Little evidence of transdifferentiation of bone marrow-derived cells into pancreatic beta cells
Bone marrow cells contain at least two distinct types of stem cells which are haematopoietic stem cells and mesenchymal stem cells. Both cells have the ability to differentiate into a variety of cell types derived from all three germ layers. Thus, bone marrow stem cells could possibly be used to generate new pancreatic beta cells for the treatment of diabetes. In this study, we investigated the feasibility of bone marrow-derived cells to differentiate into beta cells in pancreas.
Using green fluorescent protein transgenic mice as donors, the distribution of haematogenous cells in the pancreas was studied after bone marrow transplantation.
In the pancreas of green fluorescent protein chimeric mice, green fluorescent protein-positive cells were found in the islets, but none of these cells expressed insulin. Previous data has suggested that tissue injury can recruit haematopoietic stem cells or their progeny to a non-haematopietic cell fate. Therefore, low-dose streptozotocin (30 or 50 mg/kg on five consecutive days) was injected into the mice 5 weeks after bone marrow transplantation, but no green fluorescent protein-positive cells expressing insulin were seen in the islets or around the ducts of the pancreas.
Our data suggests that bone marrow-derived cells are a distinct cell population from islet cells and that transdifferentiation from bone marrow-derived cells to pancreatic beta cells is rarely observed.
KeywordsRegeneration regeneration therapy insulin islet stem cell haematopoietic stem cell mescenchymal stem cell bone marrow
enhanced green fluorescent protein
von Willebrand Factor
green fluorescent protein
Intraperitoneal glucose tolerance test
The pancreas is composed of exocrine and endocrine compartments. The endocrine compartment consists of the islets of Langerhans, which contain clusters of four cell types: glucagon-producing alpha cells, somatostatin-producing delta cells, pancreatic polypeptide-producing PP cells, and insulin-producing beta cells. An inadequate mass of functional pancreatic beta cells is found in both Type 1 and Type 2 diabetes. Thus, beta-cell replacement therapy is thought to be a possible curative treatment for diabetes. In order to make beta-cell replacement therapy more widely available, since islet transplantation is currently the only method of beta-cell replacement therapy, new sources of beta cells need to be explored.
Each tissue or organ is believed to contain a small sub-population of cells that is capable of self-renewal and has the ability to give rise to each mature cell type . Thus, one of the most promising sources of beta cells might be pancreatic stem cells. While several studies have shown the existence of pancreatic stem cells [2, 3, 4, 5], these cells have not yet been isolated from the pancreas in a pure form.
Cells derived from bone marrow have been used to replace haematopoietic stem cells in the treatment of various haematological malignancies. These cells include both haematopoietic stem cells and mesenchymal stem cells, which can differentiate into various types of cells derived from the mesenchyme . Recent studies using methods for tracking cell lineage have allowed us to identify the differentiation of bone marrow stem cells and mescenchymal stem cells into specific cells residing in various tissues [7, 8, 9]. Isolated haematopoietic stem cells might contribute to the epithelium of multiple organs of endodermal and ectodermal origin [10, 11, 12]. In contrast, mesenchymal stem cells can have a wider range of fates including transformation into endothelial, myogenic, hepatic, and neural cells .
Several lines of evidence suggest that tissue injury enhances the recruitment of haematopoietic stem cells, mescenchymal stem cells, or their progeny towards a non-haematopoietic fate [11, 14, 15, 16]. This might be because migration of bone marrow stem cells throughout the body essentially act as a back-up system to augment each organ's intrinsic regenerative capacity.
Streptozotocin (STZ) induces diabetes by specific destruction of pancreatic beta cells. Multiple subdiabetogenic doses of STZ provoke different changes in the pancreas from a single injection of a diabetogenic dose of STZ [17, 18, 19] and enable us to observe the re-organization of pancreatic beta cells . Thus, multiple low doses of STZ are considered to create a good model for observing regeneration after beta-cell-specific injury. However, as far as we know, there has been no detailed investigation of whether bone marrow stem cells can differentiate into pancreatic beta cells after tissue injury.
Achieving the reconstitution of pancreatic beta cells by use of bone marrow-derived cells suggests that bone marrow cells are a feasible source for beta-cell replacement therapy. To elucidate the potential of bone marrow-derived cells for use as beta-cell replacement therapy, we established chimeric mice with the bone marrow of transgenic mice constitutively expressing enhanced green fluorescent protein (EGFP) [16, 21, 22, 23]. As we showed previously, the bone marrow of these chimeric mice is almost completely reconstituted with EGFP-positive cells 5 weeks after transplantation . These chimeric mice allow us to track the progeny of transplanted bone marrow cells, by using green fluorescence as a marker.
Materials and methods
Among the primary antibodies, rabbit anti-Pdx1 antibody was generated as described previously , while mouse anti-nestin antibody was kindly provided by Dr. S. Hockfield (Yale University, New Haven, Conn., USA). All other antibodies used for this study were purchased from commercial sources: guinea-pig (GP) anti-human insulin antibody (Linco, St. Charles, Mo., USA), rabbit anti-glucagon antibody (Dako, Carpinteria, Calif., USA), anti-CD45 antibody (Pharmingen, Franklin Lakes, N.J., USA), rabbit anti-Factor VIII related antigen/von Willebrand Factor (vWF) (Dako, Glostrup, Denmark), rat anti-bromodeoxyuridine (BrdU)(OBT, Oxford, UK), mouse anti-green fluorescent protein (GFP) antibody (Sigma-Aldrich, St. Louis, Mo., USA), and mouse anti-E-cadherin antibody (BD Bioscience, Flanklin Lakes, N.J., USA). All of the secondary antibodies came from commercial sources: biotinylated goat anti-rabbit IgG (Vector, Burlingame, Calif., USA), biotinylated goat anti-mouse IgG (Biomeda, Foster, Calif., USA), biotinylated goat anti-rat IgG (Cedarlane, Ontario, Canada), goat anti-GP IgG conjugated Texas-red (Cortex, Sanleandro, Calif., USA), and goat anti-mouse IgG conjugated with Alexa 568 (Molecular Probes, Eugene, Ore., USA)
Animals and creation of GFP chimeric mice
Male C57BL/6 mice (18–20 g) were purchased from Japan SLC at 8 weeks of age and maintained in the animal facility of Juntendo University School of Medicine. The animals were allowed free access to a standard laboratory diet and water. GFP transgenic mice, in which EGFP expression was under the control of the cytomegalovirus enhancer and the chicken β-actin promoter, were generously provided by Dr. M. Okabe (Osaka University, Osaka, Japan) . Using these transgenic mice, we generated GFP chimeric mice [16, 22], in brief, bone marrow transplantation was carried out with 8-week-old C57BL/6,GFP mice as the donors and 8-week-old C57BL/6 mice as the recipients. Donor bone marrow cells were obtained from the femurs and tibias of GFP mice after the injection of 5-fluorouracil (150 mg/kg) at 48 h before transplantation, and were injected into the tail veins of irradiated (5 Gy×2) non-transgenic recipient mice. All the experiments were conducted in accordance with the rules and regulations of the animal committee of Juntendo University School of Medicine.
Induction of diabetes with STZ
At 5 weeks after transplantation, mice were injected intraperitoneally with a low dose of STZ (30 mg/kg  or 50 mg/kg  body weight) dissolved in citrate buffer (pH 4.5). Injections were given daily for five consecutive days.
Glucose tolerance test
An intraperitoneal glucose tolerance test (IPGTT) was carried out before STZ injection and at 3 and 5 weeks after STZ injection. After an overnight fast, mice were injected intraperitoneally with glucose (1.0 g/kg body weight). Blood samples were taken from the tail vein at 0, 30, 60, 120, and 180 min and the plasma glucose concentration was measured with a Freestyle glucose meter (Kissei Corporation, Nagano, Japan).
Preparation of pancreatic sections and immunohistochemistry
Before and at 1, 3, and 5 weeks after STZ injection, mice were thoroughly perfused with PBS followed by 4% paraformaldehyde under pentobarbital sodium anesthesia. The pancreas was removed and fixed with 4% paraformaldehyde for 2 days at 4°C. After then, tissues were placed in 30% sucrose in PBS for 1 day at 4°C and embedded in OCT compound for 2 days at 4°C. After being frozen on dry ice, the tissues were stored at −80°C until examination. Cryostat sections (4 µm thick) were cut at −20°C and dried on slides overnight at room temperature. The sections were blocked with 10% goat serum for 30 min at room temperature, and then incubated with each primary antibody overnight at 4°C. The primary antibodies were diluted to the following dilutions in 2% goat serum: 1:104, for rabbit anti-Glucagon, GP anti-human insulin and rabbit anti-Pdx1; 1:106, for mouse anti-Nestin and rabbit anti-vWF; 1:105, for rat anti-mouse CD45; 1:4000, for mouse anti-GFP; 1:2500, for mouse anti E-cadherin and 1:200 for rat anti-BrdU. After being washed with PBS three times, the sections were incubated with the appropriate secondary antibodies for 30 min at room temperature. For detection of Glucagon, Pdx-1, Nestin, vWF, CD45, BrdU, the streptavidin-biotin complex method was used, so biotinylated goat anti-rabbit IgG (1:200), biotinylated goat anti-mouse IgG (1:200), or biotinylated goat anti-rat IgG (1:200) was used as the secondary antibody respectively. For the detection of insulin, goat anti-GP IgG conjugated with Texas-Red (1:200) was used as the secondary antibody. For the detection of GFP and E-cadherin, goat anti-mouse IgG conjugated with Alexa 568 (1:200) was used as the secondary antibody. Except in the case of staining for insulin, GFP, and E-cadherin after being washed twice with PBS, the sections were incubated with streptavidin (1:200, conjugated Alexa 594, Molecular Probes, Eugene, Ore., USA) for 30 min at room temperature. Fluorescence from these samples was observed using a Zeiss Axioskop 2 plus microscope (Carl Zeiss, Jena, Germany), and digital images were captured using Axiovision 3.0 software. For the investigation of co-staining with GFP and insulin, we examined 50 to 100 pancreatic sections that contained tissue from the head to the tail and were selected from six pancreata in each of the control and STZ-treated groups. For the detection of other proteins, we examined 3 to 7 sections per protein from each of six mice.
To identify the proliferating cells in the pancreas, injection of BrdU was carried out according to the previous protocol [27, 28] with some modification. Briefly, mice were injected intraperitoneally with BrdU (100 mg/kg: Sigma-Aldrich, dissolved in 0.007 N NaOH in PBS) at 20, 16, 6, and 2 h before being killed. Cryostat sections for immunohistochemical analysis of BrdU underwent the following treatment before incubation with the primary antibody: incubation with 2 N HCl for 30 min at 37°C, incubation with 0.1 mol di-sodium tetraborate for 10 min at room temperature, washing with PBS, and rinsing in 50 mmol/l Tris-bufferred saline (pH 7.6) twice for 5 min each.
After overnight incubation with the primary antibody at 4°C, the sections were incubated with biotinylated goat anti-rat IgG (1:100) as the secondary antibody for 30 min at room temperature. After washing three times with PBS, the sections were incubated with streptavidin conjugated with horseradish peroxidase (Dako, Carpenteria, Calif., USA) for 30 min at room temperature. Positive reactions were visualized by incubation with the peroxidase substrate solution containing 3,3'-diaminobenzidine tetrahydrochloride after washing three times with PBS, and the mean number of BrdU-positive cells per islet was calculated from 70 to 100 islets in each group.
Differences between groups were analyzed using Student's t test with correction for different variance whenever appropriate. A p value of less than 0.05 was considered statistically significant.
Characterization of the fate of transplanted bone marrow cells in the pancreas
To explore the possibility of transdifferentiation from bone marrow-derived cells into pancreatic endocrine cells, we investigated the expression of insulin and glucagon in GFP-positive cells. We investigated 50 to 100 pancreatic sections from each of six mice which were stained with insulin antibodies. We carefully selected sections from each pancreas that should contain tissue from the pancreatic head to the pancreatic tail. However, we could not find any insulin or glucagon expression in GFP-positive cells (Fig. 2C,D). Furthermore, we could not identify any GFP-positive cells expressing E-cadherin, which is a marker of endodermal epithelial cells (Fig. 2E). This suggested that almost all of the GFP-positive cells in the islets might be mesenchymal cells .
Previous studies have reported transdifferentiation events between different types of somatic stem cells. In the pancreas, no obvious stem cells have been identified so far. If the stem cells from transplanted bone marrow underwent differentiation into islet stem cells, it might be possible to observe GFP-positive cells expressing nestin within the islets. However, we could not find any GFP-positive cells in islets that also expressed nestin (Fig. 2F).
Pdx1 is a transcription factor involved in pancreatic development, its expression is mainly observed in pancreatic beta cells and occasionally in exocrine cells and duct cells in the adult pancreas. We could not find any GFP-positive cells expressing Pdx1 in the islets in agreement with the lack of GFP-positive cells expressing insulin (Fig. 2G). In contrast, there were a few GFP-positive cells expressing Pdx1 outside the islets (Fig. 2G,H). These findings suggested that stem cells derived from transplanted bone marrow could differentiate into pancreatic cells.
The lineage of bone marrow-derived cells after destruction of pancreatic beta cells
In this study, we focused on the fate of transplanted bone marrow cells in the pancreas. At 5 weeks after bone marrow transplantation, we could not find any cells derived from the bone marrow expressing insulin despite intensive observation.
Several groups have already generated chimeric mice or carried out transplantation using isolated haematopoietic stem cells or mescenchymal stem cells and have tried to identify the cells derived from bone marrow in various tissues and found that bone marrow-derived stem cells differentiated into various tissue specific cells [12, 13]. During the preparation of this manuscript, another study showed that bone marrow-derived cells differentiated into insulin expressing cells . This report showed that bone marrow cells could differentiate into beta cells. Through a genetic approach, the authors ruled out cell fusion as the mechanism for EGFP-positive cells with islet-like characteristics. If this is the case, why did we not find transdifferentiation into pancreatic beta cells? A recent report contradicted an earlier study  and suggested that neural stem cells rarely transdifferentiated into blood cells . Another study reported that a single haematopoietic stem cell robustly reconstituted peripheral blood leukocytes, but did not contribute appreciably to non-haematopoietic tissues, including the brain, kidney, gut, liver, and muscle. Furthermore, they used parabolic mice to enable the massive transfer of haematopoietic stem cells, but few transdifferentiational events were observed . We cannot point out the solid reason for these conflicts. Only the difference of experimental condition could explain the conflicting results. Thus, in this paper, we would like to emphasize that under the experimental condition described here, we could not show the contribution of bone marrow-derived cells to differentiate into islet cells.
Previous studies have shown that tissue injury enhances the recruitment of haematopietic stem cells, mescenchymal stem cells or their progeny to non-haematopoietic fate [11, 14, 15, 16]. Intravenous injection of adult bone marrow cells in FAH(-/-) mice, an animal model of tyrosinemia type I, rescued these animals and restored the biochemical functions of the liver. In these mice, surprisingly, as few as 50 highly purified haematopoietic stem cells could be induced to generate large colonies of functional hepatocytes . In our study, using mice treated with low-dose STZ as a model of selective pancreatic beta-cell injury, we explored the transformation of cells derived from the bone marrow to insulin-positive cells. In the pancreas, we observed an increase of cell proliferation in islet cells and a few insulin-positive cells surrounding the pancreatic ducts, but we could not find any insulin-expressing cells derived from bone marrow cells.
We used low-dose STZ as a tissue injury model. However, several other models of pancreatic injury, such as partial pancreatectomy  or cellophane wrapping , have been established. Each model might involve a different mechanism of regeneration of the pancreas, so, we cannot deny the possibility that transdifferentiation into pancreatic beta cells might be observed in a different model of tissue injury.
In conclusion, with regard to beta-cell replacement therapy for diabetes, our data suggests that bone marrow-derived stem cells cannot differentiate into beta cells automatically. However, our data does not deny the feasibility of bone marrow-derived cells as a host cells for beta-cell replacement therapy. Recently several lines of evidence have suggested that the expression of Pdx1 into various cells can provoke differentiation into cells similar to pancreatic beta-cells [39, 40, 41, 42], so introducing a beta-cell differentiation factor like Pdx1 into bone marrow-derived stem cells seems to be worth investigating.
We would like to thank to M. Okabe (Osaka University) for GFP transgenic mice, S. Hockfield (Yale University) for mouse anti-nestin antibody, and also Mrs. N. Daimaru for excellent technical assistance. This study was supported in part by grants from the Juvenile Diabetes Research Foundation International (to H.W.), the Ministry of Education, Culture, Sports, and Technology of Japan (to H.W.), Takeda Medical Research Foundation (to H.W.) and a High Technology Research Center Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H.M.).
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