Formation of Pancreatic Duct Epithelium from Bone Marrow During Neonatal Develop - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:11 |只看该作者 |倒序浏览 |打印

作者：Xiuli Wanga, Shundi Gea, Ignacio Gonzalezb, George McNamarac, C. Barth Rountreed, Kenny Kezhe Xia, Grace Huanga, Anil Bhushane, Gay M. Crooksa作者单位：a Division of Research Immunology/Bone Marrow Transplantation, : Y3 ^9 r; v" S- {




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      【摘要】) f) [- K. u( y0 c
   Recent reports suggest that bone marrow¨Cderived cells engraft and differentiate into pancreatic tissue at very low frequency after pancreatic injury. All such studies have used adult recipients. The aim of our studies was to investigate the potential of bone marrow to contribute to the exocrine and endocrine components of the pancreas during the normal rapid growth of the organ that occurs during the neonatal period. Five to ten million bone marrow cells from adult, male, transgenic, green fluorescent protein (GFP) mice were injected into neonatal nonobese diabetic/severely compromised immunodeficient/&szlig;2microglobulin-null mice 24 hours after birth. Two months after bone marrow transplantation, pancreas tissue was analyzed with fluorescence immunohistochemistry and fluorescence in situ hybridization (FISH). Co-staining of GFP, with anticytokeratin antibody, and with FISH for the presence of donor Y chromosome indicated that up to 40% of ducts (median 4.6%) contained epithelial cells derived from donor bone marrow. In some of these donor-derived ducts, there were clusters of large and small ducts, all comprised of GFP  epithelium, suggesting that whole branching structures were derived from donor bone marrow. In addition, rare cells that coexpressed GFP and insulin were found within islets. Unlike pancreatic damage models, no bone marrow¨Cderived vascular endothelial cells were found. In contrast to the neonatal recipients, bone marrow transplanted into adult mice rarely generated ductal epithelium or islet cells (p < .05 difference between adult and neonate transplants). These findings demonstrate the existence in bone marrow of pluripotent stem cells or epithelial precursors that can migrate to the pancreas and differentiate into complex organ-specific structures during the neonatal period.
      【关键词】 Bone marrow transplantation Epithelial stem cells Pancreas Ducts Neonate Mice
   INTRODUCTION
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A debate has raged in recent years over whether stem cells within the bone marrow have the capacity to "transdifferentiate," i.e., to generate nonhematopoietic cell types , after bone marrow or cord blood transplantation. Although the donor cells express tissue-specific markers, the low frequency of such events has created technical difficulties in proving whether true transdifferentiation is being observed rather than contamination of circulating, mature hematopoietic cells. In addition, in almost all studies, some form of tissue injury is present, raising the possibility that trafficking of inflammatory or endothelial precursors (with or without cell fusion) is responsible for at least some of the observations. With these caveats accepted, the obvious possible therapeutic implications necessitate the further careful study of bone marrow as a source of tissue repair and regeneration.6 X6 O; R. Z& C3 D5 _( L

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 . All experiments were performed with adult donors and recipients.$ }- M* n1 r3 E- L2 o+ W) }

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.$ s! B) c2 M! S5 H

MATERIALS AND METHODS+ O* `& n( a  m8 d1 }, x

5-Bromo-2'-deoxyuridine Analysis of Cell Cycling in Neonatal Mice: x. T  [0 E" @% v5 E

To detect the presence of cycling cells in the pancreas, neonatal and adult animals were administered 5-Bromo-2'-deoxyuridine (BrdU) (25 mg/kg; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) by i.p. injection. Pancreas tissue was harvested from animals sacrificed 2 hours after BrdU and processed for fluorescence immunohistochemistry. BrdU was detected with BrdU sheep polyclonal antibody conjugated with Cy3TM, and beta cells and ductal epithelial cells were detected with fluorescein isothiocyanate (FITC)¨Cinsulin or FITC¨Ccytokeratin (CK), respectively, by using fluorescence immunohistochemistry staining as below.
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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.  h5 d7 P9 o; G/ J& f/ T, o
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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 . 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¨CImmunocytometry Systems, San Jose, CA, http://www.bdbiosciences.com/immunocytometry_systems).
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In separate experiments, adult Nod/Scid/ß2Mnull mice (8 to 10 weeks old) were transplanted via tail vein injection with 5¨C10 x 106 adult GFP  bone marrow cells without irradiation and were sacrificed for analysis 2 months post transplantation.

Histochemistry' v, V. y* Y9 a6 u, L8 p. w

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¨C300 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¨Cfree, 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.
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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¨CFITC, anti-rabbit¨CCy3, and anti-mouse¨CFITC (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¨C2/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).
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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
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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¡ãC 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)¨C 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¡ãC for 10 minutes, and hybridized overnight at 37¡ãC 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, z0 }" P7 V* ~* K! c) U
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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.) Q9 r0 d  Z, k

Statistical Analysis

Means ¡À 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 # J4 C# m4 u+ ~# B- l2 v

RESULTS

Neonatal Pancreas Contains a High Frequency of Cycling Epithelial Ductal and Beta Cells1 m+ Q  I0 r7 q2 Y4 V: e6 d

We hypothesized that the neonatal model may represent a unique scenario in which pancreatic epithelium undergoes rapid cell division without injury, thus providing signals that assist in recruitment of epithelial precursors from the bone marrow. To explore this possibility, BrdU labeling in the pancreas was analyzed 2 hours after administration to otherwise untreated neonatal and adult Nod/Scid/ß2Mnull mice.2 C  G* ~: U8 |# B: P) _* S
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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 ¡À 1.9 versus 2.7 ¡À 0.3 per microscope field, magnification 20x, p ' _" j: D* `$ M. C  I* l
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Figure 1. Cell cycling of ductal epithelium and beta cells is increased during the neonatal period. BrdU expression (shown as red nuclei) in (A) ductal cells (CK shown as green) and (B) beta cells (insulin shown as green) of pancreas tissue from neonatal (1-day-old) and adult (8-week-old) mice. DAPI (blue) stains all nuclei. Scale bars = 50 µm. The percentage of ducts (C) and islets (D) containing cycling cells (i.e., at least one BrdU  cell) was significantly increased in neonates compared with adults (*p

Hematopoietic Engraftment After Bone Marrow Transplantation into Nonirradiated Neonatal Recipients
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Hematopoietic engraftment of GFP  donor bone marrow was determined by analyzing GFP expression in peripheral blood at the time of sacrifice of recipient mice (2 months post transplantation) (Fig. 2A). Bone marrow and peripheral blood in donor GFP transgenic mice did not uniformly express the GFP marker (39.0% ¡À 0.7% of nucleated cells expressed GFP in bone marrow and 88.6% ¡À 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% ¡À 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).0 S6 e2 h7 `+ I2 |$ r* s
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Figure 2. Hematopoietic engraftment after neonatal BMT. (A): Whole BM cells from adult male GFP transgenic mice were injected intravenously into newborn Nod/Scid/ß2Mnull mice 24 hours after birth, and recipient mice were sacrificed 2 months post BMT for analysis. Detection of GFP  donor cells in PB by FACS analysis of (B) nontrans-planted Nod/Scid/ß2Mnull mouse, and (C) Nod/Scid/ß2Mnull mouse transplanted with GFP  BM. (D): Fluorescence immunohistochemistry staining of pancreas from transplanted animal demonstrating GFP  blood cells (red) inside vessel stained with PECAM (green). DAPI (blue) stains all nuclei. Scale bars = 50 µm. Abbreviations: BM, bone marrow; BMT, bone marrow transplantation; CK, cytokeratin; DAPI, 4'6-diamidino-2-phenylindole; FACS, fluorescence-activated cell sorting; FISH, fluorescence in situ hybridization; GFP, green fluorescent protein; IHC, immunohistochemistry; Nod/Scid/ß2Mnull, nonobese diabetic/severely compromised immunodeficient/ß2microglobulin-null; PB, peripheral blood; PECAM, platelet/endothelial cell adhesion molecule.

Bone Marrow Cells Contribute to Pancreatic Duct Formation When Transplanted into Neonatal Mice
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To investigate the capacity of bone marrow cells to contribute to pancreatic tissue during normal neonatal development, GFP  bone marrow cells were intravenously infused into mice 24 hours after birth without inducing pancreas injury. Pancreas tissues from all animals were analyzed at 2 months post transplantation. Numerous hollow structures lined with uniform, low columnar, tightly spaced cells were partially or completely derived from donor bone marrow cells based on GFP expression. The identity of the structures was determined using the epithelial marker CK and the vascular endothelial marker PE-CAM-1; no cross-reaction between the antibodies was seen, and pancreatic ducts were clearly distinguished from blood vessels in both transplanted and nontransplanted mice (data not shown).
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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%¨C43.1%) 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%¨C31.3%) of all ducts had more than 50% donor cells and 4.1% (range 0.9%¨C11.8%) 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¨C4C).4 e) g. v% {, L: y; h& ^/ D
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Figure 3. Formation of pancreatic ducts derived from GFP  donor bone marrow. Fluorescence immunohistochemistry of pancreas tissue from transplanted animals with CK (green), GFP (red) antibodies, and DAPI (blue) (A¨CC). Different patterns of donor engraftment are shown. (A): Clusters of ducts, all of which contain 100% of epithelium of donor origin. (B): Ducts derived completely from donor cells are seen adjacent to recipient-derived ducts. (C): Less than 50% of cells lining duct are of donor origin. (D): Pancreatic ducts from GFP transgenic donor (positive control). (E): Pancreatic ducts from nontransplanted animal (negative control) show CK expression (green) but not GFP. Scale bars = 50 µm. Abbreviations: CK, cytokeratin; DAPI, 4'6-diamidino-2-phenylindole; GFP, green fluorescent protein.
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Table 1. Frequency of donor-derived pancreatic ducts after neonatal BMT

Figure 4. Colocalization of CK and GFP in donor-derived duct epithelial cells. Fluorescence immunohistochemistry staining of pancreas section from transplanted animal with CK (green) and GFP (red) antibodies and DAPI (blue). Frames (A, B) show CK (green) and GFP (red) coexpression in pancreatic duct, respectively. (C): Merged image of (A, B). Scale bars = 50 µm. Abbreviations: CK, cytokeratin; DAPI, 4'6-diamidino-2-phenylindole; GFP, green fluorescent protein.0 e6 F1 k& Y' ]- w# i$ U& h0 C" y

FISH and CK fluorescence immunohistochemistry analysis of the sections from female recipients showed donor-derived ductal epithelial cells with Y chromosome, confirming the presence of male donor cells contributing to the epithelial lining of the pancreatic ducts (Fig. 5). Analysis of serial sections taken along the length of the pancreas showed that the bone marrow¨Cderived 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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Figure 5. Coexpression of CK and Y chromosome in donor-derived duct epithelial cells. Dual staining of CK (green) and Y chromosome (red) in the four different pancreas sections from transplanted animals showing coexpression of CK and Y chromosome in the donor-derived duct epithelial cells (A) (arrows). Pancreas sections from male animal as positive (B) and female animal as negative (C) controls for fluorescence in situ hybridization staining. Scale bars = 25 µm. Abbreviations: CK, cytokeratin; DAPI, 4'6-diamidino-2-phenylindole.' e$ ^' S- \4 z2 f6 q8 Z& I1 v1 G

To rule out the presence of contaminating hematopoietic cells, CD45 staining was applied to the sections from transplanted and nontransplanted mouse pancreas. No duct epithelial cells coexpressed CD45 (data not shown).
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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¨CDerived Cells Occurred at Low Frequency in Islets

Co-staining of pancreatic tissue sections showed occasional bone marrow¨Cderived 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).

Table 2. Frequency of donor-derived GFP insulin  cells after neonatal BMT/ L$ H* p- {; p
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Ductal Generation from Bone Marrow Is Rare After Transplantation Performed in Adult Recipients
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To test whether a similar pattern of ductal engraftment from bone marrow could be seen when transplantation occurred outside the neonatal period, GFP  bone marrow cells were infused into nonirradiated adult Nod/Scid/ß2Mnull mice. High levels of hematopoietic engraftment were seen at the time of sacrifice (33.9% ¡À 5.8% GFP  cells in peripheral blood). Among 2,352 ductal structures from four mice sacrificed after 2 months, a median of 0.5% (range 0%¨C1.6%) of ducts contained GFP  cells, 0.1% (range 0%¨C0.2%) of ducts contained more than 50% donor GFP  epithelial cells, and 0.4% (range 0%¨C1.4%) of ducts contained less than 50% donor GFP  epithelial cells (Table 1). 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
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DISCUSSION

The diverse array of the body¡¯s epithelial cells is developed and maintained by mechanisms that are largely unknown . Findings presented here demonstrate the existence of cells within the marrow with the capacity to migrate to the neonatal pancreas and differentiate at high frequency into complex epithelial structures.

Previous reports have suggested a role for bone marrow in epithelial differentiation in multiple organs . 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.6 t" Q% q2 x7 b1 f  ]4 `% \4 J

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 . 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 . 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 .
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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¨C fraction of splenic cells was able to generate pancreatic ductal epithelial cells when transplanted into diabetic NOD mice . 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.. l. k! ?6 r5 i& U

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 , 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.

ACKNOWLEDGMENTS
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We thank the animal facility of CHLA and Denise Carbonaro for assistance with animal care and transplantation, and Dr. Donald Kohn for his helpful discussions. Fluorescence microscopy was performed in the Congressman Julian Dixon Cellular Imaging Core of the Saban Research Institute, CHLA. This work was generously supported by grants from the Seaver Institute and NIH (R01-DK68719).
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DISCLOSURES

The authors indicate no potential conflicts of interest.
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