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TISSUE-SPECIFIC STEM CELLS |
a Division of Research Immunology/Bone Marrow Transplantation,
b Department of Pathology,
c Congressman Julian Dixon Cellular Imaging Core,
d Division of Gastroenterology, Hepatology, and Nutrition, and
e Developmental Biology Program, Childrens Hospital Los Angeles, Los Angeles, California, USA
Key Words. Bone marrow transplantation • Epithelial stem cells • Pancreas • Ducts • Neonate • Mice
Correspondence: Gay M. Crooks, M.D., Division of Research Immunology/BMT, Childrens Hospital Los Angeles, 4650 Sunset Blvd., M.S. #62, Los Angeles, CA 90027, USA. Telephone: 323-669-5690; Fax: 323-906-8193; e-mail: gcrooks{at}chla.usc.edu
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ABSTRACT |
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INTRODUCTION |
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Reports of bone marrow differentiation into pancreatic tissue have been few and contradictory. Some studies have shown rare cells of donor bone marrow origin that coexpress insulin lodged within the pancreatic islets, raising the possibility that bone marrow might be used to regenerate beta cells in the treatment of type 1 diabetes [14, 15]. Other studies have found little if any evidence of beta-cell differentiation [16, 17]. In all previous studies, whether negative or positive, some degree of pancreatic injury was either induced specifically (using the beta-cell toxin streptazotocin) [15–17] and/or nonspecifically and subclinically by total body irradiation prior to the bone marrow transplant [14–17]. All experiments were performed with adult donors and recipients.
In contrast to previous reports, the current study explored the potential of bone marrow to contribute to the development of the exocrine and endocrine components of the pancreas in the neonatal period during which rapid tissue growth and differentiation occur in the absence of injury. Data presented here show that bone marrow transplanted into immune-deficient neonatal mice contributed to a high percentage of epithelial cells within the pancreas, in some cases forming entire branching ductal structures. Bone marrow transplanted into adult recipients did not generate pancreatic ducts, indicating that factors present specifically during neonatal development are responsible for the recruitment of bone marrow cells with epithelial potential to areas of rapid epithelial growth. These data have important implications for the study of epithelial differentiation in the developing animal and support the existence of significant epithelial potential in the bone marrow that might be harnessed for clinical use.
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MATERIALS AND METHODS |
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Bone Marrow Transplantation into Neonatal Mice
Adult (10- to 12-week-old) male hemizygous mice transgenic for enhanced green fluorescent protein (GFP) gene (C57BL/6-TgN, ACEbEGFP 1Osb/J; The Jackson Laboratory, Bar Harbor, ME, http://www.jax.org) were used as donors for bone marrow transplantation (BMT). Bone marrow cells from femurs and tibia of donors were flushed, washed twice with phosphate-buffered saline (PBS) without Ca and Mg (Mediatech, Inc., Herndon, VA, http://www.cellgro.com), and frozen down in 10% Cryoserv (Edwards Lifesciences, Irvine, CA, http://www.edwards.com) without further manipulation.
Nonobese diabetic/severely compromised immunodeficient/ß2microglobulin-null (Nod/Scid/ß2Mnull) mice were used as recipients of transplanted bone marrow under a protocol approved by the Institutional Animal Care and Use Committee at Childrens Hospital Los Angeles (CHLA). Five to ten million thawed bone marrow cells from GFP+ mice suspended in 50 µl PBS were injected into neonatal mice within 24 hours after birth, through the superficial temporal vein as described [18]. No irradiation or other conditioning regimen was given. Animals were housed in sterile conditions and weaned at 4 weeks of age. Because gender typing of neonatal mice could not be performed at the time of transplant, both male and female recipients were transplanted and analyzed. Recipient mice were sacrificed 2 months post transplantation for analysis. Engraftment of donor-derived cells in peripheral blood was confirmed at the time of sacrifice by identifying GFP+ cells by flow cytometry (FACSCalibur; BD Biosciences–Immunocytometry Systems, San Jose, CA, http://www.bdbiosciences.com/immunocytometry_systems).
In separate experiments, adult Nod/Scid/ß2Mnull mice (8 to 10 weeks old) were transplanted via tail vein injection with 5–10 x 106 adult GFP+ bone marrow cells without irradiation and were sacrificed for analysis 2 months post transplantation.
Histochemistry
After sacrifice, pancreas tissue was dissected and fixed in 10% neutral buffered formalin (Richard-Allan Scientific, Kalamazoo, MI, http://www.rallansci.com) for 6 hours, embedded in paraffin (Leica, Nussloch, Germany, http://www.leica.com), sectioned using a microtome (Leica). Slides of 5-µm thickness were dewaxed with 100% Toluene (Sigma-Aldrich), and rehydrated. Antigen unmasking was performed with Vector unmasking buffer (Vector Laboratories, Inc., Burlingame, CA, http://www.vectorlabs.com) for 12 minutes. Nonspecific binding was blocked with 250–300 µl 100 mM Tris-Buffered Saline (TBS) (pH 7.5) containing 0.1% Tween-20, 3% bovine serum albumin, and 5% normal donkey serum (Immunoglobulin G–free, Protease-free; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, http://www.jacksonimmuno.com) for at least 30 minutes until primary antibody was added. Pancreas tissues from nontransplanted Nod/Scid/ß2Mnull mice and GFP transgenic mice were used as negative and positive controls for GFP staining, respectively.
The following anti-mouse primary antibodies were used for fluorescence immunohistochemistry: BrdU sheep polyclonal antibody conjugated with Cy3TM (Abcam Inc., Cambridge, MA, http://www.abcam.com), platelet/endothelial cell adhesion molecule-1 (PECAM-1) (CD31) goat polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com); GFP rabbit polyclonal antibody (Novus Biologicals, Inc., Littleton, CO, http://www.novus-biologicals.com), insulin guinea pig polyclonal antibody (DakoCytomation Inc., Carpinteria, CA, http://www.dakocytomation.com), pan-CK mouse monoclonal antibody (Sigma), and CD45 rat monoclonal antibody (SouthernBiotech, Birmingham, AL, http://www.southernbiotech.com). Secondary antibodies used in the study were anti-goat–FITC, anti-rabbit–Cy3, and anti-mouse–FITC (Jackson ImmunoResearch Laboratories, Inc.). Slides were mounted with Vectashield medium with 4'6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Inc.) after washing three times with TBS containing 0.1% Tween-20 (TBST). Images were viewed with a Leica DMRXA microscope (Bannockburn, IL) using a Plan Apo 20 or 40x/1.25 NA phase 3 DIC or Plan Apo 63x/1.32 oil immersion objective lens. Filter sets used were DAPI, chroma 31000; fluorescein, 41001; and Cy3, 41007a (Chroma Technology Corp., Rockingham, VT, http://www.chroma.com). An LS300W ozone-free xenon arc lamp (Sutter Instrument Co., Novato, CA, http://www.sutter.com) was coupled to the microscope with a liquid light guide. Images were acquired from EasyFISH software with a SkyVision–2/VDS-1300 12-bit digital camera (Applied Spectral Imaging Ltd., Migdal Ha’emek, Israel, http://www.spectral-imaging.com) and printed using Microsoft PowerPoint (Redmond, WA, http://microsoft.com).
Hematoxylin and eosin staining of skin and liver from transplanted and nontransplanted mice and analysis by a blinded pathologist were performed to rule out the presence of graft-versus-host disease (GVHD).
Fluorescence In Situ Hybridization
In cases in which female recipients were used, fluorescence in situ hybridization (FISH) analysis was performed to identify donor Y chromosomes in tissue. For dual staining of CK and mouse Y-paint chromosome, slides were processed with antigen unmasking, as described above, followed by digestion with 0.16% trypsin in diluent (Zymed Laboratories, San Francisco, http://www.zymed.com) for 10 minutes at 37°C and washing with TBST. After blocking, sections were incubated with antibody against mouse CK, followed by incubation with biotinylated anti-mouse antibody, and visualized by fluorescein (dichlorotriazinyl aminofluorescein)– conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc). Post fixation in 4% paraformaldehyde, slides were sequentially dehydrated in 70%, 90%, and 100% alcohol. One microliter of Y-paint probe (Cy3TM) in 9 µl hybridization buffer (Cambio Ltd., Cambridge, U.K., http://www.cambio.co.uk) was applied on a 22 x 22 mm coverslip. Slides were sealed with rubber cement, denatured in 60°C for 10 minutes, and hybridized overnight at 37°C in humidified container. After washing, the slides were mounted with Vectashield medium containing DAPI. Fluorescein and Cy3 filters were used to reveal CK and Y chromosomes.
Quantification of Donor-Derived Ducts in Recipient Pancreas
Six to 24 sections of pancreas tissue from each mouse were analyzed by fluorescence microscopy. These sections were derived from the 10th, 20th, 30th, 40th, 50th, 60th, and 70th 5-µm sections that spanned the length of the pancreatic tissue, thus providing representative analysis of the entire organ. Ducts were defined in each section based on CK staining of definite duct structures. GFP+ ducts were defined by coexpression of GFP and CK in at least two cells in each clearly defined ductal structure.
Statistical Analysis
Means ± SEM of BrdU+ cells obtained from adult and neonatal mice were determined, and a two-sample t-test was performed. Medians and confidence intervals of GFP+ ducts from recipient animals were given, as these data were nonparametric. Comparison between the neonatal and adult recipients was performed with a Mann-Whitney test. p < .05 was considered significant (two-tailed).
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RESULTS |
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Pancreas tissue from neonatal mice displayed irregularly shaped islets and very small ducts compared with their adult counterparts. BrdU+ cells could be seen in both exocrine and endocrine compartments of the pancreas and were significantly more frequent overall in the neonatal than in adult pancreas (37.1 ± 1.9 versus 2.7 ± 0.3 per microscope field, magnification 20x, p < .0001). When ducts and beta cells were specifically analyzed by BrdU staining, a low background level of cell cycling was seen in both cell types from adult mice (6.8% ± 1.4% of ducts and 3.0% ± 1.0% of islets contained at least one BrdU+ cell). The frequency of cycling was significantly higher in the neonatal pancreas in both ductal epithelium (48.1% ± 5.5% of ducts contained BrdU+ cells) and beta cells (26.8% ± 2.2% of islets contained BrdU+ cells) than in the adult pancreas (p < .0001) (Figs. 1A–1D
). Because epithelial tissue in the neonatal pancreas is rapidly dividing, we reasoned that this scenario may provide a unique stimulus required to recruit epithelial precursors from bone marrow to participate in epithelial growth and differentiation.
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). Bone marrow and peripheral blood in donor GFP transgenic mice did not uniformly express the GFP marker (39.0% ± 0.7% of nucleated cells expressed GFP in bone marrow and 88.6% ± 2.1% in peripheral blood) (data not shown). Nontransplanted mice had no background GFP staining in peripheral blood (Fig. 2B). Engraftment of donor cells was obtained in all transplanted mice, with 22.3% ± 4.9% GFP+ cells observed in the peripheral blood (n = 9 mice) (Fig. 2C). Consistent with the fluorescence-activated cell sorting data, hematopoietic cells of donor origin were also occasionally observed in the blood vessels of recipient pancreas, using fluorescence immunohistochemistry staining (Fig. 2D).
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Bone marrow contribution to epithelial ducts was analyzed in a total of 165 pancreas sections from nine mice (in different experiments) sacrificed 2 months after neonatal BMT. Among 4,034 ductal structures identified on the basis of CK staining, a median of 4.6% (range 0.9%–43.1%) of ducts contained cells that coexpressed GFP and CK. Two patterns of ductal engraftment by donor cells could be seen. In the first pattern, ducts were completely or mostly derived from GFP+ donor cells (Figs. 3A, 3B). These were recorded as ducts with greater than 50% of epithelial cells coexpressing GFP (Table 1). In these cases, clusters of large and small ducts, all expressing GFP, were often found, as if whole branching structures were derived from donor bone marrow. Coexistence of donor- and host-derived ducts in the same section could be seen (Fig. 3B), demonstrating the specificity of staining. In the second pattern, only small numbers of epithelial cells of donor origin were found scattered within each of the ducts (Fig. 3C). The existence of two or more isolated GFP+ cells per duct with this pattern was recorded as ducts with less than 50% donor cells (Table 1). Ducts containing only one GFP+ cell were not recorded. The pattern and specificity of GFP staining in ductal epithelium were validated with pancreas tissues from GFP transgenic mice (Fig. 3D) and nontransplanted Nod/Scid/ß2Mnull mice (Fig. 3E). In the nine neonatal transplanted mice analyzed at 2 months, a median of 0.2% (range 0%–31.3%) of all ducts had more than 50% donor cells and 4.1% (range 0.9%–11.8%) of all ducts had less than 50% donor cells (Table 1). The colocalization of CK and GFP in the donor-derived ductal epithelial cells can be clearly shown at higher magnification (Figs. 4A–4C).
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). Analysis of serial sections taken along the length of the pancreas showed that the bone marrow–derived ducts tended to be found toward the center of the organ. These clusters of predominantly GFP+ ducts represented interlobular ducts (large ducts typically surrounded by loose mesenchymal tissue); in contrast, the small intralobular ducts surrounded by acinar tissue rarely contained GFP+ epithelial cells. In some areas, approximately 50% of the interlobular ducts were GFP+.
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Of note, no GFP-expressing cells were found in 1,300 blood vessels (identified by expression of PECAM-1) in any of the nine animals transplanted during the neonatal period. Thus, bone marrow cells did not contribute to the formation of pancreatic blood vessels in the neonatal bone marrow transplant model.
Bone Marrow–Derived Cells Occurred at Low Frequency in Islets
Co-staining of pancreatic tissue sections showed occasional bone marrow–derived cells within the islets that expressed both GFP and insulin (data not shown). Among a total 438 islets identified in pancreas sections from five mice analyzed 2 months after BMT, 2.5% of islets appeared to contain one or more donor-derived cells coexpressing GFP and insulin. However, only rare cells (1.3%) within each positive islet expressed GFP (Table 2). To further confirm the donor origin of these GFP+insulin+ cells, pancreas sections stained with GFP/insulin were processed for FISH staining and found to have single X and Y chromosome FISH signals, demonstrating diploidy (data not shown).
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). Analysis of the data from all animals studied confirmed that the number of ducts containing donor-derived cells was significantly higher in neonatal than adult recipients (p < .05). No beta cells of donor bone marrow origin were found in animals transplanted as adults. |
DISCUSSION |
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Previous reports have suggested a role for bone marrow in epithelial differentiation in multiple organs [21]. In these studies, however, the rarity of the phenomenon, usually demonstrated as single donor cells lodged randomly within a background of recipient tissue, has made interpretation difficult. The possibility of fusion between donor hematopoietic cells and host tissue has been raised to explain the data [22]. To our knowledge, in all such reports, BMT was performed during adult life. In the case of bone marrow–to–pancreas differentiation, reports have shown either no donor contribution to pancreatic tissue [16, 17] or rare events of engraftment/differentiation from individual bone marrow– derived cells in islets [14] and/or endothelium [15, 23]. The current report is distinguished from all others by the predominance of ductal differentiation from bone marrow infused during the neonatal period. As with previous reports, we found an almost complete absence of donor engraftment in the pancreatic ducts of mice transplanted during adult life, suggesting that the neonatal period offers a unique setting for this process. It has been extensively reported that bone marrow cells are able to differentiate into cells of other organs, including the pancreas. However, all such studies used models with various tissue damage. To investigate the differentiation potential of bone marrow cells under physiological conditions, irradiation and specific pancreatic damage were eliminated from our study. Although the C57BL/6-TgN to Nod/Scid/ß2Mnull combination is not a syngeneic transplant model, GVHD-mediated tissue damage did not occur clinically or at the histologic level, suggesting that this represents a genuine nondamage setting.
As noted in some previous reports using adult models, we also found rare cells within the islets of neonatal recipients. FISH analysis confirmed that the cells were of donor origin, suggesting that bone marrow cells may also have the potential for differentiation into the endocrine components of the pancreas. The question of whether adult stem cells capable of beta-cell neogenesis exist in the pancreas is a controversial one. Insulin-producing cells can be seen integrated into the lining of ducts [24], and many investigators have concluded that pancreatic stem cells exist in and around the ducts [25–27]. Interestingly, Polak et al. reported that during human fetal development, endocrine differentiation is associated with the central, rather than peripheral, ducts [28]. The loose surrounding mesenchyme of these central ducts is similar to that of the interlobular ducts noted in our study to be the predominant epithelium generated from bone marrow.
However, recent papers concluded that all beta-cell production during adult life is generated by self-replication rather than from adult stem cells [29, 30]. Without a method for lineage tracing, it is difficult to say whether the isolated beta cells of bone marrow origin we observed were derived from interlobular ductal cells. However, the chimerism created within the pancreatic ducts using the neonatal bone marrow transplant model provides a potential experimental tool to track the lineage fate of ductal cells in the adult animal.
The complete absence of bone marrow differentiation into vascular endothelium seen in this study may be because of the lack of tissue injury used in our neonatal transplant model [23]. Even in those reports that did not induce pancreas-specific injury (e.g., from streptozotocin or pancreatectomy), total body irradiation was used to obtain a hematopoietic graft [14–17]. Irradiation, even at sublethal doses, is known to induce widespread subclinical organ toxicity [31] and thus could result in recruitment of inflammatory cells and/or endothelial precursors to injured pancreas tissue [32, 33].
The findings here neither support nor reject the possibility of "transdifferentiation" from hematopoietic stem cells as the mechanism for epithelial development from bone marrow. However, the fact that the data were achieved using unfractionated "whole" bone marrow makes it equally likely that a nonhematopoietic epithelial progenitor/precursor is responsible for the generation of ducts in this model. In support of this, in a previous report, the CD45– fraction of splenic cells was able to generate pancreatic ductal epithelial cells when transplanted into diabetic NOD mice [34]. In the current study, the development of clusters of whole branching ductal structures of marrow origin suggests that the bone marrow cells responsible have considerable proliferative and even clonogenic capacity although the possibility of cell fusion cannot be completely ruled out as an explanation for the findings.
The success of the neonatal model in revealing the epithelial potential of bone marrow suggests that factors regulating proliferation, differentiation, and/or homing of these precursors are unique to (or at least significantly more active in) the neonatal period than in the adult. A very recent study demonstrated that factors present in young serum restored the proliferation and regeneration capacity of stem or progenitor cells in aged animals, suggesting that the behavior of adult stem cells can be altered by exposure to a young systemic environment [35]. Extensive expansion of the rat beta-cell mass during the early neonatal period has been described and is produced at least in part by a high frequency of beta cells in cell cycle during the first 2 days after birth [36]. During embryonic development, soluble factors produced in the mesenchyme, such as Fibroblast Growth Factor 10 (FGF10) and follistatin, are key to the regulation of growth and differentiation of the exocrine and endocrine pancreas [37–39]. However, little information is available on the regulation of ductal growth and differentiation during the neonatal period. One recent study reported that rapidly proliferating cells that express c-kit are found lining the embryonic and neonatal pancreatic ducts, but that proliferation falls rapidly during the first week of postnatal life [40]. c-kit is also expressed on hematopoietic stem cells, and its ligand acts to stimulate proliferation and prevent apoptosis in hematopoietic stem cells [41], providing another potential link between the hematopoietic and pancreatic organs.
In summary, the neonatal transplant model demonstrates the existence in bone marrow of epithelial precursors that can migrate to the pancreas and differentiate into complex, organ-specific ductal structures. Identifying the unique factors at play in pancreatic development during neonatal life may allow us to harness the process of epithelial engraftment and differentiation after BMT in adult recipients.
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ACKNOWLEDGMENTS |
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DISCLOSURES
The authors indicate no potential conflicts of interest.
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REFERENCES |
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This article has been cited by other articles:
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  F Marrache, S Pendyala, G Bhagat, K S Betz, Z Song, and T C Wang Role of bone marrow-derived cells in experimental chronic pancreatitis Gut, August 1, 2008; 57(8): 1113 - 1120. [Abstract] [Full Text] [PDF]
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 C. B. Rountree, L. Barsky, S. Ge, J. Zhu, S. Senadheera, and G. M. Crooks A CD133-Expressing Murine Liver Oval Cell Population with Bilineage Potential Stem Cells, October 1, 2007; 25(10): 2419 - 2429. [Abstract] [Full Text] [PDF]
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 A. E. Butler, A. Huang, P. N. Rao, A. Bhushan, W. J. Hogan, R. A. Rizza, and P. C. Butler Hematopoietic Stem Cells Derived From Adult Donors Are Not a Source of Pancreatic {beta}-Cells in Adult Nondiabetic Humans Diabetes, July 1, 2007; 56(7): 1810 - 1816. [Abstract] [Full Text] [PDF]
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E. M. Bruscia, E. C. Ziegler, J. E. Price, S. Weiner, M. E. Egan, and D. S. Krause Engraftment of Donor-Derived Epithelial Cells in Multiple Organs Following Bone Marrow Transplantation into Newborn Mice Stem Cells, October 1, 2006; 24(10): 2299 - 2308. [Abstract] [Full Text] [PDF]
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 S. Ge, G. M. Crooks, G. McNamara, and X. Wang Fluorescent Immunohistochemistry and In Situ Hybridization Analysis of Mouse Pancreas Using Low-power Antigen-retrieval Technique J. Histochem. Cytochem., July 1, 2006; 54(7): 843 - 847. [Abstract] [Full Text] [PDF]
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