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Endocrinology, doi:10.1210/en.2006-1351
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Endocrinology Vol. 148, No. 5 2006-2015
Copyright © 2007 by The Endocrine Society

Bone Marrow (BM) Transplantation Promotes ß-Cell Regeneration after Acute Injury through BM Cell Mobilization

Yutaka Hasegawa1, Takehide Ogihara1, Tetsuya Yamada, Yasushi Ishigaki, Junta Imai, Kenji Uno, Junhong Gao, Keizo Kaneko, Hisamitsu Ishihara, Hironobu Sasano, Hiromitsu Nakauchi, Yoshitomo Oka and Hideki Katagiri

Division of Advanced Therapeutics for Metabolic Diseases (Y.H., T.O., J.I., K.U., J.G., K.K., H.K.), Center for Translational and Advanced Animal Research, Division of Molecular Metabolism and Diabetes (Y.H., T.Y., Y.I., J.I., K.U., J.G., K.K., H.I., Y.O.), and Department of Pathology (H.S.), Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan; and Laboratory of Stem Cell Therapy (H.N.), Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan

Address all correspondence and requests for reprints to: Hideki Katagiri, M.D., Ph.D., Division of Advanced Therapeutics for Metabolic Diseases, Center for Translational and Advanced Animal Research, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. E-mail: katagiri{at}mail.tains.tohoku.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is controversy regarding the roles of bone marrow (BM)-derived cells in pancreatic ß-cell regeneration. To examine these roles in vivo, mice were treated with streptozotocin (STZ), followed by bone marrow transplantation (BMT; lethal irradiation and subsequent BM cell infusion) from green fluorescence protein transgenic mice. BMT improved STZ-induced hyperglycemia, nearly normalizing glucose levels, with partially restored pancreatic islet number and size, whereas simple BM cell infusion without preirradiation had no effects. In post-BMT mice, most islets were located near pancreatic ducts and substantial numbers of bromodeoxyuridine-positive cells were detected in islets and ducts. Importantly, green fluorescence protein-positive, i.e. BM-derived, cells were detected around islets and were CD45 positive but not insulin positive. Then to examine whether BM-derived cell mobilization contributes to this process, we used Nos3–/– mice as a model of impaired BM-derived cell mobilization. In streptozotocin-treated Nos3–/– mice, the effects of BMT on blood glucose, islet number, bromodeoxyuridine-positive cells in islets, and CD45-positive cells around islets were much smaller than those in streptozotocin-treated Nos3+/+ controls. A series of BMT experiments using Nos3+/+ and Nos3–/– mice showed hyperglycemia-improving effects of BMT to correlate inversely with the severity of myelosuppression and delay of peripheral white blood cell recovery. Thus, mobilization of BM-derived cells is critical for BMT-induced ß-cell regeneration after injury. The present results suggest that homing of donor BM-derived cells in BM and subsequent mobilization into the injured periphery are required for BMT-induced regeneration of recipient pancreatic ß-cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEVERAL LINES OF evidence indicate that bone marrow (BM)-derived cells are capable of transdifferentiating into various cell types, including endothelial cells, arterial smooth muscle cells, myoblasts, myocardium, and epithelia of the gastrointestinal tract (1, 2, 3, 4, 5, 6). In the field of regenerative medicine for diabetes treatment, BM cells are seen as promising pancreatic ß-cell sources (7, 8, 9). However, whether BM cells can transdifferentiate into ß-cells and/or stimulate ß-cell differentiation is controversial.

A previous study (10) showed that BM-derived cells can directly transdifferentiate into ß-cells. In that report, 4–6 wk after BM transplantation (BMT; i.e. lethal irradiation of recipient mice and subsequent BM cell infusion from other mice), donor BM-derived insulin-positive cells were detected in 1.7–3% of pancreatic islet cells. However, in subsequent similar studies (11, 12, 13), very few or no donor BM-derived insulin-positive cells were detected in recipient islets, suggesting that if direct transdifferentiation from BM-derived cells into ß-cells occurs, it would involve only a very small percentage of cells. BM-derived cells also reportedly initiate recipient ß-cell regeneration rather than directly transdifferentiating into ß-cells (14). In that study, BMT increased recipient ß-cells with the appearance of donor-derived endothelial cells in the pancreas, resulting in improvement of hyperglycemia in streptozotocin (STZ)-induced diabetic mice. Other studies also demonstrated that BMT improves hyperglycemia in diabetic animals such as STZ-treated mice (15) and rats (16), E2f1/E2f2 mutant mice (17), and KKAy mice (18). However, several studies obtained contradictory results, i.e. no improvement in hyperglycemia after BMT (12, 19). Whether BMT promotes ß-cell regeneration and improves hyperglycemia in diabetic mice and, if so, how ß-cells are regenerated remains essentially unknown. Herein we attempted to address these questions.

First, we observed that BMT, but not simple BM cell infusion without preirradiation, restored islet numbers and improved hyperglycemia in STZ-treated mice. Donor-derived cells were detected around post-BMT islets and were CD45 (pan-hematopoietic marker) positive, suggesting that mobilization of BM-derived cells to the pancreas induces ß-cell regeneration. To examine this hypothesis, we performed BMT experiments using endothelial nitric oxide synthase (eNOS)-deficient (Nos3–/–) mice, in which mobilization of BM-derived cells after myelosuppression is impaired (20). In STZ-treated Nos3–/– mice, BMT effects on ß-cell regeneration and improvement of hyperglycemia were very limited. Thus, BM-derived cell mobilization is apparently involved in BMT-induced ß-cell regeneration after acute injury.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
C57BL/6J mice were purchased from Clea Japan, Inc. (Tokyo, Japan). Green fluorescent protein (GFP) transgenic mice with the C57BL/6J background were kindly provided by Dr. M. Okabe (Osaka University, Osaka, Japan) (21). Enhanced GFP is under transcriptional control of the chicken ß-actin promoter and the cytomegalovirus enhancer in this strain, resulting in high-level expression in most tissues. Nos3–/– mice were purchased from Jackson Laboratories (Bar Harbor, ME). Age- and sex-matched wild-type (Nos3+/+) littermates served as controls. These animals were generated and have been maintained with a C57BL/6J background by backcrossing of hemizygous carriers to C57BL/6J for more than six generations. Mice were housed in an air-conditioned environment, with a 12-h light, 12-h dark cycle, and fed a regular unrestricted diet. Hyperglycemia was induced by ip infusion of 35 mg/kg body weight STZ (Sigma-Aldrich, St. Louis, MO) daily for 8 d [modification of method reported by Wang et al. (22)]. STZ was solubilized in citrate sodium buffer (pH 4.5) and injected, within 15 min after preparation, into 6-wk-old mice. All animal experiment procedures were approved by our Institutional Review Board, Tohoku University School of Medicine, and conducted according to institutional guidelines for animal experiments.

Measurements
Blood glucose was measured after a 10-h fast and assayed using Antsense II (Horiba Industry, Kyoto, Japan). Plasma insulin was determined with an ELISA kit (Morinaga Institute of Biological Science, Yokohama, Japan). Insulin content was measured as described previously (23).

BMT
BM cells were flushed in bulk from the medullary cavities of femurs and tibias. BM donors were young (6 wk old) sex-matched GFP transgenic mice and Nos3–/– or Nos3+/+ mice. Recipient mice were lethally irradiated (10 Gy) and reconstituted a single iv infusion of 2 x 106 BM cells, from donor mice through the tail vein. Tissues were analyzed 30–40 d after BMT. The percentage of GFP-positive cells among recipient BM cells was determined by fluorescence-activated cell sorting (FACS), using a FACS Caliber with CellQuest software (BD PharMingen, Franklin Lakes, NJ).

Bromodeoxyuridine (BrdU) in situ detection
To identify proliferating cells in the pancreas, BrdU was injected according to the BrdU in situ detection kit protocol (BD Bioscience, San Jose, CA). Mice were injected ip with 1 mg BrdU 24 h before pancreas extraction at 0, 3, 7, 10, 15, or 25 d after BMT. The labeled cells were immunostained with anti-BrdU antibody. To calculate numbers of islets and cells per islet and the percentage of BrdU-positive cells among islet cells, we microscopically examined the whole pancreas in 30-µm sections and counted the numbers of islets, islet cells, and BrdU-positive nuclei in islets.

Immunohistochemistry
Mouse pancreases were excised and fixed overnight in 10% paraformaldehyde. Fixed tissues were processed for paraffin embedding and 3-µm sections were prepared. The streptavidin-biotin method was performed with a Histofine streptavidin-biotin-PO kit (Nichirei, Tokyo, Japan) for immunostaining using antibody against insulin (Sigma-Aldrich) or GFP (Santa Cruz Biotechnology, Santa Cruz, CA). Slides were deparaffinized and immediately exposed to the blocking solution. Sections were incubated for 18 h at 4 C with antibody against human insulin or GFP diluted 1:1000 in PBS. Slides were incubated with the biotinylated IgG for 1 h and then peroxidase-conjugated streptavidin for 30 min at room temperature. Finally, immunoreactivity was visualized by incubation with a substrate solution containing 3,3'-diaminobenzidine tetrahydrochloride. For double staining of insulin and BrdU, the streptavidin-peroxidase method was applied, followed by incubation with Simple stain 3-amino-9-ethyl carboxazole solution (Nichirei).

Fluorescent immunohistochemistry
For double staining of insulin with glucagon, keratin/cytokeratin, or CD45, the 3-µm sections of paraffin-embedded pancreases were incubated overnight with the respective antibodies at 4 C. Antibodies against insulin, glucagon (Dako Corp., Carpinteria, CA), keratin/cytokeratin (Nichirei, and CD45 (Santa Cruz Biotechnology) were diluted 1:1000 in PBS. For platelet endothelial cell adhesion molecule (PECAM)-1 staining, sections were immunostained with rat anti-CD31 (1:10; BD Biosciences). Labeled cells were visualized with a biotin-conjugated secondary antibody with streptavidin, TX red conjugate (Vector Laboratories, Burlingame, CA). For double staining of insulin with glucagon or keratin/cytokeratin, the sections were incubated for 1 h at room temperature in a mixture of Alexa Fluor 488 goat chicken antimouse IgG (Molecular Probes, Eugene, OR) diluted 1:100 and Alexa Fluor 594 donkey antirabbit diluted 1:50 in PBS. For double staining of insulin and CD45, the sections were incubated in a mixture of Alexa Fluor 488 chicken antimouse IgG and Alexa Fluor 546 goat antirabbit IgG diluted 1:1000 in PBS. Sections were observed under a fluorescence microscope, LSM 5 PASCAL (Carl Zeiss, Oberkochen, Germany) and the image was analyzed using the PASCAL system.

Statistical analysis
Data are expressed as means ± SE. Differences between experimental groups were evaluated using the unpaired Student’s t test for several independent observations. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Recipient BM was replaced with donor cells after irradiation followed by BM cell infusion but not after simple BM cell infusion without preirradiation
Six-week-old C57BL/6J mice were given STZ daily for 8 d, followed by lethal irradiation and subsequent infusion of BM cells (STZ+BMT mice). In these experiments, BM cells were obtained from GFP transgenic mice (Fig. 1AGo). A group of STZ-treated mice was simply infused with the same number (2 x 106) of BM cells without preirradiation (STZ+BM-infused mice). First, we confirmed replacement of recipient BM with that of donor mice using fluorescence microscopy and FACS analysis. As shown in Fig. 1BGo, there were no GFP-positive cells in the BM of C57BL/6J mice, whereas nearly all BM cells from GFP mice were GFP positive. BM cells of STZ+BMT mice showed high donor chimerism, indicating the recipient BM to have essentially been replaced with donor BM cells. In contrast, STZ+BM-infused mice had no donor-derived GFP cells in their BM, suggesting that preirradiation is necessary for BM replacement.


Figure 1
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  		FIG. 1. BMT in STZ-treated mice. A, Experimental protocol. B, Bone marrow chimerism. Bright field (left upper panels), FITC (left lower panels), and representative FACS analyses of bone marrow chimerism from C57BL/6J mice (a), GFP transgenic mice (b), STZ-treated mice receiving lethal irradiation and BMT from GFP transgenic mice (c), and STZ-treated mice infused with BM cells of GFP transgenic mice, without preirradiation (d) (right panels) are shown. C, Fasting blood glucose of STZ-treated mice with or without BMT. {blacktriangleup}, STZ-treated mice without BMT (hyperglycemic control); {blacksquare}, STZ-treated mice receiving BMT (lethal irradiation and subsequent BM cell infusion from GFP transgenic mice); bullet, STZ-treated mice infused with BM cells of GFP transgenic mice, without preirradiation; {circ}, mice with neither STZ nor BMT (normoglycemic control). *, P < 0.05 for {blacksquare}, compared with {blacktriangleup} group (n = 5–6 in each group). D, Fasting plasma insulin on d 40. Cont, Mice with neither STZ nor BMT (normoglycemic control); STZ, STZ-treated mice without BMT (hyperglycemic control); STZ+BMT, STZ-treated mice receiving BMT. *, P < 0.05 for STZ+BMT, compared with STZ group (n = 5–6 in each group).

 
BMT, but not simple BM cell infusion without preirradiation, improved hyperglycemia in STZ-treated mice
As shown in Fig. 1CGo, STZ-treated mice receiving neither irradiation nor BM cell infusion (hyperglycemic controls) showed markedly higher fasting blood glucose than mice without STZ treatment (normoglycemic controls). Notably, blood glucose levels of STZ+BMT mice were significantly lower than those of hyperglycemic controls. Forty days after the first STZ administration, blood glucose levels of STZ+BMT mice were similar to those of normoglycemic controls. However, blood glucose levels of STZ+BM-infused mice did not decrease, instead remaining similar to those of hyperglycemic controls for 50 d after STZ administration. We additionally examined the effects of BMT, performed 30 d after STZ treatment. This late BMT did not significantly decrease blood glucose levels (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Together, these findings suggest that BMT improves hyperglycemia after acute injury of pancreatic ß-cells with STZ treatment.

Next, we measured fasting plasma insulin levels on d 40 (Fig. 1DGo) in STZ+BMT mice. STZ administration markedly decreased plasma insulin levels, whereas BMT partially but significantly restored these levels by d 40.

STZ administration followed by BMT increased pancreatic islets in the vicinity of pancreatic ducts
We histologically analyzed pancreatic islets in the four groups. With hematoxylin-eosin staining on d 35, islet number and size were markedly decreased in hyperglycemic (Fig. 2AGo, b and f), as compared with normoglycemic (Fig. 2AGo, a and e), controls. Whereas simple BM infusion without preirradiation did not reverse the diminished number and size of islets (Fig. 2AGo, c and g), islet number and size were both restored in STZ+BMT mice (Fig. 2AGo, d and h). Several islet populations were enlarged as compared with those in normoglycemic controls (Fig. 2AGo, a and e vs. d and h).


Figure 2
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  		FIG. 2. Pancreatic islets of STZ-treated mice receiving subsequent BMT. A, Hematoxylin-Eosin staining of pancreases on d 35. Pancreases from normoglycemic control mouse (a and e), hyperglycemic control mouse (b and f), STZ-treated mouse simply infused with BM cells without preirradiation (c and g), and STZ-treated mouse receiving lethal irradiation and BMT (d and h). a–d, Magnification, x40; e–h, x100. B, Antiinsulin immunostaining of pancreases. Pancreases from normoglycemic control mouse (a), hyperglycemic control mouse (b), and STZ-treated mouse receiving BMT (c and d). a–c, Magnification, x40; d, x200. C, Double immunostaining of pancreas with antiinsulin and antikeratin/cytokeratin antibodies. Green indicates insulin-positive and red keratin/cytokeratin-positive cells, i.e. pancreatic ductal epithelium. D, Double immunostaining of pancreases with antiinsulin and antiglucagon antibodies. Pancreases from normoglycemic control mouse (a), hyperglycemic control mouse (b), and STZ-treated mouse receiving BMT (c). In C and D, to avoid overlapping staining of GFP with FITC, BM cells obtained from wild-type C57BL/6J mice, but not from GFP transgenic mice, were transplanted. Representative histological findings among six independent experiments are presented.

 
Antiinsulin staining of pancreatic specimens is shown in Fig. 2BGo. In hyperglycemic controls, insulin-positive cells were markedly diminished (Fig. 2BGo, b) as compared with normoglycemic controls (Fig. 2BGo, a). In contrast, in STZ+BMT mice, islet numbers were restored and sizes varied with some being enlarged (Fig. 2BGo, c). Notably, in the large view (Fig. 2BGo, d), a major population of insulin-positive cells in STZ+BMT mice is located in the vicinity of pancreatic ducts, which were stained with antikeratin/cytokeratin antibody (Fig. 2CGo).

Next, we performed double immunostaining using antibodies against insulin and glucagon. In immunofluorescent experiments (Figs. 2Go, C and D), to avoid overlapping staining of GFP with fluorescein isothiocyanate (FITC), BM cells obtained from wild-type C57BL/6J mice, but not GFP transgenic mice, were transplanted. Compared with islets of normal and STZ-treated mice (Fig. 2DGo, a and b), islets in STZ+BMT mice exhibited normal architecture with slightly fewer ß-cells surrounded by {alpha}-cells (Fig. 2DGo, c).

To exclude the possibility that irradiation suppresses inflammation in response to STZ and prevents ß-cell injury, STZ-treated mice were exposed to lethal irradiation (10 Gy) without subsequent BM cell infusion. Lethal irradiation alone did not lower blood glucose in STZ-treated mice. Pancreatic islets were diminished in size, as in hyperglycemic controls, 9 d after irradiation (mice died 10–14 d after lethal irradiation without BMT in our experiment; data not shown). Next, to examine prolonged effects of irradiation, mice were sublethally irradiated (5 Gy). Sublethal irradiation alone likewise did not significantly improve hyperglycemia in STZ-treated mice (data not shown), suggesting that irradiation does not exert protective effects against STZ-induced ß-cell injury.

To further examine whether these islets in STZ+BMT mice were regenerated or only protected from STZ injury, BrdU staining was performed. In islets of normoglycemic (Fig. 3AGo) and hyperglycemic (Fig. 3BGo) controls, there were very few BrdU-positive cells. In contrast, islets of STZ+BMT mice (10 d after BMT) contained substantial numbers of BrdU-positive cells in and around islets, and some were detected among the pancreatic ductal cells (Fig. 3CGo). In other sections as well, islets containing BrdU-positive cells were mostly located near ducts and blood vessels. Most BrdU-positive cells in islets were insulin positive, whereas those outside the islets, mostly in the ductal structure, did not express insulin. (Fig. 3DGo). Given reports that pancreatic stem/progenitor cells exist among ductal cells (24, 25, 26), BMT after STZ treatment might stimulate the generation of new islets from ductal progenitor cells as well as proliferation of ß-cells in this model.


Figure 3
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  		FIG. 3. BrdU-positive proliferating cells in pancreases of STZ-treated mice receiving subsequent BMT. A–C, Pancreases from normoglycemic control mouse (A), hyperglycemic control mouse (B), and STZ-treated mice receiving BMT (C) (10 d after BMT). Brown cells are BrdU positive. D, Double immunostaining of pancreases from STZ-treated mice receiving BMT (10 d after BMT) with antiinsulin and anti-BrdU. Brown and red cells are BrdU and insulin positive, respectively.

 
We also quantitatively examined the time courses of islet numbers, cell number per islet and percentage of BrdU-positive cells among islet cells after BMT (Table 1Go). Although decreased by STZ, islet number was significantly increased 10 and 15 d after BMT. The peak islet number was greater than in normoglycemic control mice by 47%. The islet number and percentage of BrdU-positive cells among islet cells were also increased through 10 d after BMT and then fell to normoglycemic control levels. These findings clearly indicate that BMT induces ß-cell regeneration, resulting in pancreatic islet restoration in this model.


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  		TABLE 1. Islet numbers and BrdU-positive cells per pancreatic islet cells of STZ-treated mice receiving subsequent BMT

 
Although no BM-derived insulin-positive cells were detected, the regenerated islets were surrounded by BM-derived CD45-positive cells
To investigate whether BM-derived cells transdifferentiated into insulin-producing cells in our model, pancreases from STZ+BMT mice on d 35 were immunostained with antiinsulin antibody, followed by an intensive search for both insulin- and GFP-positive cells using confocal fluorescence microscopy. However, no double-positive cells were detected (Fig. 4AGo), suggesting that regenerated ß-cells in STZ+BMT mice are derived from recipient cells. In contrast, intriguingly, GFP-positive, i.e. BM-derived, cells were located around islets (Fig. 4AGo). In STZ+BM-infused mice, no GFP-positive cells were detected around islets (data not shown). Immunostaining with anti-GFP antibody confirmed that GFP-positive cells exist around islets of STZ+BMT mice (Fig. 4BGo, black arrows indicate islets). To identify the lineage of BM-derived cells around islets, we used several antibodies to immunostain lineage markers. GFP-positive cells around islets were CD45-positive (Fig. 4CGo, white arrows indicate islets), although these cells were not positively stained with F4/80, CD68 (macrophage lineage), CD3/CD5 (T cell lineage), or CD20 (B cell lineage) (data not shown), suggesting immature hematopoietic cells. We additionally examined whether these BM-derived cells are positive for an endothelial cell marker, CD31 (PECAM-1). Although a few GFP-positive cells were positive for CD 31 (Fig. 4DGo, red arrow), most BM-derived cells in or around islets were not positively stained with this endothelial marker. Taken together, these observations suggest that donor immature hematopoietic cells, which may be expanded and mobilized to peripheral blood after BMT, initiate ß-cell regeneration.


Figure 4
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  		FIG. 4. BM-derived cells in pancreases of STZ-treated mice receiving subsequent BMT. A, BM-derived cells and insulin-positive cells in pancreas of STZ-treated mouse receiving BMT from GFP transgenic mice. Pancreases of STZ-treated mouse receiving subsequent BMT from GFP transgenic mice (35 d after the first STZ). Left panel, Insulin-positive cells; middle panel, GFP-positive, i.e. BM-derived cells; right panel, merged image of the left and middle panels. B, BM-derived cells in pancreases of STZ-treated mice receiving BMT from GFP transgenic mice. Brown cells are GFP positive, i.e. BM-derived cells, and arrows indicate islets. C, CD45-positive and BM-derived cells in pancreas of STZ-treated mice receiving BMT from GFP transgenic mice. Left panel, Immunostaining with anti-CD45 antibody. Red indicates CD45-positive cells. Middle panel, Green indicates GFP-positive cells. Right panel, Merged image of the left and middle panels. Yellow indicates GFP and CD45 double-positive cells. Arrows indicate islets. D, CD31 (PECAM-1)-positive and BM-derived cells in pancreases of STZ-treated mice receiving BMT from GFP transgenic mice. Left panel, Immunostaining with anti-CD31 antibody. Red indicates CD31-positive cells. Middle panel, Green indicates GFP-positive cells. Right panel, Merged image of left and middle panels.

 
Mobilization of BM-derived cells is necessary for the glucose-lowering effect of BMT after STZ administration
To determine whether BM-derived cell mobilization is pivotal in this process, we investigated the effects of BMT on ß-cell regeneration using eNOS-deficient (Nos3–/–) mice as a model for impaired BM-derived cell mobilization. In Nos3–/– mice, mobilizations of hematopoietic stem cells and endothelial progenitor cells from BM were reportedly impaired after myelosuppression. Deficiency in eNOS reportedly reduces hematopoietic recovery in response to 5-fluorouracil treatment due to impaired progenitor cell mobilization (20). Therefore, we performed similar experiments using Nos3–/– mice. First, we compared two BMT groups, i.e. Nos3+/+ donors to Nos3+/+ recipients (Nos3+/+ to Nos3+/+ mice) and Nos3–/– donors to Nos3–/– recipients (Nos3–/– to Nos3–/– mice).

Peripheral white blood cells (WBCs) were counted after lethal irradiation and subsequent BM infusion (Fig. 5AGo). Myelosuppression after irradiation was profound and recovery of peripheral WBC counts was markedly delayed in Nos3–/– to Nos3–/– mice vs. Nos3+/+ to Nos3+/+ mice. Thus, eNOS deficiency impairs hematopoietic reconstitution after not only 5-fluorouracil treatment but also BMT. We measured the blood glucose levels after STZ administration followed by BMT (Fig. 5BGo). In Nos3+/+ to Nos3+/+ mice, STZ-induced hyperglycemia was improved to nearly normoglycemic control levels 40 d after the first STZ administration, consistent with the findings shown in Fig. 1CGo. In contrast, in Nos3–/– to Nos3–/– mice, BMT did not improve STZ-induced hyperglycemia (Fig. 5BGo). Thus, eNOS function is essential for improving hyperglycemia after BMT.


Figure 5
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  		FIG. 5. BMT experiments using Nos3+/+ and Nos3–/– mice. A, Time courses of peripheral WBC counts in Nos3+/+ and Nos3–/– mice receiving BMT. {blacksquare}, STZ-treated Nos3+/+ mice receiving BMT from Nos3+/+ mice; {blacktriangleup}, STZ-treated Nos3–/– mice receiving BMT from Nos3–/– mice; bullet, STZ-treated Nos3+/+ mice receiving BMT from Nos3–/– mice. *, P < 0.05 for {blacktriangleup}, compared with {blacksquare} group; #, P < 0.05 for bullet, compared with {blacktriangleup} group, respectively (n = 5–6 in each group). B, Fasting blood glucose levels of Nos3+/+ and Nos3–/– receiving BMT. {circ}, Normoglycemic control Nos3+/+ mice with neither STZ nor BMT; {triangleup}, STZ-treated Nos3+/+ mice without BMT (hyperglycemic control); {blacksquare}, STZ-treated Nos3+/+ mice receiving BMT from Nos3+/+ mice; {blacktriangleup}, STZ-treated Nos3–/– mice receiving BMT from Nos3–/– mice; bullet, STZ-treated Nos3+/+ mice receiving BMT from Nos3–/– mice. *, P < 0.05 for bullet, compared with {triangleup} group; # P < 0.05 for {blacksquare}, compared with bullet group, respectively (n = 5–6 in each group). C, Pancreatic insulin contents. STZ, STZ-treated Nos3+/+ mice without BMT; Cont, Nos3+/+ or Nos3–/– mice with neither STZ nor BMT; STZ+BMT, STZ-treated Nos3+/+ mice receiving BMT from Nos3+/+ mice and STZ-treated Nos3–/– mice receiving BMT from Nos3–/– mice. *, P < 0.05 between STZ-treated Nos3+/+ mice receiving BMT from Nos3+/+ mice and STZ-treated Nos3–/– mice receiving BMT from Nos3–/–mice. D, Time courses of islet numbers after BMT. E, Time courses of BrdU-positive cell percentage per islet cells after BMT. In D and E, the thick line indicates STZ-treated Nos3+/+ mice receiving BMT from Nos3+/+ mice, and the dotted line STZ-treated Nos3–/– mice receiving BMT from Nos3–/– mice. *, P < 0.05 between STZ-treated Nos3+/+ mice receiving BMT from Nos3+/+ mice and STZ-treated Nos3–/– mice receiving BMT from Nos3–/–mice at the same time points. F, Immunostaining of pancreases with antiinsulin and anti-CD45 antibodies. Pancreases from normoglycemic control mice (Nos3+/+ mouse in a and Nos3–/– mouse in f), hyperglycemic control mice (Nos3+/+ mouse in b and Nos3–/– mouse in g), STZ-treated Nos3+/+ mice receiving BMT from Nos3+/+ mice (7 d after BMT in c and d, 14 d after BMT in e), and STZ-treated Nos3–/– mice receiving BMT from Nos3–/– mice (7 d after BMT in h and i, 14 d after BMT in j). Green indicates insulin-positive, red CD45-positive cells. Red arrows indicate CD45-positive cells in and around islets and white arrows pancreatic ducts and blood vessels.

 
In Nos3–/– to Nos3–/– mice, not only mobilization of BMT-derived progenitor cells but also pancreatic endothelial function may be impaired due to systemic eNOS deficiency. Therefore, we performed an additional BMT, i.e. Nos3–/– donors to Nos3+/+ recipients (Nos3–/– to Nos3+/+ mice), whose eNOS is intact in pancreatic blood vessels. In Nos3–/– to Nos3+/+ mice, myelosuppression was profound and subsequent recovery of the WBC count was delayed, compared with Nos3+/+ to Nos3+/+ mice, but this delay in recovery was significantly less severe than that seen in Nos3–/– recipients (Fig. 5AGo). In Nos3–/– to Nos3+/+ mice, blood glucose levels also reached midrange values; the glucose-lowering effects of BMT did occur but were significantly blunted (Fig. 5BGo). These findings indicate that the lack of hyperglycemia improvement in Nos3–/– to Nos3–/– mice is not attributable solely to the impaired pancreatic endothelial function of recipients. The glucose-lowering effect of BMT inversely correlates with the severity of myelosuppression and delayed recovery, which apparently reflects impaired mobilization of BM cells to peripheral blood.

BMT-induced ß cell regeneration was impaired in STZ-treated Nos3–/– mice
To quantify BMT-induced ß-cell regeneration in Nos3+/+ to Nos3+/+ and Nos3–/– to Nos3–/– mice, pancreatic insulin contents 40 d after STZ (30 d after BMT) were measured (Fig. 5CGo). Compared with Nos3+/+ controls without STZ treatment, STZ-treated Nos3+/+ mice had markedly lower pancreatic insulin contents. In Nos3+/+ to Nos3+/+ mice, BMT partially restored pancreatic insulin contents, consistent with our findings that plasma insulin levels were partially restored by BMT (Fig. 1DGo). In contrast, in Nos3–/– to Nos3–/– mice, BMT effects on pancreatic insulin contents were very limited; pancreatic insulin contents were significantly lower in Nos3–/– to Nos3–/– mice than in Nos3+/+ to Nos3+/+ mice (Fig. 5CGo).

Next, changes in islet numbers and percentage of BrdU-positive cells among islet cells in response to BMT were compared between Nos3+/+ to Nos3+/+ mice and Nos3–/– to Nos3–/– mice. Whereas islet numbers were increased in STZ-treated Nos3+/+ to Nos3+/+ mice during the period 7–15 d after BMT, islet numbers were significantly less in STZ-treated Nos3–/– to Nos3–/– mice (Fig. 5DGo). In addition, whereas percentages of BrdU-positive cells among islet cells were markedly increased in STZ-treated Nos3+/+ to Nos3+/+ mice 7–10 d after BMT, there were significantly fewer such cells in STZ-treated Nos3–/– to Nos3–/– mice (Fig. 5EGo). These results suggest that impaired BM-derived cell mobilization in Nos3–/– mice suppresses BMT-induced ß-cell regeneration after acute injury.

BM-derived CD45-positve cells around islets are important for ß-cell regeneration-induced by BMT
To examine whether impaired mobilization of BM-derived cells in Nos3–/– mice affects hematopoietic cell assembly around islets and ß-cell regeneration, we compared pancreases from Nos3+/+ to Nos3+/+ and Nos3–/– to Nos3–/– mice using antiinsulin and CD45 antibodies (Fig. 5FGo). In Nos3+/+ to Nos3+/+ mice, substantial numbers of CD45-positive cells were detected in and around the regenerated islets (red arrows in Fig. 5FGo, c–e). No such cells were detected around islets in Nos3+/+ or Nos3–/– mice treated with STZ alone (Fig. 5FGo, b and g), suggesting that STZ-induced inflammation alone is not responsible for recruiting these cells. In pancreases from Nos3+/+ to Nos3+/+ mice, regenerated islets were located near pancreatic ducts and blood vessels (white arrows in Fig. 5FGo, c–e). In contrast, ß-cell regeneration was markedly impaired in Nos3–/– to Nos3–/– mice (Fig. 5FGo, h–j), and far fewer CD45-positive cells were present in and around islets in Nos3–/– to Nos3–/– than in Nos3+/+ to Nos3+/+ mice. These results support the notion that BMT-induced BM-derived cell mobilization is critical for regeneration of recipient ß-cells from stem/progenitor cells in pancreatic ducts.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently considerable research attention has focused on pancreatic ß-cell regeneration. In particular, several previous studies examined the role of BM-derived cells in ß-cell regeneration using BMT (10, 11, 12, 13, 14, 15, 16, 17, 18), but no definitive conclusions have yet been reached. In this study, we clearly demonstrate that BMT can regenerate recipient ß-cells under certain conditions. Our data supported those of a previous report (14) showing BMT to improve hyperglycemia in STZ-induced diabetic mice via regeneration of recipient pancreatic ß-cells. Herein we attempted to elucidate the mechanisms whereby BMT induces ß-cell regeneration.

First, we demonstrated that BMT, but not simple BM cell infusion without preirradiation, promotes ß-cell regeneration after STZ-induced injury. What are the differences between these procedures? BMT involves lethal irradiation and subsequent BM cell infusion. We confirmed, using FACS analysis, that recipient BM is essentially replaced with that of donor mice after BMT. In contrast, mice receiving BM cell infusion alone without preirradiation showed no BM replacement with donor-derived cells. Myelosuppression and subsequent expansion of donor BM cells take place in BMT. During this process, donor BM cells home to the BM microenvironment and progenitor cells mobilize and expand in the peripheral blood (27). In contrast, simple BM cell infusion does not induce homing or expansion of donor BM cells. Therefore, expansion of immature BM cells, which are rarely detected in peripheral blood in normal circumstances, is likely to be important for ß-cell regeneration after BMT. We ruled out the possibility that irradiation suppresses inflammation in response to STZ administration and prevents ß-cell injury. STZ-treated mice were exposed to lethal (10 Gy) and sublethal (5 Gy) irradiation without subsequent BM cell infusion. Irradiation alone had no effect on hyperglycemia or ß-cell number in STZ-treated mice. Furthermore, in STZ+BMT mice on d 2 after BMT, islet numbers and cell numbers per islet were both significantly decreased by STZ but were restored by d 10. Thus, it is unlikely that irradiation itself protects ß-cells.

Next, we found that a major population of post-BMT islets were located near pancreatic ducts and blood vessels. This observation raises possibilities regarding the origins of post-BMT islets. In general, multipotent adult stem cells are located in somatic tissues, which maintain and regenerate impaired tissues (28, 29). However, there is considerable controversy regarding the existence and location of pancreatic tissue stem cells (30, 31). Previous studies have shown pancreatic stem/progenitor cells in ductal epithelium (24, 25, 26). However, recent reports suggest that ß-cells arise only from self-duplication of preexisting ß-cells, i.e. ß-cells cannot be derived from non-ß-cell progenitors (32, 33). In this study, post-BMT islets were located near pancreatic ducts. In addition, BrdU-positive cells were detected in the vicinity of pancreatic ducts in STZ+BMT mice. After islet numbers had been decreased by STZ, a rise above normoglycemic control levels was seen, indicating new islet formation. In addition, BM-derived cells accumulated in and around post BMT-islets. Thus, BM-derived cells are likely to stimulate proliferation and differentiation of pancreatic stem/progenitor cells in ductal epithelium, resulting in new islet formation. Given the observation that BrdU-positive cells in islets expressed insulin, these cells must still have been proliferative after differentiation into pancreatic ß-cells. However, further studies, focusing on the origin of newly generated islets, are needed to support this speculation. Whereas BM-derived cells that accumulated around the islets in STZ+BMT mice were CD45 positive, immunohistochemical studies revealed that these cells do not express mature T or B lymphocyte or macrophage markers. Taken together with the finding that simple BM infusion without preirradiation induced neither ß-cell regeneration nor accumulation of BM-derived cells (data not shown), we speculate that these immature BM-derived cells send signals triggering proliferation and differentiation of stem/progenitor cells into ß-cells. Our next goal is identification of these signals.

To examine the causal relationship between BM-derived cell mobilization and BMT-induced ß-cell regeneration, we performed similar experiments using a model of impaired BM-derived cell mobilization. Mechanisms underlying mobilization of hematopoietic and endothelial progenitor cells from BM after myelosuppression have been studied in detail (34). After myelosuppression, secreted cytokines/chemokines, such as granulocyte-colony stimulating factor, stromal cell-derived factor, and vascular endothelial growth factor, activate matrix metalloproteinase (MMP)-9 in the BM microenvironment. Activated MMP-9 processes membrane-bound kit-ligand, releases it as soluble kit-ligand (sKitL), followed by binding of sKitL to c-kit on the stem cell surface and stimulation of its mobilization from the BM. Because nitric oxide from BM is necessary for MMP-9 activation, sKitL production and the resultant mobilization of BM-derived cells are impaired in Nos3–/– mice (20). Therefore, using Nos3–/– mice, we examined the effects of BMT on blood glucose levels and glucose ß-cell regeneration. We first confirmed that recovery of the WBC count after BMT was significantly delayed in Nos3–/– to Nos3–/– mice, compared with Nos3+/+ to Nos3+/+ mice. Judging from the doubling time of hematopoietic cells, a 1-wk delay in WBC recovery indicates marked impairment of BM cell mobilization by approximately 2 orders of magnitude. In Nos3–/– to Nos3–/– mice, BMT had virtually no effects on blood glucose levels, pancreatic insulin contents, islet numbers, or percentage of BrdU-positive cells among islet cells. In addition, far fewer CD45-positive cells were detected in and around islets. These results support the notion that BMT-induced BM-derived cell mobilization plays a pivotal role in ß-cell regeneration from ductal progenitor cells.

Neovascularization in ischemic regions is also impaired in Nos3–/– mice because of decreased mobilization of BM-derived endothelial progenitor cells (20). The microvasculature is well developed in pancreatic islets (35). Endothelial signals are reportedly important for islet development (36), insulin gene expression, and ß-cell proliferation (37). In the present study, a small population of BM-derived cells around regenerated islets was positively stained with CD31, although these cells were largely CD31 negative. This observation is consistent with the results of previous report (14). Therefore, in addition to hematopoietic progenitor cells, endothelial progenitor cells mobilized from BM may contribute to ß-cell regeneration after BMT by promoting islet microvasculature formation. In addition, BM-derived endothelial progenitor cells have been shown to contribute to neovascularization in impaired tissues, including myocardial (38) and hind limb ischemia (39). Recruitment of these cells reportedly occurs in response to acute injury of ß-cells (19). Taken together with the finding that, when BMT was performed 30 d after STZ treatment, hyperglycemia-improving effects were far smaller, acute STZ injury might trigger migration of immature BM-derived cells to the injured pancreas.

We do not rule out the importance of eNOS in pancreatic blood vessels for BMT-induced ß-cell regeneration. However, in Nos3–/– to Nos3+/+ mice, which have decreased BM eNOS (because of BM replacement with eNOS-deficient cells) with intact pancreatic eNOS, the blood glucose-lowering effects of BMT were significantly blunted, compared with Nos3+/+ to Nos3+/+ mice. Thus, glucose-lowering effects correlated inversely with the severity of myelosuppression and delayed recovery of the peripheral WBC count, suggesting the importance of BM-derived cell mobilization, rather than eNOS activity in pancreatic blood vessels, in BMT-induced ß-cell regeneration.

In summary, BMT promotes ß-cell regeneration after STZ-induced injury. A series of BMT experiments using Nos3–/– mice demonstrated BM-derived cell mobilization to be essential for BMT-induced ß-cell regeneration. Acute injury with STZ treatment may trigger recruitment of immature BM-derived cells to the injured pancreas. Recruited BM-derived cells may then stimulate stem/progenitor cells located in the recipient pancreas, resulting in islet regeneration. To our knowledge, this is the first report showing mobilization of BM-derived cells to be involved in ß-cell regeneration in diabetic animals. From the viewpoint of clinical application, it is important to study the effects of myelosuppression-inducing reagents, such as antitumor drugs, on pancreatic islet regeneration.


   Acknowledgments
 
The authors thank Dr. M. Okabe for providing GFP transgenic mice and are indebted to M. Hoshi, I. Sato, K. Kawamura, and J. Fushimi, who assisted with various aspects of this study.


   Footnotes
 
This work was supported by a Grant-in-Aid for Young Scientists, B (16790503) (to T.O.), a Grant-in-Aid for Scientific Research (B2, 18390267) (to H.K.) from the Ministry of Education, Science, Sports, and Culture of Japan, and a Grant-in-Aid for Scientific Research (H16-genome-003) (to Y.O.) from the Ministry of Health, Labor, and Welfare of Japan. This work was also supported by grants from the 21st Century COE Program "CRESCENDO" (to H.K.) and "the Center for Innovative Therapeutic Development for Common Diseases" (to Y.O.) from the Ministry of Education, Science, Sports, and Culture of Japan.

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 25, 2007

1 Y.H. and T.O. contributed equally to this work. Back

Abbreviations: BM, Bone marrow; BMT, BM transplantation; BrdU, bromodeoxyuridine; eNOS, endothelial nitric oxide synthase; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; GFP, green fluorescence protein; MMP, matrix metalloproteinase; PECAM, platelet endothelial cell adhesion molecule; sKitL, soluble kit-ligand; STZ, streptozotocin; WBC, white blood cell.

Received October 5, 2006.

Accepted for publication January 18, 2007.


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 Results
 Discussion
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