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TISSUE-SPECIFIC STEM CELLS |
aPituitary Research Unit, Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia;
bBernard O'Brien Institute of Microsurgery and Department of Surgery, The University of Melbourne, St. Vincent's Hospital, Fitzroy, Victoria, Australia;
cSchool of Molecular & Biomedical Science, The University of Adelaide, South Australia, Australia
Key Words. Colony-forming cells • Pituitary • Growth hormone
Correspondence: Paul Thomas, Ph.D., School of Molecular & Biomedical Science, University of Adelaide, Adelaide, South Australia 5005, Australia. Telephone: +613 8303 7047; Fax: +613 8303 4362; e-mail: paul.thomas{at}adelaide.edu.au; or Diana Lepore, Ph.D., Pituitary Development and Disease, Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Victoria 3052, Australia. Telephone: +613 8341 6205; Fax: +613 9348 1391; e-mail: diana.lepore{at}mcri.edu.au
Received January 10, 2007;
accepted for publication March 20, 2007.
First published online in STEM CELLS EXPRESS March 29, 2007.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionSummaryDisclosures of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosures of Potential...AcknowledgmentsReferences |
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Recently, our laboratory identified cells within the pituitary gland that possess both colony-forming capacity and an apparent ability to differentiate into GH(+) cells, which we termed pituitary colony-forming cells (PCFCs) [5]. The stem-like characteristics of this cell type were reported in a subsequent study, showing that PCFCs have high proliferative potential in vitro and can be highly enriched by selecting for cell subpopulations that express stem cell-associated markers angiotensin converting enzyme and stem cell antigen-1 [6]. The identification of a stem-like cell within the pituitary offers the possibility of manipulating these cells to produce somatotropes, notably in conditions of growth hormone cell deficiency. However, the behavior of PCFCs in vivo has not yet been tested.
The Bernard O'Brien Institute of Microsurgery has developed a unique in vivo mouse model to test transplanted cells in a physiological chamber system [7]. The principle of this model is that vascularized tissue is created by injecting the cells of interest together with a supporting extracellular matrix into a split cylindrical chamber that envelops the epigastric artery and vein. The circulating blood that flows through the chamber supports the growth and survival of the chamber contents and perfuses the chamber through branching blood vessels that sprout from the central artery and vein. This tissue engineering model has been used successfully to grow adipose tissue, connective tissue [7], and skeletal muscle [8]. This model has also been successful in supporting the viability of transplanted pancreatic islets [9, 10], pancreatic precursor cells [11], and bone marrow-derived mesenchymal stem cells [12]. The aim of the current study was to test the ability of transplanted PCFCs to survive and differentiate in vivo using this murine microchamber model.
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MATERIALS AND METHODS |
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Recipient Mice. Male severe combined immune deficiency (SCID) mice (19–25 g) were used to set up the microchamber model into which donor cells were implanted. SCID mice were purchased from Animal Resources Centre (Perth, Western Australia, http://www.arc.wa.gov.au/).
Donor Mice. Donor PCFCs were obtained from three different strains of mice described below, and these ranged in weight from 15–25 g and in age from 7–16 weeks. Strain (1) ROSA 26 mice ubiquitously express the LacZ gene [13] and were obtained from the Animal Resources Centre. Strain (2) LacZ-enhanced green fluorescent protein (eGFP) transgenic mice have X-linked expression of eGFP to tag expression of the LacZ gene [14–17] and were obtained from Professor Patrick Tam, Children's Medical Research Institute, Sydney, Australia. Strain (3) GH-eGFP transgenic mice were generated from GH-eGFP transgenic mouse sperm generously donated by Professor Iain Robinson (Department of Neurophysiology, National Institute for Medical Health Research, Mill Hill, London, U.K.). This transgenic mouse line was originally generated in Professor Robinson's laboratory, and it expresses eGFP under the control of the growth hormone promoter [18].
Isolation and ß-Ala-Lys-N
-7-Amino-4-methyl-coumarin-3-acetic Acid Labelling of Pituitary Cells
Female 5–6-week-old mice (ROSA 26, GFP-LacZ or GH-eGFP as described above) were culled by cervical dislocation, and the pituitary gland was removed. The neural lobe was carefully dissected using a dissection microscope. The remaining anterior portions of the pituitary were digested [5] and incubated in the presence of the dipeptide-fluorophore ß-Ala-Lys-N
-7-amino-4-methyl-coumarin-3-acetic acid (AMCA) [19], donated and synthesized by Professor Karl Bauer (Max Planck Institute, Hanover, Germany) as previously described [20].
Fluorescence-Activated Cell Sorting Analysis
Fluorescence-activated cell sorting (FACS) was performed on a FACS Vantage SE with DIVA option (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Propidium iodide (0.8 µg/ml; Molecular Probes, Eugene, OR, http://probes.invitrogen.com) was used to exclude nonviable cells. Cell doublets were excluded by a combination of high forward scatter height versus forward scatter area characteristics [21]. Live, single nucleated pituitary cells were sorted by size and their exclusion of propidium iodide. AMCA(+) cells were excited using an I90 laser (Coherent Scientific, 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 band pass filter. eGFP cells were excited using an I90 Sapphire 200 mW laser (Coherent Scientific) tuned to 488 nm and run at 100 W. The emission was then collected with a 530/30 nm filter.
Surgery
The Vascularized Microchamber Model.
The vascularized microchamber mouse model used has been previously described [7]. This method uses chambers made from laboratory silicone tubing (Dow Corning Corporation, Midland, MI, http://www.dowcorning.com) that were cut into segments (5-mm long, 3.35-mm internal diameter, volume 
45 µl) with a lateral split. With mice under general anesthesia (chloral hydrate, 4 mg/g, intraperitoneal injection), chambers were wrapped around the inferior epigastric artery and vein in the groin region. The chamber was sealed at the proximal end and along the lateral split using melted Ethicon Bone Wax (Johnson & Johnson International, Brussels, Belgium, http://www.jnj.com). Care was taken not to apply the heated wax directly to the vessel, and the chamber was then filled with 50,000 total donor cells suspended in 40 µl of Matrigel support matrix [7] supplemented with basic fibroblast growth factor (bFGF) (100 ng/ml) and heparin (80 U/ml). The open end was then sealed with bone wax. The whole chamber was anchored to underlying muscle with 10/0 nylon microsutures to prevent the pedicle from being dislodged during the animal's postoperative mobilization. The construct was carefully placed in the groin and the wound closed using metal clips. The animal was then allowed to recover from the anesthesia. Control grafts received Matrigel alone (without cells) representing a vehicle-alone control and the background environment that the cells would be placed in [7].
Tissue Harvesting. Six weeks after implantation and with the mice again under general anesthesia, tissue specimens were harvested from the microchambers. This time point was chosen based on previous studies that demonstrated that, at 6 weeks, the microchamber was fully vascularized and contained surviving cells [7]. The tissue was fixed in 4% wt/vol paraformaldehyde in phosphate-buffered saline (PBS) for 15 minutes on ice. Specimens were then embedded in Optimum Cutting Temperature (Tissue-Tek, Hatfield, PA, http://www.gmi-inc.com) in a 1 cm x 1 cm Cryomold (Tissue-Tek) then rapidly frozen in isopentane cooled on frozen carbon dioxide. Whole specimens were cut into transverse 5-µm thick cryosections on a Leica CM1900 cryostat (Heerbrugg, Switzerland, http://www.leica.com). Sections were selected from five points along the length of the graft and histologically stained.
Histopathology and Immunohistochemistry
Beta-Galactosidase Staining for LacZ Expression. Donor cells were detected in grafts by LacZ expression. Paraformaldehyde-fixed sections were chemically stained in X-gal solution overnight at 36°C. X-gal solution is comprised of 0.1% vol/vol X-Gal (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com), 20 µM CaCl2, 5 mM EGTA, 0.01% wt/vol sodium deoxycholate, 0.01% Nonidet P-40, 5 mM K3Fe(CN)6, and 5 mM K4Fe(CN)6 in 20 mM Sorensen's PBS at pH 7.4. Slides were then rinsed in PBS and stained using an immunoperoxidase method to detect GH, eGFP, or phosphohistone 3.
Immunoperoxidase Stain. Sections were permeabilized and endogenous peroxidases quenched by incubating the sections in 0.5% (vol/vol) Triton X-100 in PBS in the presence of 2% (vol/vol) hydrogen peroxide (Merck & Co., Whitehouse Station, NY, http://www.merck.com) for 20 minutes. Sections were then rinsed three times in PBS for 5 minutes each. The primary antibody was diluted in antibody diluent solution (10% vol/vol fetal calf serum and 1% wt/vol bovine serum albumin in PBS) and incubated on the sections overnight at 4°C. Sections were then rinsed in PBS three times for 5 minutes. The secondary antibody was then applied, and the sections were incubated for 2 hours at room temperature. The sections were then rinsed three times for 5 minutes in PBS. Metal-enhanced diaminobenzidine substrate (Pierce, Rockford, IL, http://www.piercenet.com) was then applied to the sections for 3 minutes to develop the color-metric reaction for the immunodetection of eGFP or GH. The cell nuclei were counterstained with Harris hematoxylin (0.5% hematoxylin, 5% vol/vol ethanol, 10% vol/vol ammonium alum, and 0.25% wt/vol mercuric oxide in distilled water) for 3 minutes followed by washes in H2O (1 minute), HCl (1% vol/vol, 1 minute), ammonia water (0.3% vol/vol, 30 seconds), H2O (1 minute), and 100% ethanol (30 seconds) and then cleared with Histoclear solution (30 seconds) (Histopure; Australian Biostain, Melbourne, Victoria, Australia, http://www.australianbiostain.com.au). Slides were mounted with coverslips using Aquamount mounting medium (Aquatex, London, U.K.).
Primary Antibodies. eGFP was detected with goat anti-mouse eGFP obtained from Rockland (Gilbertsville, PA) and diluted to a final concentration of 10 µg/ml. Growth hormone was detected with rabbit anti-rat serum to growth hormone obtained from Dr. Parlow (National Hormone and Peptide Program, UCLA Medical Center, Torrance, CA). As the antibody concentration was not defined, this antisera was titered and used at a dilution of 1 in 10,000. Rabbit anti-mouse phosphohistone-H3 (PH3) antibody was obtained from Upstate Laboratories (Upstate, Charlottesville, VA, http://www.upstate.com).
Secondary Antibodies. Horseradish peroxidase-conjugated rabbit anti-goat or goat ani-rabbit immunoglobulins were obtained from DakoCytomation (Glostrup, Denmark, http://www.dakocytomation.com) and diluted by 1 in 60.
Cell Counting
Graft tissue was cut into 5-µm sections along the entire length of the graft, and five sections for immunostaining were sampled at regular intervals across the graft. Cell counting was performed on a Leica DMIRB inverse microscope with a x40 bright-field objective. The number of positive cells (LacZ, GH, eGFP, PH3) in each cross-section of the graft was counted in at least n = 6 sections for each graft. The total number of surviving donor cells per graft was then calculated using the method detailed by Weibel [22] that involves the De Hoff and Rhines equation [23]. This equation allows the estimation of the number of cells per volume by taking into account the number of cross-sections and the volumetric density. For each cross-section, the area and volume were calculated using the known length of the section (5 µm) and the dimensions of the section obtained using an eyepiece graticule (Leica). Given that the average length of the grafts was 3.2 mm (n = 15 grafts), this mean length was used to calculate the total volume of individual grafts. Using the De Hoff and Rhines equation, the total number of donor cells recovered in each graft was calculated as follows: Recovery (R) = volume of graft (V) x (mean number of nucleated cells per section [n]/mean maximal nucleus diameter [d]) where V = area of graft x length of graft. The cells recovered were then expressed as a percentage of the initial number seeded.
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RESULTS |
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Differentiation of Donor Cells In Vivo
In order to determine whether PCFC cells have the ability to differentiate into GH(+) cells in vivo, donor cells were obtained from transgenic mice that expressed eGFP driven by the growth hormone promoter (GH-eGFP). Previous immunofluorescence analysis of pituitary tissue has shown that somatotropes from this transgenic line coexpress GH and eGFP [18]. We further confirmed that eGFP and GH could be readily detected in the anterior lobe of transgenic adult pituitary tissue, first by detecting immunofluorescence expression of eGFP (Fig. 4Ai, 4Aii) and second by using the colorimetric diaminobenzidine reaction to detect eGFP (Fig. 4Bi, 4Bii).
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Cells that stained positive for either eGFP or GH using the diaminobenzidine method were detected in sections of the same grafts that were sampled within 50 µM of each other, first in positive control grafts that had been seeded with GH(+) somatotropes (Fig. 5) and second in grafts that had been seeded with AMCA(+)eGFP(–) cells (Fig. 6). Positive cells were invariably located near blood vessels throughout the grafts, and we observed the typical globular nature of GH (Fig. 6Aii, 6Bii) that has been previously published for somatotropes derived from GH-eGFP transgenic mice [18]. Control grafts that received Matrigel alone did not stain for eGFP or GH (Fig. 5Aii, 5Bii), whereas eGFP(+) cells were detected in all grafts that received donor cells, indicating that differentiation had occurred in vivo.
The immunohistochemical observations in grafts were confirmed using FACS analysis. Six grafts were digested into single cell suspensions and FACS-sorted to find eGFP cells (Fig. 7A). These were compared with single-cell digests of Matrigel control grafts (Fig. 7B). eGFP(+) cells were found in grafts initially seeded with AMCA(+)eGFP(–) cells, confirming that differentiation had occurred in vivo. We quantified by FACS the number of eGFP(+) cells recovered from the graft as a percentage of initially seeded cells, even given that enzymatic digestion of the grafts caused some loss of cells. We found that the eGFP(+) cells recovered by FACS represented on average 3.3% of initially seeded cells (n = 5 grafts). In grafts where growth hormone-releasing hormone (GHRH) and/or pituitary environment support cells were added to supplement the donor cells, we did not observe any increase in the number of eGFP/GH(+) cells detected in digested grafts (data not shown).
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DISCUSSION |
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Survival of Donor Cells
The presence of LacZ(+) cells in the grafts indicated that donor cells survived for at least 6 weeks after transplantation (Fig. 3). At this time, the microchamber grafts indicated good vascularization, with blood vessels evenly distributed throughout the graft (Fig. 2). This suggested good perfusion of the graft and exposure of implanted cells to circulating blood-borne nutrients, growth factors, and oxygen. At the time of implantation, cells are suspended in Matrigel extracellular matrix, which provides associated matrix products and growth factors that encourage anchoring of cells and subsequent cellular signaling [24].
Recovery of Donor Cells
Of the total LacZ(+)AMCA(+) cells transplanted, approximately one-third was recovered. This recovery is consistent with the number of PCFCs being only a small proportion of the AMCA(+) population (12%) [5]. In addition, proliferation of surviving cells contributed to the recovery, as cell-cycling (phosphohistone 3(+)) cells were also observed among surviving (LacZ(+)) cells. This ability to divide in vivo is supported by the high proliferation potential of PCFCs recently reported in vitro [6].
Interestingly, mature somatotropes (GH(+)) that were seeded in GH positive control grafts also survived 6 weeks and could be detected expressing GH (Fig. 5). Since somatotropes can live for up to 80 days [3], this is consistent with the survival of these cells for 6 weeks in a well vascularized graft. However, transplanted somatotropes are unappealing for long-term treatment strategies, as their long-term proliferative potential is limited.
Differentiation of PCFCs In Vivo
Differentiation of PCFCs in vivo was suggested by the LacZ(+) cells that were also GH(+) (Fig. 3). However, to confirm that differentiation had occurred in vivo, experiments were performed using donor cells from transgenic mice that coexpress eGFP and GH (GH-eGFP) [18]. Donor PCFC cells from the GH-eGFP transgenic model enabled, first, the exclusion of cells that were already GH(+) prior to transplantation and, second, the detection of cells that switch on the expression of eGFP with the onset of growth hormone expression. In grafts implanted with AMCA(+)eGFP(–)/GH(–) cells, eGFP(+)GH(+) cells were consistently found near blood vessels in 6-week-old grafts. The finding that differentiated cells were frequently located near blood vessels suggests that the surviving cells are supported by blood-borne factors and/or endothelial cell-related signals. The histological data were then corroborated further by FACS analysis, where the eGFP(+) cells were detected in single-cell digests of the 6-week-old grafts (Fig. 7). The differentiation capacity of PCFCs, a subpopulation of AMCA(+) cells, into growth hormone(+) cells in vivo fits also with the earlier observation (Fig. 4) that the AMCA(+) population has a subset of cells that expresses eGFP and, therefore, also GH (Fig. 7). The histological and FACS data together indicate that differentiation is occurring in vivo in the microchamber, albeit at low levels (only 3.3% of donor cells). To augment differentiation in vivo, it may be necessary to add specific factors or signals of the pituitary environment that are critical to somatotrope differentiation. We have not observed any augmentation in differentiation in pilot experiments using the coculture in vivo of donor cells supplemented with pituitary cell suspensions with and without GHRH. On-going experiments to investigate the specific environmental signaling niche of PCFCs may shed light on the specific components required to augment PCFC differentiation in vivo. It is also possible that PCFCs may have the capacity to generate pituitary hormones other than GH in vivo, given that prolactin-positive cells can be detected in cultures of PCFCs in vitro [5].
Although the levels of PCFC differentiation into GH(+) cells in the current microchamber model are modest, the question arises: How did the transplanted cells receive the appropriate signals to differentiate into GH cells, given that the chamber is positioned in the groin region, an area that is remote from the pituitary gland or its direct hypothalamic stimuli? It is possible that growth factors present in Matrigel may affect both the expansion and differentiation of donor cells. The composition of Matrigel includes extracellular membrane proteins, laminin, collagen IV, entactin, and heparin sulfate proteoglycan. In addition, Matrigel has endogenous growth factors, including tumor-derived growth factor-beta (TGF-ß), bFGF, epidermal growth factor (EGF), neural growth factor (NGF), platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF-1) [25, 26]. The expansion effect of PDGF, EGF, and bFGF on stem cells of neural lineage is well documented [27, 28]. Similarly to neural stem cells, PCFCs are located in both the luminal and subluminal zones of the gland [6, 27, 28], and they express glial fibrillary acidic factor [5, 28]. Since PCFCs share properties with neural stem cells, it is tempting to speculate that they also respond to similar factors for expansion. There is evidence suggesting pituitary progenitor cell proliferation and differentiation in response to FGF-8 and FGF-10 [29]. However, the effect of EGF, NGF, PDGF, TGF-ß, or IGF-1 on pituitary progenitor cells is not well documented. In addition to growth factors present in the Matrigel, the cells within the microchambers were likely to have been exposed to circulating levels of GHRH. GHRH, required for somatotrope maturation during pituitary development, also causes proliferation of somatotrope cells [30, 31] and the synthesis/secretion of growth hormone [1, 18, 32]. Circulating levels of GHRH can be derived not only from the hypothalamus but also from the central nervous system, gut, and endocrine pancreas [1].
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SUMMARY |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSummaryDisclosures of Potential...AcknowledgmentsReferences |
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DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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This article has been cited by other articles:
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  T. Fauquier, K. Rizzoti, M. Dattani, R. Lovell-Badge, and I. C. A. F. Robinson SOX2-expressing progenitor cells generate all of the major cell types in the adult mouse pituitary gland PNAS, February 26, 2008; 105(8): 2907 - 2912. [Abstract] [Full Text] [PDF]
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