From the Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida
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ABSTRACT |
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Type 1 diabetes is an insulin-dependent, autoimmune disorder characterized by the destruction of insulin-producing ß-cells (1). Hence, a reversal of type 1 diabetes could be afforded by replacement of functional ß-cells. Unfortunately, islet transplantation has historically been hampered by immune rejection and/or a recurrent attack by underlying autoimmunity against islets, as well as the scarcity of donor islets (2,3). One theoretical alternative for islet transplantation would involve the use of a renewable source of stem cells capable of self-renewal and differentiation, as well as that of insulin production. Indeed, the development of a simple, reliable procedure to obtain autologous stem cells having the ability to differentiate into functional insulin-producing cells would provide a potentially unlimited source of islet cells for transplantation and alleviate the major limitations of availability and allogeneic rejection.
Recent studies have shown that bone marrow (BM)-derived stem (BMDS) cells have the ability to differentiate into a number of neuroectodermal, endothelial, mesenchymal, epithelial, and endodermal cell types (410). The ability of hepatic stem cells, such as oval cells and functional hepatocytes, to derive from BM cells has also been suggested in several in vivo (1113) and in vitro (10) studies. The observation that human BMDS cells can differentiate into mature hepatocytes (14,15) confirms the close interrelationship of BMDS cells and hepatocytes. Previously, we demonstrated (16) that highly purified rat hepatic oval cells can be induced to differentiate into functional insulin-producing cells when cultured long term in a high-glucose environment, that these differentiated oval cells express insulin, glucagon, and pancreatic polypeptide, and that they respond (i.e., produce insulin) to a high-glucose challenge. A key question that remained following those studies was whether BMDS cells could be induced to become functional insulin-producing cells. Because the pancreas and liver have common precursor cells during embryogenesis (17), stem cells of these two organs may have the same origin, that being from BM.
In this present study, we isolated murine BMDS (mBMDS) cells and obtained single-cellderived cell clones that were subsequently induced to transdifferentiate into insulin-producing cells under culture conditions containing high concentrations of glucose and the addition of ß-cellstimulating factors. The functionality of these cells was confirmed by insulin production and release in a glucose-responsive manner and by their reversal of hyperglycemia after being transplanted into mice rendered diabetic by treatment with streptozotocin (STZ). Taken together, our results indicate that under suitable conditions, mBMDS cells can be induced in vitro to differentiate into functional insulin-producing cells capable of normalizing hyperglycemia in a diabetic animal model. This study provides support for continuing efforts aimed at utilizing adult stem cells as a steady and renewable source of autologous insulin-producing cells for transplantation in patients with type 1 diabetes.
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RESEARCH DESIGN AND METHODS |
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Antibodies.
Rabbit anti-insulin polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA), guinea pig anti-insulin (Dako, Carpinteria, CA), rabbit anti-rat C-peptide antibody (Linco Research, St. Charles, MO), anti-rabbit IgG, guinea pig serum, and Cy3-coupled antiguinea pig IgG (RDI, Research Diagnostics, Flanders, NJ) were obtained and utilized as indicated and in accordance with the manufacturers recommendations. Antibodies directed against CD34, CD45, C-kit, and Sca-1 (BD Pharmingen, San Diego, CA) were used for the flow cytometric analysis.
Flow cytometric analysis.
The mBMDS cells at three to four passages were released by trypsinization. The cells were incubated with anti-mouse fluorescent dyelabeled hematopoietic antibodies, with 10,000 events acquired for analysis of fluorescence intensity, as previously described (18). Isotype-matched mouse immunoglobulins served as controls for autofluorescence.
In vitro differentiation cultures.
To induce the mBMDS cells to undergo pancreatic endocrine cell differentiation, the cloned cells were cultured (37°C, 5% CO2) in basic medium composed of RPMI 1640 medium (10% FCS) for 24 months in the presence of low (5.5 mmol/l) or high (23 mmol/l) concentrations of glucose. Cellular differentiation was monitored by observation of three-dimensional, islet-like cell cluster formation and by the expression of genes related to pancreatic ß-cell development and insulin production. To promote cellular maturation, the cells were cultured (37°C, 5% CO2) for 7 days in RPMI 1640 medium containing 5.5 mmol/l glucose, 5% FCS, and 10 mmol/l nicotinamide (Sigma, St. Louis, MO). The cells were then cultured for an additional 57 days in the presence of 10 nmol/l exendin 4 (Sigma).
Cell line culture.
The rat INS-1 cell line (clone 832/13), a cell line capable of insulin release in response of glucose stimulation, was a generous gift from Dr. Christopher B. Newgard (Duke University, Durham, NC). This cell line was derived from stable transfection of a plasmid containing the human proinsulin gene driven by a cytomegalovirus promoter and has the capacity to express and process both rat and human insulin. The cells were maintained in RPMI 1640 medium with 11.1 mmol/l D-glucose supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 10 mmol/l HEPES, 2 mmol/l L-glutamine, 1 mmol/l sodium pyruvate, and 50 µmol/l ß-mercaptoethanol at 37°C/5% CO2 in a humidified atmosphere (19). This cell line was used as a positive control for studies of insulin content and insulin release.
RT-PCR.
Total RNA was prepared from BMDS cell cultures maintained in low- or high-glucose culture for 4 months using TRIzol reagent. To eliminate genomic DNA contamination, mRNAs were purified using oligo-dT cellulose (Micro-FastTrack 2.0 Kit; Invitrogen, Carlsbad, CA) according to the manufacturers protocol. Transcriptional gene expression related to pancreatic endocrine development and function as well as other lineage markers (neuronal, intestine, and liver) from these cultures was determined by RT-PCR according to a published protocol (16) with minor modifications. The forward and reverse primers of each PCR set were designed to be located in different exons based on sequences obtained from GenBank to distinguish the PCR products from DNA contamination. Key PCR products of genes related to pancreatic development were confirmed by sequence analysis. The name and sequences of the primers, the sizes of PCR products, cycles, and annealing temperature for each pair are listed in Table 1.
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Immunocytochemistry and immunofluorescence.
Cytospin slides from differentiated mBMDS (D-mBMDS) cells were made for insulin and C-peptide protein expression. The cells were fixed with 4% formaldehyde for 30 min at room temperature, and immunocytochemistry performed with polyclonal guinea pig anti-insulin (1:500) (Dako) and guinea pig anti-rat C-peptide antibody (1:100) (Linco Research) for 1 h. After washing, the cells were incubated with Cy3-coupled anti-guinea pig (1:1,000) secondary antibodies (RDI) for 30 min. Guinea pig serum was used as a negative control. Cells were then examined by fluorescence microscopy (Olympus BX51).
Deconvolution microscopy.
Cells were stained with Cy3-conjugated secondary antibodies after they were incubated with antibodies specific for insulin or C-peptide. The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) and imaged by deconvolution microscopy using an Olympus OMT inverted fluorescent microscope system equipped with Delta Vision deconvolution analysis software. The images depict three-dimensional projections of 25 optical slices (each 0.2-µm thick) through the cell, with the center focused on the DAPI-stained chromatin in the nuclei. All images used in this report were scale adjusted, including images of staining with nonspecific isotype antibody conjugates as a negative control.
Mouse insulin enzyme-linked immunosorbent assay.
D-mBMDS cells were cultured (37°C, 5% CO2) in the presence or absence of exendin-4 for 7 days after 1 week of 10 mmol/l nicotinamide treatment in RPMI 1640 containing 5% fetal bovine serum and 5.5-mmol/l glucose. Following this, the cells were confirmed to express insulin genes by RT-PCR. In a parallel experiment, the cells were cultured in the presence of exendin 9-39 for 7 days. The cells were switched to serum-free medium containing 0.5% BSA for 12 h, washed twice with PBS, then stimulated by the addition of 23 mmol/l glucose for 2 h. The culture media was collected and frozen at 70°C until assay for insulin release. Importantly, serum-free culture medium containing 0.5% BSA was used as a control for secreted insulin measurements. Insulin release was detected by using an ultrasensitive mouse insulin enzyme-linked immunosorbent assay (ELISA) kit (Alpco Diagnostics, Windham, NH) following the manufacturers protocols. According to the manufacturers instructions, this assay does not detect proinsulin.
Electron microscopy with immunogold labeling.
For immunogold localization of insulin, the cells were embedded in Lowicryl K4M resin (EM Sciences, Fort Washington, PA). Ultrathin sections were blocked with 5% BSA/5% normal goat serum in PBS and then incubated overnight at 4°C in rabbit anti-insulin antibody (Santa Cruz Biotechnology) diluted 1:50 in PBS containing 0.2% BSA and 10 mmol/l NaN3. After washing, the samples were incubated for 1.5 h at room temperature with the secondary goat anti-rabbit IgG antibody conjugated to 0.8-nm colloidal gold particles (Aurion EM Grade Ultra Small, EM Sciences), washed, treated with 1.25% glutaraldehyde in PBS, and washed again. The gold particles were silver enhanced for 45 min at room temperature (Aurion R-Gent SE; EM Sciences). The samples were counterstained using uranyl acetate and lead citrate, then viewed using a Zeiss EM-10A transmission electron microscope.
Transplantation studies in mice.
Balb/c male mice received two intraperitoneal injections of STZ at 250 and 50 µg/g body wt, 3 days apart, according to published procedures (20,21) with minor modification. Blood glucose levels were monitored using an AccuChek glucose detector (Roche Diagnostics, Indianapolis, IN). Within 12 days of the first injection, all Balb/c mice became hyperglycemic, with blood glucose levels >350 mg/dl. The D-mBMDS cells (5 x 106/mouse) were transplanted into the left renal capsule and the distal tip of the spleen of six diabetic mice. Five diabetic mice received sham surgery without implants as a control. The nonfasting blood glucose levels were monitored at 1600 every 2 days following transplantation. Most of the diabetic mice with sham surgery died between 15 and 20 days because they did not receive insulin treatment. The diabetic mice with D-mBMDS cell transplants were killed 26 days after transplantation. The pancreas tissue was harvested for morphologic analysis. For the intraperitoneal glucose tolerance (IPGT) test, normal nondiabetic Balb/c male mice (n = 5) and diabetic mice (n = 3) with normalized glucose levels following the D-mBMDS cell transplantation received intraperitoneal injections of glucose (2 mg/g body wt) according to published procedures (20). Blood glucose levels were monitored at 0, 30, 60, 90, 120, and 150 min for each mouse.
Measurement of apoptosis.
The mBMDS cells were cultured in medium containing 23 mmol/l glucose for various times including 1 week, 2 weeks, 1 month, and 2 months. Cells were passaged when they reached 8090% confluency. Cultured mBMDS cells in the medium containing a 5.5-mmol/l glucose concentration served as a baseline control for no treatment. Cells were released from culture dishes by trypsinization and incubated in the same medium for an additional 2 h in suspension at 37°C. The cells them were then subjected to testing for apoptosis using an Annexin V-PE apoptosis detection kit I (BD Biosciences Pharmingen) following the manufacturers protocol.
Statistics.
Evidence of statistical significance was determined by Fishers exact testing. A P value of <0.05 was deemed significant.
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RESULTS |
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Gene expression of mBMDS and D-mBMDS cells.
To determine whether the mBMDS cells had undergone pancreatic differentiation, gene expression profiles for pancreatic ß-cell differentiation markers and hormones were assessed using RT-PCR. Four of the six mBMDS clones underwent pancreatic endocrine differentiation as evidenced by expression of Pdx-1 and insulin genes. As illustrated in Fig. 2, cells cultured under high-glucose concentrations (23 mmol/l) for 4 months expressed multiple genes characteristic of endocrine ß-cell development, including insulin I and II, Glut-2, glucose kinase, islet amyloid polypeptide, nestin, Pdx-1, and Pax6. However, gene expression of Pax4, NeuroD, and islet-1 (Fig. 2, upper panel) was not detected. The Oct-4 gene, typical for pluripotent embryonic stem cells, was not detected in mBMDS or D-mBMDS cells. In contrast, mBMDS cells cultured under a low-glucose concentration (5.5 mmol/l) for 4 months expressed no detectable levels of the aforementioned genes with the exception of nestin (Fig. 2, lower panel). Further differentiated mBMDS cells (late stage), similar to the mouse ß-cell line (ß-TC) expressed the glucagon-like peptide (GLP)-1 receptor gene (Fig. 3D), yet this gene expression was not observed in undifferentiated mBMDS cells or in early stages of the D-mBMDS cells (data not shown).
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To confirm that the D-mBMDS cells are indeed derived from Balb/c mice and that there is no cross-contamination from different cell lines or species, we analyzed cellular DNA for five mouse microsatellite molecular markers: D2Mit30, D3Mit15, D6Mit15, D11Mit4, and D2Nds3. Tissues from Balb/c mice as well as rat and human were obtained for these analyses. Genomic DNA from Balb/c mice served as a positive control in these analyses. These mouse microsatellite markers have the ability to distinguish tissues among different species (mouse, rat, and human), but in addition, can also be used to distinguish different strains within the same mouse species (i.e., NOD/MrKTac, Balb/cJ, C57BL/6J CAST, etc.) due to polymorphisms at a particular site of the host chromosome. The PCR results assigning the specific polymorphism (Fig. 4A) showed that the sizes of the PCR products of the five markers (D2Mit30, D3Mit15, D6Mit15, D11Mit4, and D2Nds3) were precisely located at 136, 143, 195, 242, and 400 bp, respectively, and that the pattern was identical to that of a Balb/c mouse (Fig. 4B, left panels). There was no cross-contamination among species present (Fig. 4B).
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Insulin content and release in response to glucose stimulation.
To determine whether the D-mBMDS cells were responsive to a glucose challenge, insulin release from undifferentiated and D-mBMDS cells was measured using an ultrasensitive mouse insulin ELISA. In order to enhance the sensitivity of these cells to high-glucose challenge, the differentiated cells were switched to low-serum, low-glucose medium plus nicotinamide for 1 week. Since the GLP-1R gene was expressed in the latter stages of D-mBMDS cells, as shown in Fig. 3D, we then cultured cells in the presence of either exendin 4, or its antagonist, exendin 9-39, for 7 days before analysis. The cells were then washed twice with PBS and switched to serum-free low-glucose medium containing 0.5% BSA (overnight incubation), then stimulated by the addition of 23 mmol/l glucose for 2 h. The cell culture media was then collected for the analysis. All studies were performed in triplicate. The results shown in Fig. 6 indicate that approximately fourfold more insulin was released in exendin 4treated cells in comparison to the cells treated with exendin 9-39 under long-term high-glucose conditions. In contrast, control mBMDS cells cultured in low concentrations of glucose (5.5 mmol/l) showed no significant release of insulin in the presence or absence of glucose challenge, even in the presence of exendin 4. These data suggest that high-glucose culture plays an indispensable role in the transdifferentiation of BMDS cells into insulin-producing cells and that differentiated BMDS cells were responsive to glucose challenge. Moreover, the results also indicated that the D-mBMDS cells might represent a precursor to ß-like cells and that further induction might be needed to reach a high degree of differentiation and maturation, as would be observed in the in vivo hyperglycemic environment of diabetic animals.
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DISCUSSION |
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To overcome these limitations, we explored the possibility of using human and mouse BMDS cells as sources for transdifferentiation into insulin-producing cells under specific in vitro culture conditions. BM has been known for years to represent a safe and abundant source for large quantities of adult stem cells. In the present study, we isolated, cloned, and characterized mBMDS cells. We also generated functional insulin-producing cells from the mBMDS cells under an in vitro differentiation procedure and confirmed the presence of insulin production by RT-PCR, immunofluorescence, and electron microscopy combined with gold anti-insulin labeling. Furthermore, we tested the functionality of the in vitrogenerated insulin-producing cells from mouse BM by measuring insulin release in response to a glucose challenge and by demonstrating a reversal of diabetes upon subsequent implantation of these cells into diabetic mice. In addition to these studies, we have derived islet-like, functional insulin-producing cells from human BMDS cells (D.-Q.T., B.R.B., L.-Z.C., S.A.L., M.A.A., L.-J.Y., unpublished data). Taken collectively, these studies provide direct evidence that the BM contains pluripotent cells capable of being reprogrammed in vitro to become functional insulin-producing cells.
Several in vivo studies (2830) demonstrate that BM cells contribute to pancreatic ß-cell regeneration at a low frequency, ranging from 1 to 2.7%. Ianus et al. (29) provided in vivo evidence of adult mouse BM harboring cells that could transdifferentiate into glucose-competent pancreatic endocrine cells using a cAMP response elementLoxP system, as assessed in cross-sex BM transplant experiments. The above findings indicate that this in vivo process is likely due to transdifferentiation of BM-derived cells into insulin-producing ß-cells, rather cell fusion, as the main source of BM-derived hepatocytes repopulating the liver of the mice, as was the case with fumarylacetoacetate hydrolase deficiency, suggested by other studies (15,31). In our current study, the homogeneous mBMDS cells were used to induce in vitro differentiation into insulin-producing cells; hence, cell fusion is likely not the answer for the presence of competent insulin-producing cells. Other studies confirm that allogeneic BM transplantation with as low as 1% chimerism in pancreatic islets can reverse the diabetogenic process in pre-diabetic mice (28). Hess et al. (30) have provided a theory based on their observations in a recent study to explain the possible mechanism of reversing hyperglycemia in diabetic mice after BM transplantation. They suggest that pancreatic engraftment of donor BMderived cells expressing endothelial markers after BM transplantation initiate endogenous ß-cell regeneration, whereas donor BMderived insulin-positive ß-cells represent a rare event. Kojima et al. (32) recently found extrapancreatic proinsulin-producing cells present in the liver, BM, spleen, adipose tissue, and thymus in hyperglycemic animals and that the majority of these proinsulin-producing cells were derived from the donor BM, as evidenced by BM transplantation experiments. These studies support our observation that BM contains stem cells capable of differentiation into insulin-producing cells.
One of the key questions to address is which cell type in the BM is responsible for pancreatic endocrine transdifferentiation. Unfortunately, it is difficult to extrapolate the cell phenotype from currently available studies using an in vivo approach. Our results from both humans and mice suggest that CD45-negative adherent pluripotent mesenchymal stem cells are capable of transdifferentiation into insulin-producing cells in vitro under high-glucose culture conditions. The common phenotype of the BMDS cells between humans and mice is CD45 negative, CD34 negative, and C-kit negative, indicating that they are unlikely to be hematopoietic stem cells. However, the possibility of circulating pancreatic stem cells cannot be completely excluded. A recent study published by Kodama et al. (33) indicated that injection of splenocytes into pre-diabetic NOD mice reversed diabetes and promoted pancreatic ß-cell regeneration. Their results further indicated that CD45-negative splenocytes (presumably mesenchymal precursor cells) were responsible for islet ß-cell regeneration. This result indirectly supports our conclusion that the BM-derived stem cells capable of generating islet precursor cells have an immunophenotype and biologic characteristics similar to those of BM mesenchymal cells.
There are two key steps in our cell culture conditions that appear important for inducing the differentiation of BMDS cells into insulin-producing islet-like cells. First, the mBMDS cells initially require culture in medium containing a high-glucose concentration (23 mmol/l) for various durations of time until certain genes, such as Pdx-1, insulin I and II, Glut-2, and islet amyloid polypeptide, become detectable. Second, in order for the D-mBMDS cells to become glucose responsive, further differentiation and maturation are required through either in vitro culture with ß-cellpromoting factors, such as nicotinamide and exendin 4, or transplantation of the cells into diabetic animals. In this study, we demonstrated that mBMDS cells cultured under low-glucose conditions did not express the aforementioned genes, and in addition, they did not secrete insulin upon glucose stimulation, even in the 7-day presence of the ß-cellstimulating factors exendin 4 and nicotinamide. Our data indicate that long-term culture in a high-glucose medium reprogrammed these cells toward a pathway of pancreatic endocrine cell differentiation. At a certain period of time (24 months) and via a still unclear mechanism, this switch occurred.
It is well known that glucose is a growth factor for ß-cells (34). It promotes ß-cell replication in vitro and in vivo at a 20- to 30-mmol/l concentration (35) and increases insulin content in cell lines derived from embryonic stem cells (20) at a 5-mmol/l concentration. The effect of chronic hyperglycemia on pancreatic ß-cells, however, remains controversial. In an in vivo study, Jonas et al. (36) showed that the expression of several genes important for glucose-stimulated insulin secretion (glucose metabolism enzymes and ion channels/pumps) was gradually decreased with increasing levels of blood glucose. They also suggested a link between stimulation of ß-cell growth and a reduced state of differentiation in hyperglycemic animals. However, these observations were primarily focused on pancreatic ß-cells. The effects of long-term high-glucose culture on stem cells (adult or embryonic) were unclear. In a previous study, we demonstrated that a long-term culture of purified hepatic oval stem cells in high-glucose (23 mmol/l) medium promoted the oval cells to transdifferentiate into functional insulin-secreting cells (16). In addition, we have observed that overexpression of Pdx-1 in a hepatic stem cell line (WB cells) only results in the generation of pancreatic precursor cells (unpublished observations). These cells did not respond to a glucose challenge in vitro by releasing insulin. Rather, these precursor cells became fully functional under two conditions: one involving long-term culture in high-glucose medium and the other being the transplantation of these cells into diabetic (i.e., hyperglycemic) animals.
The notion that in vitro high-glucose culture (or in vivo hyperglycemia) represents a critical factor for adult stem cell transdifferentiation into insulin-producing cells has been supported by recent two publications. Zalzman et al. (37) demonstrated that culture of immortalized human fetal Pdx-1expressing hepatocytes in media containing 25 mmol/l glucose activated multiple ß-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner. In another work, Kojima et al. (32) showed that it was hyperglycemia produced by a 25% glucose injection into nondiabetic mice as well as in three other types of hyperglycemic animal models that led to the appearance of proinsulin-positive cells within 3 days in the liver, fat, spleen, BM, and thymus, as well as insulin-positive cells within 15 days in those organs. These studies support our observation that both liver and BM-containing stem cells can be induced under high-glucose conditions to differentiate into insulin-producing cells, and that insulin-producing cells can be derived from liver and BM cells. A sharp difference between our in vitro observations (in months) and those of the in vivo works of Kojima et al. (in days) (32) most likely resides in the required duration of exposure in terms of the need for high-glucose conditions to generate insulin-producing cells. Possible explanations may include that 1) in vivo three-dimensional structure and cell-cell contact and interaction may play a vital role in promoting cell differentiation, 2) other soluble factors in addition to high glucose in vivo may also play a role in accelerating cell differentiation, and 3) it takes a long time for a single-cellderived cell clone to form three-dimensional cell clusters under high-glucose culture conditions. The above theory is supported by our observations involving our detection of an increase in insulin in culture medium taken from 3-week cultures of whole-marrow adherent cells under high-glucose conditions.
One possible explanation of the constant viable cells in the high-glucose cultures is that cycle events occur between immature stem-like cells and D-mBMDS cells. The immature stem-like cells, which may be more resistant to high-glucose culture conditions, give rise to the D-mBMDS cells, which may be more susceptible to high-glucose-induced apoptosis. These early apoptotic cells were removed following either medium change or cell splits. This assumption helps us explain the strange behavior of the cells we have repeatedly observed, namely the loss of pancreatic endocrine gene expression and insulin production when these cells were quickly expanded to a large quantity for cell transplantation experiments. It has been proposed that rapid growth will reduce cell differentiation. In our system, rapid proliferation may be only a part of the story for the loss of cell differentiation, the other facet being related to the high-glucoseinduced cell apoptosis of the D-mBMDS cells and the subsequent loss of the differentiated cells. The latter may explain the variations between experiments and the loss in expression of genes related to pancreatic endocrine differentiation. Second, our experience with transdifferentiation of the human BMDS cells also indicated the requirement for subsequent culture with maturation factors, such as exendin-4 and nicotinamide, in a medium containing low FCS and low glucose to promote cell maturation and to restore the sensitivity to a glucose challenge (D.-Q.T., L.-Z.C., S.A.L., M.A.A., L.-J.Y., unpublished results). GLP-1 is an incretin hormone capable of restoring normal glucose tolerance in aging glucose-intolerant Wistar rats and inducing differentiation of islet Pdx-1positive ductal cells into insulin-secreting cells (38). GLP-1 stimulates insulin secretion and augments ß-cell mass via activation of ß-cell proliferation and islet neogenesis (13). A recent study by Suzuki et al. (25) demonstrated that GLP-1 converts intestinal epithelial cells into functional insulin-producing cells. Exendin-4 is a potent GLP-1 agonist that has previously been shown to stimulate both ß-cell replication and neogenesis from ductal progenitor cells (39). We have shown that the late stage of D-mBMDS cells expressed the GLP-1 receptor gene and that this expression may correlate with glucose-responsive insulin release.
Nicotinamide is a poly(ADP-ribose) synthetase inhibitor known to differentiate and increase cell mass in cultured human fetal pancreatic cells (40) and to protect cells from desensitization induced by prolonged exposure to large amounts of glucose. Sjoholm, Korsgren, and Andersson (41) demonstrated that nicotinamide promoted formation of fetal porcine islet-like cell clusters and increased the rates of proinsulin biosynthesis in these clusters. They concluded that the stimulatory effects of nicotinamide on insulin production and content by fetal porcine islet-like cell clusters result from neoformation of ß-cells through differentiation. Finally, a report by Ramiya et al. (24) described how nicotinamide-treated islets derived from the pancreatic progenitor cell had more insulin and secreted significantly more insulin than cultures treated with glucose alone. Our previously published study (16) showed that nicotinamide promotes in vitro transdifferentiation and maturation of the liver stem cells into insulin-producing cells. Taken together, a combination of exendin 4 and nicotinamide effectively promotes further D-mBMDS cell differentiation in our experimental system.
Our present study demonstrates the potential for cell-based therapy of diabetes involving the generation of autologous insulin-producing cells in vitro from BMDS cells. These in vitrogenerated insulin-producing cells could, in theory, provide a potentially unlimited source of islet-like cells without the limitation of immune rejection based on alloimmunity. However, because there are multifactorial influences in the transdifferentiation of BM-derived stem cells into competent insulin-producing cells, there are many questions left unanswered and unresolved issues remain. Among those are answers to the questions of what are the decisive steps (e.g., addition of the glucose, exogenous factors, and timing of factor addition) for the transdifferentiation process to take place? In our experience, these differentiated cells are unlike ß-cellderived cell lines, such as ß-TC and INS-1 cells, in terms of their gene expression profiles, cell maturity, and capacity to release insulin in response of glucose stimulation (data not shown). Hence, one can also question whether these cells can really be pushed to the level of maturity like true ß-cells by changing the in vitro culture conditions. Another relevant clinical question involves the issue of autoimmunity. Will the immune response to ß-cell antigens recognize and destroy the newly generated insulin-producing cells obtained from BMDS cells? Obviously, further research is required to address these important questions. Yet we believe the results demonstrated in this study provide direct evidence supporting the notion that transdifferentiation of adult stem cells to insulin-producing cells may represent a viable therapeutic option for type 1 diabetes.
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ACKNOWLEDGMENTS |
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We thank Dr. Jill Verlander Reed and Kim Ahren for technical assistance.
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FOOTNOTES |
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Address correspondence and reprint requests to Dr. Li-Jun Yang, 1600 SW Archer Rd., P.O. Box 100275, Gainesville, FL 32610. E-mail: yanglj{at}pathology.ufl.edu
Received for publication October 2, 2003 and accepted in revised form March 25, 2004
BM, bone marrow; DAPI, 4',6-diamidino-2-phenylindole; D-mBMDS, differentiated murine BM-derived stem cells; ELISA, enzyme-linked immunosorbent assay; GLP, glucagon-like peptide; IPGT, intraperitoneal glucose tolerance; mBMDS, murine BM-derived stem cells; Pdx, pancreatic duodenal homeobox; STZ, streptozotocin
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REFERENCES |
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