinsed away with PBS, and sections were blocked again as above followed by incubation with secondary antibody (goat anti-rabbit IgGs conjugated to Alexa Fluor 488 at a final concentration of 1.3 ␮g/ml) for 2 hours at room temperature. Images were captured using an Olympus FV1000 laser scanning confocal microscope or on an inverted IX81 microscope (Olympus, Tokyo, http://www.olympus-global.com). AMCA was excited with a 405-nm laser and Alexa-488 with a 488-nm argon ion laser.

RESULTS ACE and Sca-1 Are Expressed by a Subpopulation of AMCAⴙ Cells We have previously shown that approximately 3.7% of viable adult pituitary cells specifically import the fluorescent dipeptide ␤-Ala-Lys-N␧-AMCA and that PCFCs are contained exclusively within this population [5]. To determine whether stem cell associated markers were expressed by the AMCA⫹ population, we performed FACS analysis using Sca-1 and ACE monoclonal antibodies (Fig. 1). Leukocytes, comprising 10% of the AMCA⫹ population, were excluded using CD45, and endo-

Lepore, Jokubaitis, Simmons et al.

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Table 1. Summary of CFA, recovery, and relative percentages of subpopulations of AMCA⫹ cells

Percentage of all pituitary cells

Percentage of AMCAⴙ cells

AMCA⫹

3.3

100

AMCA⫹ACE⫹

2.0

61

AMCA⫹ACE⫺

1.3

39

AMCA⫹Sca-1⫹ACE⫹

1.3

38

AMCA⫹Sca-1⫹ACE⫺

0.6

17

AMCA⫹Sca-1⫺ACE⫺

0.5

16

AMCA⫹Sca-1⫹

1.8

55

AMCA⫹Sca-1⫺

1.3

39

Cell population

Number of colonies per single cells plated 126 in 671 (n ⫽ 3) 162 in 480 (n ⫽ 2) 38 in 480 (n ⫽ 2) 263 in 672 (n ⫽ 3) 93 in 576 (n ⫽ 3) 40 in 575 (n ⫽ 3) 31 in 192 (n ⫽ 1) 21 in 192 (n ⫽ 1)

Colony frequency (%)

Fold-enriched PCFCs over unselected pituitary cells

18.8

94

100

170

110

34 7.9 39 16.1 7

39.5 195 80.5 35

Recovery of CFA from AMCAⴙ fraction (%)

16.4 78.8 14.6 5.9

16.1

80.5

47

10.9

54.5

22.8

AMCA⫹CD45⫺CD31⫺-selected cells are compared with selection for ACE⫹/⫺, Sca-1⫹/⫺, or dual ACE⫹/⫺Sca-1⫹/⫺ in terms of their CFA and total recovery of PCFCs. The most efficient enrichment and recovery of PCFCs are obtained using AMCA⫹ACE⫹ or AMCA⫹ACE⫹Sca-1⫹ selection. The AMCA⫹ subpopulations are also shown as a percentage of the total pituitary population and of the total AMCA⫹ population (n ⫽ number of experiments). Abbreviations: ACE, angiotensin-converting enzyme; AMCA, 7-amino-4-methylcoumarin-3-acetic acid; CFA, colony-forming activity; PCFC, pituitary colony-forming cell; Sca-1, stem cell antigen-1.

thelial cells (3.5%) were excluded using CD31 [5]. ACE and Sca-1 staining was detected on 61% and 55% of AMCA⫹CD45⫺CD31⫺ cells, respectively (Table 1; Fig. 1). Colabeling with ACE and Sca-1 showed that the AMCA⫹ population contains ACE⫹Sca-1⫹ (38.2% ⫾ 6.5%), ACE⫹Sca-1⫺ (22.9% ⫾ 3.9%), ACE⫺Sca-1⫹ (16.6% ⫾ 4.9%), and ACE⫺Sca-1⫺ (16.4% ⫾ 4.2%) cells (Fig. 1).

Significant Enrichment of PCFCs by ACE but Not Sca-1 Selection To determine whether PCFCs express ACE and/or Sca-1, we performed clonogenicity assays on AMCA⫹ subpopulations coselected for these antigens. Labeled cells were sorted using the selection parameters shown in Figure 1F and seeded individually into 96-well plates. Approximately 18% (one in six cells) of AMCA⫹ cells (n ⫽ 671) exhibited CFA (Fig. 2), but this increased significantly when sorted for the ACE⫹ population (n ⫽ 480). One-third of AMCA⫹ACE⫹ cells exhibited CFA, a significant 1.8-fold increase compared with selection based on AMCA⫹ alone (p ⬍ .05, ANOVA, Tukey’s). Conversely, CFA significantly decreased in the AMCA⫹ACE⫺ fraction with only one in 13 cells (n ⫽ 480) giving rise to colonies (p ⬍ .05, ANOVA, Tukey’s). Sca-1 selection increased the CFA of AMCA⫹ACE⫹ cells by 5% and that of AMCA⫹ACE⫺ cells by 8% (Fig. 2; Table 1). To confirm these results, limiting dilution growth analysis [5, 32] was used to compare the CFA of ACE⫹, ACE⫺, ACE⫹Sca-1⫹, ACE⫺Sca-1⫹, and AMCA⫹ populations. Consistent with single-cell assays, ACE selection of AMCA⫹ cells increased the frequency of colony-forming cells from approximately one in six to one in three cells, whereas ACE⫺ selection exhibited a marked reduction in CFA (Fig. 3A). Sca-1 coexwww.StemCells.com

Figure 2. CFA of AMCA⫹ pituitary cells. Single cells were cultured following selection as shown in Figure 1F. The number of colonies derived from single-cell clones is expressed as a percentage of the total number of cells seeded for each cell phenotype. Total numbers of cells plated: AMCA⫹ (n ⫽ 671), ACE⫹ (n ⫽ 480), AMCA⫹ACE⫺ (n ⫽ 480), AMCA⫹ACE⫹Sca-1⫹ (n ⫽ 672), AMCA⫹ACE⫺Sca-1⫺ (n ⫽ 575), AMCA⫹Sca-1⫹ (n ⫽ 192), and AMCA⫹Sca-1⫺ (n ⫽ 192). Data are derived from three independent experiments. CFA for AMCA⫹ACE⫹ cells is significantly greater than for AMCA⫹ cells (p ⬍ .05, analysis of variance, Tukey’s). CFA for AMCA⫹ACE⫺ cells is significantly smaller than for AMCA⫹ (p ⬍ .05). Abbreviations: ACE, angiotensin-converting enzyme; AMCA, 7-amino-4methylcoumarin-3-acetic acid; CFA, colony-forming activity; Sca-1, stem cell antigen-1.

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Figure 3. Limiting dilution analysis. The clonogenic activity of AMCA⫹ cells that expressed ACE and/or Sca-1 was compared with control AMCA⫹ cultures using limiting dilution analysis. Cells were sorted into 96-well plates (n ⫽ 24 per cell concentration), and the clonogenicity was determined using the Poisson distribution analysis method [32]. The data are derived from three independent experiments. (A): One in six AMCA⫹ gives rise to colonies, versus one in three AMCA⫹ACE⫹. There was no detectable clonogenic activity in the ACE⫺ fraction of AMCA⫹ cells. (B): ACE⫹ cultures subfractionated according to Sca-1 expression compared with ACE⫺. Clonogenic activity for ACE⫹SCA⫹-selected cells was one in two cells. (C): Phase-contrast microscopy view of a single-cell colony derived from AMCA⫹ACE⫹-selected pituitary colony-forming cells, composed mainly of cells that were stellate-like with long cytoplasmic processes and scattered with round refractile cells. Magnification: ⫻200. (D): Single-cell colonies derived from AMCA⫹ACE⫹ or ACE⫺ cultures were fixed on day 10 of culture. Nuclei were detected by staining with 4⬘,6-diamidino-2-phenylindole, and the nuclei were counted to determine the total cell number per colony. The scatter plots show the individual sizes of each colony. The total numbers of colonies counted were n ⫽ 124 and n ⫽ 59, respectively. The mean colony size ⫾ SEM is shown beside each scatter plot. ACE⫺ cultures were significantly smaller that ACE⫹ cultures (p ⬍ .01). Abbreviations: ACE, angiotensin-converting enzyme; AMCA, 7-amino-4-methylcoumarin3-acetic acid; Sca-1, stem cell antigen-1.

pression further enriched the CFA of AMCA⫹ACE⫹ cells to approximately one in two cells (Fig. 3B). Together, these data indicate that the major determinant of clonogenicity is ACE expression and that maximum enrichment (195-fold) of CFA with high recovery (79%) can be obtained based on selection of ACE⫹Sca⫹ cells (Table 1). A comparison of the morphology of colonies derived from single-cell cultures of AMCA⫹ACE⫹ and AMCA⫹ACE⫺ cells revealed marked differences. Colonies grown from individually sorted AMCA⫹ACE⫹ cells resembled our previously described AMCA⫹-derived colonies [5]. These colonies were comprised of cells that were mainly stellate in shape with long cytoplasmic processes (Fig. 3C). Also scattered throughout the colonies were small, round refractile cells. Rare GH⫹/prolactin (PRL)⫹ stellate or round cells were detected in some colonies, demonstrating that single AMCA⫹ACE⫹ cells are capable of differentiating into multiple cell types. In comparison, ACE⫺-derived colonies were poorly arranged and were less compact. These colonies lacked the small round cells and rare GH⫹/PRL⫹ cells that were present in the ACE⫹-derived colonies.

ACE Expression in PCFCs Correlates with Greater Colony Expansion To investigate the effect of ACE expression on the proliferative potential of PCFCs, the size of single-cell colonies derived from AMCA⫹ACE⫹ cells versus AMCA⫹ACE⫺ cells was compared. The average size of ACE⫹ or ACE⫺ colonies at day 10

was 102 ⫾ 10 cells (n ⫽ 124) and 59 ⫾ 9 cells (n ⫽ 30), respectively (p ⬍ .01; p ⫽ .003; Fig. 3D). ACE expression in PCFCs therefore correlates with greater colony size.

Reduction in PCFC Colony Size in the Absence of ACE To further investigate the importance of ACE activity in PCFC colony expansion, we compared the CFA and the sizes of AMCA⫹ single cell-derived colonies generated from ACEnull and wild-type littermates. ACEnull mice carry an insertional mutation that inactivates the protein [33]. No difference was detected in the percentage frequency of CFA between ACEnull and wild-type mice. However, the size of single cell-derived colonies arising from AMCA⫹ cells isolated from ACEnull mice was considerably smaller than those from wild-type mice: mean colony size on day 15 of culture 301 ⫾ 70 (n ⫽ 25) compared with 832 ⫾ 244 (n ⫽ 26) in wild-type mice (p ⬍ .05, Fig. 4). These data indicate that loss of ACE protein results in reduced expansion of PCFC colonies in vitro. Like that of their AMCA⫹ACE⫹-derived counterparts, the morphology of colonies arising from the pituitaries of wild-type mice was heterogeneous in nature, comprising mainly stellate cells scattered with round cells and rare GH⫹/PRL⫹ cells. The smaller, diffuse colonies from ACEnull mice did not contain the small, round nor the GH⫹/PRL⫹ cells.


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Figure 4. Size of single-cell colonies derived from ACEnull mice. Pituitary cell suspensions derived from ACEnull mice (n ⫽ 5) and wild-type controls (n ⫽ 5) were loaded with 7-amino-4-methylcoumarin-3-acetic acid. The size of colonies arising from single-cell cultures was determined by counting 4⬘,6-diamidino-2-phenylindole nuclei on day 15 of culture. Twenty-four and 25 single cell-derived colonies, respectively, were counted, and the individual sizes are shown as a scatter plot. The average size is shown as the mean ⫾ SEM. The mean size of ACEnull colonies was significantly less than that of wild-type mice (p ⬍ .05). Abbreviations: ACE, angiotensin-converting enzyme;

AMCAⴙACEⴙ Cells in the Adult Pituitary Gland Are Located in the MEC Zone

To determine the location of ACE⫹AMCA⫹ cells in the pituitary, AMCA-loaded pituitary glands were immunostained with antibody to cell surface ACE. As reported in our previous study [5], the MECs that line the pituitary cleft import AMCA (Fig. 5A). AMCA⫹ cells that coexpressed ACE were detected on the luminal surfaces that lined both the anterior and intermediate lobes (Fig. 5B, arrow a). Dual-positive cells were also found in the lateral MEC region of the lumen (Fig. 5C) and in the subluminal zone (Fig. 5C, arrow b). In addition, ACE⫹ cells could be found scattered throughout the anterior lobe in a coronal arrangement around the lumen; some were stellate in shape. However, we were unable to detect any overlap of AMCA and ACE expression in this area (data not shown).

DISCUSSION We have previously described a rare pituitary cell type, termed PCFCs, that has progenitor cell hallmarks, including CFA and apparent potential to differentiate into cells that are immunopositive for pituitary hormones. In the present study, we investigated whether PCFCs express cell surface antigens that could be www.StemCells.com

Figure 5. Immunofluorescence of AMCA-loaded pituitary showing the location of ACE⫹AMCA⫹ cells. (A): AMCA-loaded pituitary showing AMCA⫹ cells in blue. These can be found both in the marginal epithelial lining of the gland (arrow a), including the lateral region (arrow L), and in the anterior lobe (arrow b). (B): MECs of the pituitary luminal lining that import AMCA and express ACE: (bi) AMCA⫹ cells (blue), (bii) ACE⫹ (green), and (biii) merged image showing dual positive cells detected both in the epithelial layer (arrow a) and subluminal zone (arrow b). (biv) Confocal image of MEC showing cell surface ACE (green) and intracellular AMCA (blue). (C): Cells located in the lateral regions of the pituitary gland that are (ci) AMCA⫹ and (cii) ACE⫹. (ciii) Merged image showing dual positive cells. Arrow b indicates dual positive cells that are located in the subluminal zone. Magnifications: ⫻100 (A), ⫻400 (Bi–iii), ⫻600 (Biv), ⫻200 (C). Abbreviations: ACE, angiotensin-converting enzyme; AMCA, 7-amino4-methylcoumarin-3-acetic acid; MEC, marginal epithelial cell.

used to enrich for PCFCs and to study their anatomical location within the pituitary gland. We found that PCFCs express the stem cell markers ACE and Sca-1 and can be enriched 195-fold through selection of cells that coexpress these markers and


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possess AMCA-dipeptide importation activity. Further, we demonstrated that in the absence of ACE activity PCFC colony size is significantly reduced, suggesting that ACE is functionally important for PCFC proliferation activity. Finally, we identified a subpopulation of AMCA⫹ cells in the anterior pituitary that express ACEs that are predominantly located in the epithelial lining of the pituitary cleft and in subluminal areas. The expression of stem cell-associated antigens ACE and/or Sca-1 on subpopulations of AMCA⫹ cells further supports our previous study that the AMCA⫹ population is heterogenous, containing not only typical folliculo-stellate cells but also a cell subtype with progenitor cell properties (PCFCs) [5]. The expression of ACE on AMCA⫹ cells correlated with increased clonogenicity in both single-cell cloning and titration curve assays, indicating the importance of ACE as a marker for the PCFC subpopulation. Sca-1, although found not to be critical for the colony-forming potential of PCFCs, was often coexpressed with ACE, consistent with the progenitor-like nature of PCFCs. Because the majority of PCFCs were recovered in the ACE⫹ fraction, the expression of cell surface ACE can be used to distinguish the clonogenic subpopulation from the remainder of AMCA⫹ cells, allowing highly enriched preparations of PCFCs to be generated. This will facilitate the preparation of purified PCFC populations for additional functional and expression profiling studies. The size of single-cell colonies derived from ACE⫹ AMCA⫹ cells was significantly larger than ACE⫺AMCA⫹ selected colonies, indicating that ACE is an important regulator of PCFC proliferation. This conclusion is further supported by the significantly reduced colony size observed for ACEnull versus wild-type mice. ACE, however, does not appear to be critical for PCFC detection or attachment per se, given that colonies are still able to be generated from ACEnull mice. How does ACE regulate PCFC colony size? Two possible pathways by which ACE could act in controlling PCFC proliferation are through its role in the RAS and/or through the degradation of circulating cell cycle inhibitor, AcSDKP. Firstly, via the RAS system, ACE may exert its effect through the ACE product angiotensin II to stimulate proliferation of PCFCs. Angiotensin II may alone stimulate PCFC proliferation or synergize with putative PCFC expansion factors in a way similar to which Angiotensin II synergizes with erythropoietin to expand the erythroid progenitor compartment [22, 24, 34 –36]. Secondly, or alternatively, ACE may control PCFC proliferation through its ability to inactivate serum AcSDKP. AcSDKP, a highly specific substrate for the Nterminal catalytic site of ACE, is well known to prevent the recruitment of hemopoietic stem and progenitor cells into S-phase of the cell cycle and to inhibit cycling in proliferating cells of several other tissue types [19 –21, 25, 37– 41]. Given that PCFCs are cultured in serum-supplemented medium, cell surface ACE may promote PCFC proliferation in vitro by degrading AcSDKP. Taken together, the increased clonogenicity observed in ACE⫹AMCA⫹ cultures and decreased colony size for those derived from ACEnull mice observed in the current study suggest that ACE activity may play a role in controlling PCFCpro-

liferation. Hence, it is possible that the ACE-related control mechanisms of stem/progenitor cell proliferation previously described in the hemopoietic system may also operate in other organ systems, including the pituitary gland. Although the exact role of PCFCs in the pituitary is not known, our recent study suggests that PCFCs may act as progenitors for hormone-secreting cells [5]. A progenitor cell for GH-secreting cells was also directly implicated in the study by Borrelli et al. [4], but the identity and location of this cell type are unknown. We previously showed that hemopoietic and endothelial cells within the pituitary do not contribute to the CFA of the AMCA⫹ PCFC compartment and are therefore not candidates for PCFCs [5]. The coronally situated ACE⫹ cells in the anterior lobe identified in this report are also unlikely to be PCFCs; we did not detect AMCA signal in these cells. Indeed, these ACE⫹ cells are likely to represent the population of nonleukocytic and nonendothelial ACE⫹AMCA⫺ cells that can be identified by FACS analysis. Therefore, the most likely candidates for PCFCs are the AMCA⫹ACE⫹ cells that are located in the pituitary cleft (MECs) and in the subluminal regions of the anterior lobe. Interestingly, the luminal zone of the pituitary has previously been proposed to be a source of stem/progenitor cells for the adult gland [42– 45]. The MECs that line the lumen in the adult are the remnants of Rathke’s pouch, the embryonic primordium that develops into the anterior and intermediate lobes of the pituitary. In the adult, the MECs appear to retain a primitive phenotype given that these cells do not generally contain hormones and in some cases express Nestin, a marker of neural stem/progenitor cells [42, 43, 46]. MECs appear to differentiate into folliculo-stellate cells [42– 44]. This is supported by time course studies that indicate that folliculo-stellate cells first emanate from the lateral regions of the MECs in the postnatal period before migrating into the gland [47]. Differentiation of MECs into hormone-secreting lineages has not been shown. However, the identification of rare GH⫹/ PRL⫹/Pit-1⫹ cells in single cell-derived PCFC colonies [5] is consistent with the possibility that the ACE⫹AMCA⫹ MECs and/or subluminal counterparts may have somatotrope/lactotrope differentiation potential. Interestingly, subluminal AMCA⫹ cells share characteristics (stellate morphology, glial fibrillary acidic protein expression) with glial-like cells found in the subluminal zone of the central nervous system, which have been shown to act as progenitors for neurons, oligodendrocytes, astrocytes, and ependymal cells [48, 49]. The differentiation potential of ACE⫹AMCA⫹ cells found within the subluminal zone and MEC region of the pituitary gland remains to be explored.

CONCLUSION Our data indicate that PCFCs reside within the MEC zone of the adult pituitary and express cell surface markers typical of stem/ progenitor cells, Sca-1 and ACE, the latter of which appears to be functionally important for PCFC proliferation. These findings will facilitate further investigations into the niche of PCFCs, the control of their proliferation, gene expression analysis, and the preparation of enriched putative progenitor cell populations for in vitro and in vivo differentiation studies.


ACKNOWLEDGMENTS We thank Dr. Michael McKinley (Howard Florey Institute, Melbourne, Australia) for providing the ACEnull mice. This work was funded by the Australian National Health and Medical Research Council. P.Q.T. is an RD Wright Fellow of the Australian National Health and Medical Research Council. P.J.S. is

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DISCLOSURES The authors indicate no potential conflicts of interest.

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