Original Paper Accepted after revision: January 6, 2014 Published online: March 27, 2014
Cells Tissues Organs DOI: 10.1159/000358383
Transplantation of Bone Marrow Mesenchymal Stem Cells for the Treatment of Type 2 Diabetes in a Macaque Model Xing-hua Pan a Qiao-qiao Song b Jie-jie Dai c Xiang Yao a Jin-xiang Wang a Rong-qing Pang a Jie He a Zi-an Li a Xiao-mei Sun c Guang-ping Ruan a a
Stem Cell Engineering Laboratory of Yunnan Province, Kunming General Hospital of Chengdu Military Command, Clinical College of Kunming General Hospital of Chengdu Military Command, Kunming Medical University, and c The Institute of Medical Biology, The Chinese Academy of Medical Science and Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Center of Tree Shrew Germplasm Resources, Kunming, China b
Abstract Bone marrow mesenchymal stem cells (BMSCs) are self-renewing, multipotent cells that can migrate to pathological sites and thereby provide a new treatment in diabetic animals. Superparamagnetic iron oxide/4′,6-diamidino-2phenylindole (DAPI) double-labeled BMSCs were transplanted into the pancreatic artery of macaques to treat type 2 diabetes mellitus (T2DM). The treatment efficiency of BMSCs was also evaluated. After successful induction of the T2DM model, the treatment group received double-labeled BMSCs via the pancreatic artery. Six weeks after BMSC transplantation, the fasting blood glucose and blood lipid levels measured in the treatment group were significantly lower (p < 0.05) than in the model group, although they were not reduced to normal levels (p < 0.05). Additionally, the serum Cpeptide levels were significantly increased (p < 0.05). An intravenous glucose tolerance test and C-peptide release test had significant changes to the area under the curve. Within 14 days of the transplantation of labeled cells, the pancre-
© 2014 S. Karger AG, Basel 1422–6405/14/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/cto
atic and kidney tissue of the treatment group emitted a negative signal that was visible on magnetic resonance imaging (MRI). Six weeks after transplantation, DAPI signals appeared in the pancreatic and kidney tissue, which indicates that the BMSCs were mainly distributed in damaged tissue. Labeled stem cells can be used to track migration and distribution in vivo by MRI. In conclusion, the transplantation of BMSCs for the treatment of T2DM is safe and effective. © 2014 S. Karger AG, Basel
Introduction
Type 2 diabetes mellitus (T2DM) is a complex and heterogeneous metabolic disorder that is characterized by multiple etiologies, including high blood sugar levels, insulin resistance and impaired insulin secretion. With improved standards of living and an aging population, the incidence of diabetes is increasing. However, current
Dr. Xiao-mei Sun The Institute of Medical Biology, The Chinese Academy of Medical Science Kunming 650118 (China) E-Mail sxm @ imbcams.com.cn Co-corresponding author: Dr. Guang-ping Ruan Stem Cell Engineering Laboratory of Yunnan Province Kunming General Hospital of Chengdu Military Command Kunming 650032 (China) E-Mail ruangp @ 126.com
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Key Words Bone marrow mesenchymal stem cells · Superparamagnetic iron oxide · Type 2 diabetes · Macaque
ACR AUC BMSCs DAPI FBG FBS HE IVGTT MR MRI PAS PLL SPIO STZ TEM T2DM
acute C-peptide response area under the curve bone marrow mesenchymal stem cells 4′,6-diamidino-2-phenylindole fasting blood glucose fetal bovine serum hematoxylin and eosin intravenous glucose tolerance test magnetic resonance magnetic resonance imaging periodic acid-Schiff poly-L-lysine superparamagnetic iron oxide streptozotocin transmission electron microscopy type 2 diabetes mellitus
drug treatments are only used to manage blood sugar levels and do not prevent the occurrence of complications or provide a cure for diabetes. Bone marrow mesenchymal stem cells (BMSCs) are self-renewing, multipotent cells that can migrate to pathological sites and thereby provide a new treatment in regenerative medicine [Takemiya et al., 2010; Pourrajab et al., 2013]. BMSCs have been used to treat diabetes in many animal models [Kwon et al., 2008; Urban et al., 2008]. However, the distribution and migration of BMSCs in the injured pancreatic area as well as the outcome have not been effectively identified and tracked. The development of strategies to track BMSCs and visualize their distribution is an emerging field of research [Dong et al., 2008]. A noncytotoxic fluorescent dye, 4′,6-diamidino-2-phenylindole (DAPI), does not affect cell growth. The distribution and migration of DAPIlabeled BMSCs in lesions or injured tissues can be tracked with a fluorescence microscope. Superparamagnetic iron oxide (SPIO) particles are magnetic resonance (MR) contrast agents approved by the FDA in the USA. BMSCs can be labeled with SPIO particles in vitro, which can be tracked by MR in vivo. In our experiments, we used a macaque, which is biologically, genetically and behaviorally similar to a human, to improve the visualization of the distribution, migration and outcome of transplanted BMSCs in vivo. We induced T2DM in a macaque model. BMSCs were double-labeled with SPIO and DAPI in vitro and were then transplanted to treat T2DM in the macaque model. Our results provide evidence and an experimental basis for the application of transplanted BMSCs to treat T2DM. 2
Cells Tissues Organs DOI: 10.1159/000358383
Materials and Methods Materials and Animals The following materials and equipment were used in our study: SPIO particles (Sigma, USA), DAPI (Invitrogen Corp., USA), DMEM/F12 medium, fetal bovine serum (FBS), 0.25% trypsin (Gibco, USA), streptozotocin (STZ; Sigma, USA), egg-yolk powder (Dalian Greensnow Egg Products Development Co. Ltd., China), a Heal Force CO2 incubator (Shanghai Li Shen Scientific Instruments Ltd., China), an inverted phase-contrast optical microscope, a fluorescence microscope (Olympus, Japan) and a trace blood glucose meter (Johnson, USA). Healthy 5-year-old macaques were provided by the Animal Center of Kunming Medical Biology Institute. All the experimental protocols were approved by the Experimental Animal Ethics Committee of Kunming General Hospital of Chengdu Military Command. BMSC Culture and SPIO/DAPI Double Label BMSC Isolation, Culture and Expansion Healthy 5-year-old macaques were anesthetized with an intravenous injection of 3% sodium pentobarbital (1 mg/kg). Under sterile conditions, a bone marrow biopsy needle was used to puncture the posterior superior iliac spine and extract 15–20 ml bone marrow into a 50-ml syringe containing 1 ml of heparin saline (1,200 U/ml). Collected bone marrow was diluted with 30 ml physiological saline, mixed by pipetting and filtered through a 200mesh sieve. Cells were centrifuged for 5 min at 400 g. The supernatant was removed and 10 ml of 0.38% NH4Cl was added and thoroughly mixed for 10 min. The solution was centrifuged at 400 g for 4 min and the supernatant was removed. This was repeated 3 times. Cells were resuspended and counted. The collected cells were cultured in DMEM/F12 medium (from Hyclone) containing 20% FBS (from Biological Industries) at a cell concentration of 3 × 105/ml. Cells were seeded into 175-cm2 culture flasks and cultured in an incubator at 37 ° C with 5% CO2 and saturated humidity. The medium was changed every 3–4 days. Cells were 80% confluent after 8–12 days. Cells were detached with 0.25% trypsin and passaged 1: 2 for further subculture. Cell dynamics were observed under an inverted phase-contrast microscope.
Detection of Cell Surface Markers Passage-3 cells were cultured to 80% confluence. Cells were collected by digestion with 0.25% trypsin and 5 × 105 cells were resuspended in 100 μl FB (PBS + 2% FBS + 0.1% NaN3) solution. The appropriate amount (10 μl) of fluorescein isothiocyanate-labeled CD29, CD31, CD34, CD44, CD90 or phycoerythrin-labeled CD105 antibody was added to the cell suspension (100 μl) and the samples were stained in the dark for 30 min. The appropriate amount of sheath fluid was added and the stained cell suspension was analyzed by flow cytometry. BMSCs Labeled with DAPI Passage-3 BMSCs were digested with 0.25% trypsin and centrifuged at 400 g for 4 min. The supernatant was removed and BMSCs were resuspended in DMEM/F12 medium containing 20% FBS. Single-cell suspensions were diluted to a concentration of 5 × 105/ ml and 100 μl were seeded in each well of a 6-well plate. Once cells were 80% confluent, the culture medium was removed and 10 μl DAPI working solution (100 μg/ml) was added per milliliter of culture medium for a final concentration of 1 μg/ml. Cell morphol-
Pan /Song /Dai /Yao /Wang /Pang /He /Li / Sun /Ruan
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Abbreviations used in this paper
ogy, proliferation and labeling efficiency were observed for 12, 24, 48 and 72 h. Labeled cells were washed 6 times with PBS and the intensity of DAPI-labeled cells was examined under a fluorescence microscope. Every 2–3 days, the medium was changed and the fluorescence intensity was evaluated. The DAPI labeling efficiency was determined by evaluating at least 500 cells with fluorescence microscopy: the number of positively labeled cells/the total number of cells × 100%. SPIO-Labeled BMSCs Following a previously established protocol [Kraitchman et al., 2011], 500 μl SPIO particles (1 mg/ml) and 500 μl poly-L-lysine (PLL; 0.06 mg) were mixed at room temperature and incubated for 60 min. DMEM/F12 medium (20 ml) containing 20% FBS was then added for a final concentration of 25 μg/ml SPIO and 1.5 μg/ ml PLL. The SPIO solution was added to 80%-confluent, passage-3 BMSCs, which were then cultured at 37 ° C and 5% CO2. Cell morphology, proliferation and labeling efficiency were observed at 12, 24, 48 and 72 h.
DAPI/SPIO-Double-Labeled BMSCs According to the methods described above, passage-3 BMSCs were double-labeled by SPIO and DAPI. BMSCs were incubated for 24 h at 37 ° C in an incubator with 5% CO2. Cell morphology and proliferation were observed. The DAPI labeling efficiency was observed under a fluorescence microscope. Prussian blue was used to determine the SPIO labeling efficiency of the BMSCs. At least 500 cells from each group of BMSCs were evaluated, and the labeling efficiency was determined by: the number of positively stained cells/the total number of cells × 100%.
Establishment of the Diabetic Macaque Model In this study, a high-sugar, high-fat diet combined with a low dose of STZ was used to induce T2DM in a macaque model. The following conditions were used as model standards: a fasting blood glucose (FBG) level ≥11.1 mmol/l that continued for 4 weeks and a C-peptide level 0.05). Labeling BMSCs with SPIO and DAPI did not significantly affect the proliferative activity (data not shown). Pan /Song /Dai /Yao /Wang /Pang /He /Li / Sun /Ruan
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id and C-peptide, were detected prior to diabetes induction and then at 3 days and 4, 8 and 12 weeks after induction also by radioimmunoassay.
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Fig. 1. Cultivation of macaque BMSCs. a The morphology of MSCs after 8 days of culture. ×200. b The MSCs approached 80% confluence by passage 3. ×200. c Phe-
notype of passage-3 BMSCs analyzed by flow cytometry (with isotype). The cells expressed CD29 (95.1%), CD44 (97.1%), CD90 (100.0%) and CD105 (99.9%), but did not express CD31 (0.0%) or CD34 (1.0%). FITC = Fluorescein isothiocyanate; PE = phycoerythrin.
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Evaluation of the Diabetic Model In this experiment, diabetes was successfully induced in the model group. No animals died during the experiment. The levels of FBG (fig. 3a, b) and serum C-peptide (fig. 3d) in the model group were significantly different from the levels in the control group (p < 0.05). The area under the curve (AUC) of the IVGTT (fig. 3c) and ACR experiments (fig. 3d) show that the model group reacted to glucose stimulation; the insulin secretion of islet β-cells
Glucose Levels A high-sugar, high-fat diet combined with a low dose of STZ induced diabetes and significantly increased the FBG of macaques in the model group. This hyperglycemic state was maintained for 12 weeks (fig. 3a). Seven days after BMSC transplantation, the FBG of the treatment group
BMSCs for Treatment of T2DM in a Macaque Model
Cells Tissues Organs DOI: 10.1159/000358383
in the model group was significantly different from that in the control group.
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Fig. 2. Labeled BMSCs. a BMSCs visualized in bright field. b Nuclei of BMSCs labeled
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Cells Tissues Organs DOI: 10.1159/000358383
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with DAPI appeared blue with a fluorescence microscope in dark field. Bluestained nucleoli were observed in nearly all cells with a labeling efficiency of approximately 98%. c Unlabeled BMSCs. d SPIO/ DAPI-labeled BMSCs showed normal cell growth and the distribution of brown iron particles in their cytoplasm. An inverted phase-contrast microscope was used to visualize BMSCs. e Prussian blue staining of unlabeled BMSCs. f Prussian blue staining of labeled BMSCs shows efficient intracellular uptake of particles into endosomes. Cells were labeled for 24 h with 25 μg Fe/ml Feridex and 1.5 μg/ml PLL, which resulted in a 95% labeling efficiency. a–f ×200. g TEM image illustrates the cellular uptake and intracellular distribution of iron particles. ×20,000. Arrows indicate Fe particles in cells.
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began to decrease, but that of the model control group did not change significantly (fig. 3b). At 6 weeks after transplantation, the FBG of the treatment group decreased from 25.9 ± 2.7 to 12.2 ± 2.1 mmol/l. The FBG of the treatment group was significantly different from that of the model group (p < 0.05). However, the FBG levels did not decrease to normal levels when compared to the control group. The difference was statistically significant (p < 0.05).
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Intravenous Glucose Tolerance Test At 6 weeks after BMSC transplantation, IVGTT were performed on 3 macaques each from the control group, the model control group and the treatment group (fig. 3c).
The FBG levels of the model group were significantly higher at all the time points. The AUC of the IVGTT graph for the model group increased significantly compared to the control group (p < 0.05). The FBG levels of the treatment group were significantly lower at each time point than those of the model control group (p < 0.05), as measured by IVGTT. These levels did not decrease to normal levels compared to the control group, and the difference is statistically significant (p < 0.05; fig. 3c). As observed in the figure, the AUC for the treatment group was significantly less than that of the model control group. This indicates that the impaired glucose tolerance was restored and that the insulin resistance was gradually reduced.
BMSCs for Treatment of T2DM in a Macaque Model
Cells Tissues Organs DOI: 10.1159/000358383
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the control group (n = 3) and in the model group (n = 9). Changes were induced by a high-glucose, high-fat diet combined with a low dose of STZ. FBG levels remained high in the model group for 12 weeks. b After transplantation, the FBG levels in the BMSC treatment group (n = 6) had significantly decreased when compared to the model group. c IVGTT of each group 6 weeks after BMSC transplantation (mean n = 3). The serum glucose levels and the AUC in response to intravenous glucose stimulation were significantly different for each group. d ACR of each group at 6 weeks after BMSC transplantation (mean n = 3). The serum C-peptide levels and the AUC in response to intravenous glucose stimulation were significantly different for each group.
C-peptide (ng/ml)
Fig. 3. Changes in the FBG, IVGTT and ACR of the study groups. a FBG levels in
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Table 1. Blood lipid levels (mean ± SD)
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2.49 ± 0.08a 3.71 ± 0.50a, b
2.59 ± 0.18 3.18 ± 0.29b
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p < 0.05. The difference between the model group and the control group was statistically significant. p < 0.05. After treatment with BMSCs for 6 weeks, the levels had significantly decreased compared to pretreatment levels. b
Lipid Levels Model group animals were fed the high-sugar, high-fat diet for 4 weeks. The blood lipid levels (triglycerides and total cholesterol) increased, and a state of high cholesterol was maintained for 12 weeks after STZ injection (a total of 16 weeks after starting the high-sugar, high-fat diet). The difference between the model group and the control group was statistically significant (p < 0.05; table 1). After treatment with BMSCs, the lipid levels significantly decreased (p < 0.05) compared to the levels before treatment. In vivo Tracking of Cells by MR Imaging No change in image signal (i.e. reduction in signal area) was observed in the pancreas, kidney, liver or other organs after intravenous injection of unlabeled cells or saline solution. However, 3 days after the infusion of SPIO-labeled BMSCs, a regional area with a low-intensity signal was observed in the pancreas and kidney of T2DM monkeys in T2W1 sequence (fig. 4). At 7 days, the 8
Cells Tissues Organs DOI: 10.1159/000358383
signal was significantly reduced. At 14 days, a scattered, low-intensity signal with spotty distribution was observed in the kidney. At 21 days, the low-intensity signal had disappeared. The change in the MR scan signal 3 days after the transplantation of labeled cells showed that SPIO-labeled BMSCs had accumulated in the injured pancreas, kidney and other injured areas. Therefore, cells can be traced by MR with the optimal concentration of SPIO particles. The weakening of the MR signal indicates that the number of intercellular iron particles decreases with time; this can be attributed to cell proliferation and a sign of cells disappearing. The negative signal from the MR scan began to decline and at 21 days, it was so weak that it was difficult to scan. Pathological Examination Six weeks after BMSC transplantation in the treatment group, HE staining of the pancreas from the model group revealed a decreased number of islet cells and an increased number of atrophied islet cells. The morphology of the islet cells was damaged; they displayed irregular and jagged edges that lacked a boundary (fig. 5d). PAS staining exposed a large amount of glycogen deposition (fig. 5g). Masson staining showed a few residual islet cells. Hyperplastic islets were surrounded by fibrous tissue that was stained with a green, transparent-like substance, which is indicative of a typical diabetic pancreas (fig. 5j). The renal biopsy of the model group revealed a significantly thicker mesangium and glomerular hypertrophy with HE staining. A glass-like substance was deposited on the glomerular wall and eosinophilic material was deposited in the glomerular capillaries. We observed glomerular deposits and intracapsular fibrosis, labeled with a red dye (fig. 5e). PAS staining showed significant deposition of sugars and proteins in the renal glomerular matrix and under the capillary endothelium (fig. 5h). Masson staining revealed glomerular hyaline degeneration and wall thickening of Pan /Song /Dai /Yao /Wang /Pang /He /Li / Sun /Ruan
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C-peptide Release Test At 6 weeks after BMSC transplantation, an ACR test was completed on 3 macaques from the control group, the model control group and the treatment group. At each time point, the serum C-peptide levels of the model group were significantly lower than those of the control group with intravenous glucose stimulation. The AUC was significantly reduced. After BMSC transplantation, the serum C-peptide levels were significantly greater at each time point in the treatment group than in the model control group with intravenous glucose stimulation. The AUC increased significantly compared to the model group. However, serum C-peptide levels were not at normal levels compared to the control group; the difference was statistically significant (fig. 3d). This demonstrates the gradual reduction in insulin resistance.
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the afferent, efferent and small arteries (fig. 5k). In addition, blue nodular substances were found in the glomerular capillary and mesangial cell zone (fig. 5k). Unstructured, red, cellulose-like substances were found deposited in the glomerular capillary lumen (fig. 5k). The glomerular cells were hypertrophic and showed typical diabetic nephropathy and mesangial thickening (fig. 5k). HE (fig. 5f) and PAS (fig. 5i) staining revealed lobular structural damage, a disordered hepatic cord, narrowing of the sinus space and increased liver cell volume in the model group. Cell necrosis, fat droplets and an increased amount of glycogen deposition and fatty liver cells were observed. Six weeks after the transplantation of double-labeled BMSCs in treatment group A, fluorescence microscopy showed a large number of fluorescent signals in the frozen sections of the pancreas (fig. 5l) and the kidney (fig. 5o), mainly in the seriously damaged areas. Prussian blue staining of the pancreas (fig. 5n) and kidney (fig. 5q) la-
beled the cells with blue dye and indicated the oriented migration of BMSCs to more severely injured diabetic tissues.
BMSCs for Treatment of T2DM in a Macaque Model
Cells Tissues Organs DOI: 10.1159/000358383
Discussion
Advantages of BMSCs as Seed Cells for Diabetes Treatment In 2011, according to the National Institutes of Health (USA) public database of clinical trial sites (http://clinical trials.gov), Trounson et al. [2011] analyzed recent clinical trials of stem cell therapy. The results revealed that there are already 123 applications of MSCs being tested in clinical studies. BMSCs are derived from the mesoderm and have the ability to self-renew and self-differentiate into MSC lineages. They are abundant and relatively easy to obtain and culture in vitro, and there are no ethical issues 9
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Fig. 4. MR images of a T2DM model in macaques injected with 2 × 106/kg SPIO-labeled BMSCs. Labeled BMSCs were tracked in horizontal slices of T2-weighted MR images in vivo. After injection, regions with a hypointense signal indicated the location of SPIO-labeled cells. The hypointense signal was still present after 14 days and spread towards the injury site. Furthermore, MRI showed a real-time reduction in the signal of labeled BMSCs after 21 days. Arrows indicate cell location. Horizontal position of the kidney 3 days after injection (a), 7 days after injection (b), 14 days after injection (c) and 21 days after injection (d). e Horizontal position of the pancreas 7 days after injection.
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concerning the use of MSCs. In addition, they can migrate to pathological sites to repair or reconstruct damaged tissues. They are ideal seed cells for tissue engineering applications and provide a new direction for clinical regenerative medicine. In recent years, improved living standards have caused and increased the incidence of diabetes in the world. The statistics show that in the USA in 10
Cells Tissues Organs DOI: 10.1159/000358383
(For legend see next page.)
2011 [Herman, 2011], 6% of the total population were diabetic patients, and the treatment of diabetes accounted for one fifth of medical and health care costs. According to Yang et al. [2010], 9.7% of China’s population aged ≥20 years have diabetes; this is approximately 92 million people. Current methods of treatment include managing diabetes with hypoglycemic medications, diet control, rehaPan /Song /Dai /Yao /Wang /Pang /He /Li / Sun /Ruan
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Fig. 5. Histopathological examination of pancreatic, kidney and liver tissue of macaques 6 weeks after BMSC transplantation. a– c HE staining of the control group confirmed normal pancreatic (a), kidney (b) and liver (c) sections. Arrows show the normal islet (a), glomerular (b) and liver (c) cells. ×400. d–k Pathological sections of the model group showed lesioned pancreatic (d, g, j), kidney (e, h, k) and liver (f, i) tissue. ×400. d–f HE staining of pancreatic (d), kidney (e) and liver (f) tissue from the model group, respectively. Arrows show the abnormal islet (d), glomerular (e) and liver (f) cells. g–i PAS staining of pancreatic (g), kidney (h) and liver (i) tissue from the model group, respectively. Arrows show glycogen deposition. Masson staining of pancreatic (j) and kidney (k) tissue from the model group, respectively. Arrows show the
BMSCs were tracked in the pancreas (l, m) and kidney (o, p) with a fluorescence microscope. ×100. Blue dots represent DAPI-labeled BMSCs. l After injection with double-labeled BMSCs, a high distribution of DAPI-labeled cells was observed in the pancreas. m After injection with unlabeled BMSCs, few blue dots were observed in the pancreas. o After injection, many DAPI-labeled BMSCs were distributed in the kidney. p After injection of unlabeled BMSCs, few blue dots were observed in the kidney. Prussian blue staining of pancreatic (n) and kidney (q) tissue sections. ×200. Arrows indicate the location of cells positively labeled with SPIO. Histology of the BMSC-treated diabetic macaques: kidney (r) and pancreatic (s) tissue. ×400. After BMSC treatment, the glomerular and islet cells appear normal.
bilitation exercises and symptomatic treatments; however, no radical measures exist. There is an urgent need for improved diabetes treatments, such as a new method that can treat or cure diabetes and its complications. BMSCs
can repair cell damage, adjust immune responses and promote angiogenesis and the secretion of cell growth factors. Milanesi et al. [2012] reported that stem cells in the microenvironment of pancreatic tissue induced the
BMSCs for Treatment of T2DM in a Macaque Model
Cells Tissues Organs DOI: 10.1159/000358383
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tissue fibrosis. Six weeks after transplantation, double-labeled
Selection and Induction of the Diabetic Animal Model The establishment of an animal model is the prerequisite and basis for preventive research on T2DM. Rat and mouse models, which are established animal models, are commonly used for experimental studies on T2DM. Diabetes can be spontaneously induced in animal models by a variety of methods, such as a high-fat diet, chemical factors or genetic modifications [Kitamura et al., 2003; Kim et al., 2010; Singh et al., 2010; Kitada et al., 2011; Nakamaki et al., 2011]. These animal models are developed to evaluate T2DM treatments and to screen potential drugs. However, in order to be more relevant, treatment evaluation studies need to be performed in animals that are more closely related to humans, such as primate models. Few such studies have been conducted because of a lack of standardized protocols and models [Qiao et al., 2009; Graham et al., 2011]. Monkeys were the first cloned nonhuman primates, and their sequenced genome has up to 93% similarity to the human genome [Pennisi, 2007]. In terms of biology and the genetic and behavioral aspects, monkeys are highly similar to humans. Monkeys and humans are also immunologically, physiologically and metabolically similar. The pharmacological efficacy of treatment in preclinical evaluations performed on monkeys has more relevance for human health than any other species of experimental animals. Monkeys also spontaneously develop T2DM [Najafian et al., 2011]. Therefore, the macaque is an ideal disease model for human T2DM. Macaques are critical for the development of new diabetes drugs and in preclinical experiments. Research has shown that insulin resistance and dyslipidemia are closely related [Cooper and Jandeleit-Dahm, 2005]. Diabetic patients have abnormal lipid metabolism. A high-calorie diet is an 12
Cells Tissues Organs DOI: 10.1159/000358383
important incentive. Studies have found that a healthy, high-calorie diet [Shamekh et al., 2011] will cause dyslipidemia, reduce insulin sensitivity and induce metabolic syndromes of insulin resistance. The experimental study of a high-sugar, high-fat diet was chosen to imitate the modern lifestyle choice of high-calorie diets. STZ is a fermentation product of colorless Streptomyces. It selectively destroys the pancreatic β-cells of some species of animals and, therefore, can be used to induce diabetes. A large intravenous dose of STZ will cause a stable level of high blood sugar in many animals after 24 h. Most of the damage occurs to islet β-cells, similar to what occurs in human type 1 diabetes. However, there are some differences in the manifestation of T2DM with insulin resistance [Jin et al., 2010]. We believe that in our study, the high-sugar, high-fat diet and low dose of STZ successfully induced T2DM in a macaque model. Stem Cell Marker Selection and Safety Evaluation Conventional cell transplantation tracking methods include using fluorescent dyes, transgenic methods and labeling chromosomes or nucleic acids. These techniques may require in vitro conditions for histological analysis and identification. Therefore, it is difficult for stem cell transplantation to meet the criteria of clinical applications. In recent years, with the development of molecular imaging, SPIO particles have been used as negative MR imaging (MRI) contrast agents for a variety of in vivo cell labeling applications and tracker studies following transplantation [Huang et al., 2007; Zhou et al., 2010]. This approach has given rise to a growing and wide range of interests. MRI has high spatial resolution and enables the noninvasive visualization of SPIO-labeled cells in vitro as well as the observation of cell migration, survival and proliferation in vivo. Presently, there are many studies using magnetically labeled stem cells and in vivo tracers [Wang et al., 2011; Xie et al., 2011]. SPIO particles are nanoscale iron particles that cause changes in MR signals at very low concentrations. Cells labeled with SPIO in vitro are transplanted in vivo and used as MRI contrast agents to explicitly capture images via signals. SPIO particles modified by the positive charges of PLL [Cromer Berman et al., 2013] are endocytosed to the cell cytoplasm through electrostatic interactions with negatively charged cell membrane ligands, thereby effectively labeling the BMSCs. This experiment effectively labeled BMSCs with 25 μg/ml SPIO. TEM confirmed that cytoplasmic vesicles contained a high density of SPIO particles; the labeling efficiency was greater than 95%. An MTT assay confirmed that cell viability and proliferation were not affected, indicating that Pan /Song /Dai /Yao /Wang /Pang /He /Li / Sun /Ruan
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proliferation and differentiation of islet-like cells, which replaced damaged islet β-cells and secreted insulin to restore pancreatic endocrine function. However, stem cells can also increase the production of intracellular sugars and proteins, which can promote the binding of the insulin receptor to islet cells and decrease insulin resistance. The stem cells can repair the islet cells to produce new islet β-cells with restored insulin secretion. Stem cell repair and regeneration of these cellular functions can improve insulin secretion and play a role in hypoglycemic control. In our study, the plasmatic lipid levels decreased significantly after treatment with BMSCs compared to the levels before treatment; this suggests that treatment with BMSCs plays a role in this decrease. This demonstrates the potential of stem cells to control T2DM and its associated complications.
SPIO particles were safely and effectively endocytosed into the cell cytoplasm. DAPI is a fluorescent dye for DNA-specific binding with no significant cytotoxicity because it can penetrate the cell membrane. It quickly penetrates living cells and binds to DNA. DAPI-DNA complexes have excitation and emission wavelengths of 360 and 460 nm, respectively, and under excitation with UV light, they are visualized by blue fluorescence [Ocarino et al., 2008]. In addition, the high spatial resolution of MR permitted in vitro observations and the in vivo visualization of cell migration, survival and proliferation using noninvasive SPIO-labeled cells. The double-labeling method was both complementary and mutually authentic.
secretion of attractive chemokines. As reported in the literature [Liu and Velazquez, 2008], following tissue damage, SDF-1 and other chemokines become highly expressed, which allows damaged tissues to recruit BMSCs. In this experiment, a high-sugar, high-fat diet combined with a low dose of STZ induced pathological changes to the pancreas and kidney indicative of T2DM. Reinfusion of SPIO/DAPI-double-labeled cells in the model animal pancreas and kidney was confirmed by histopathological examination, which revealed the distribution of BMSCs. This verified that BMSCs home primarily to these damaged tissues and organs and repair the damage.
Conclusions
BMSCs for Treatment of T2DM in a Macaque Model
In summary, BMSCs were transplanted into the pancreatic artery of macaques to treat T2DM. We demonstrated that this transplantation is a safe and effective treatment for T2DM in a macaque model. It reduced the level of FBG, lipids and other metabolic parameters, increased the level of serum C-peptide and reduced insulin resistance. The transplanted BMSCs also improved numerous bodily and cellular functions and repaired damaged tissue. In addition, SPIO particles provide a safe and effective method for tracking stem cells in vivo. Tracking with MR will become a future method for the effective detection and tracking of the migration and survival of stem cells in vivo.
Acknowledgments This work was supported by the National Natural Science Foundation of China (31172170), 973 Projects (2012CB518106) and the Special Project of High-New Technology Industrial Development in Yunnan Province (201204). We thank American Journal Experts for assisting in the preparation of the manuscript.
References
Cells Tissues Organs DOI: 10.1159/000358383
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MRI of SPIO-Labeled BMSCs In this study, SPIO/DAPI-double-labeled BMSCs were transplanted in the pancreatic tail artery of macaques. Changes in the MR signal indicate that the SPIOlabeled BMSCs accumulated in the pancreatic and kidney tissue 3 days after BMSC transplantation. However, over time, the negative signal from the MR scan declined. This was attributed to cell proliferation and a sign of the disappearance of cells, which decreased the intracellular concentration of iron particles and thereby weakened the magnetic signal. The BMSCs labeled with both SPIO and DAPI that accumulated in pancreatic and kidney tissues were observed via fluorescence and Prussian blue staining. FBG, C-peptide and lipid levels detected also provided a multidirectional authentication for the use of BMSCs for tracking cell migration, survival and proliferation in vivo. In this study, the visible DAPI fluorescence signal correlated with the signal distribution captured from the SPIO particles by MR. By MR in vivo tracking and using DAPI as a fluorescent tracer, BMSCs were found mainly in the pancreas and kidney. This demonstrated the transport of labeled cells into the injured organ via the blood stream (the pancreatic artery), so the first route, presumably, is through the pancreas and a strong adhesion at the endothelial cells of the injured tissue is a first barrier (endothelial cells also secrete danger signals). The number of BMSCs observed in the pancreas increased. The labeled BMSCs migrated to the damaged pancreas and kidney tissue; over time, this directed migration will gradually increase the number of cells, which can be detected by MR imaging to a certain extent. The survival, accumulation and directed migration of transplanted BMSCs in a diabetic individual are regulated by complex mechanisms. We believe that the pancreatic and kidney damage caused by diabetes initiates the
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Cells Tissues Organs DOI: 10.1159/000358383
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Graham, M.L., L.A. Mutch, E.F. Rieke, J.A. Kittredge, A.W. Faig, T.A. DuFour, J.W. Munson, E.K. Zolondek, B.J. Hering, H.J. Schuurman (2011) Refining the high-dose streptozotocin-induced diabetic non-human primate model: an evaluation of risk factors and outcomes. Exp Biol Med (Maywood) 236: 1218–1230. Herman, W.H. (2011) The economics of diabetes prevention. Med Clin North Am 95: 373– 384,viii. Huang, Z.Y., J.B. Ge, S. Yang, S.H. Zhang, R.C. Huang, H. Jin, M.S. Zeng, A.J. Sun, J.Y. Qian, Y.Z. Zou (2007) In vivo cardiac magnetic resonance imaging of superparamagnetic iron oxides-labeled mesenchymal stem cells in swine. Zhonghua Xin Xue Guan Bing Za Zhi 35: 344–349. Jin, X., L. Zeng, S. He, Y. Chen, B. Tian, G. Mai, G. Yang, L. Wei, Y. Zhang, H. Li, et al. (2010) Comparison of single high-dose streptozotocin with partial pancreatectomy combined with low-dose streptozotocin for diabetes induction in rhesus monkeys. Exp Biol Med (Maywood) 235: 877–885. Kim, H.Y., T. Okubo, L.R. Juneja, T. Yokozawa (2010) The protective role of amla (Emblica officinalis Gaertn.) against fructose-induced metabolic syndrome in a rat model. Br J Nutr 103: 502–512. Kitada, M., A. Takeda, T. Nagai, H. Ito, K. Kanasaki, D. Koya (2011) Dietary restriction ameliorates diabetic nephropathy through antiinflammatory effects and regulation of the autophagy via restoration of Sirt1 in diabetic Wistar fatty (fa/fa) rats: a model of type 2 diabetes. Exp Diabetes Res 2011: 908185. Kitamura, T., C.R. Kahn, D. Accili (2003). Insulin receptor knockout mice. Annu Rev Physiol 65: 313–332. Kraitchman, D.L., D.A. Kedziorek, J.W. Bulte (2011) MR imaging of transplanted stem cells in myocardial infarction. Methods Mol Biol 680: 141–152.
Cells Tissues Organs DOI: 10.1159/000358383
Transplantation of Bone Marrow Mesenchymal Stem Cells for the Treatment of Type 2 Diabetes in a Macaque Model Xing-hua Pan a Qiao-qiao Song b Jie-jie Dai c Xiang Yao a Jin-xiang Wang a Rong-qing Pang a Jie He a Zi-an Li a Xiao-mei Sun c Guang-ping Ruan a a
Stem Cell Engineering Laboratory of Yunnan Province, Kunming General Hospital of Chengdu Military Command, Clinical College of Kunming General Hospital of Chengdu Military Command, Kunming Medical University, and c The Institute of Medical Biology, The Chinese Academy of Medical Science and Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Center of Tree Shrew Germplasm Resources, Kunming, China b
Abstract Bone marrow mesenchymal stem cells (BMSCs) are self-renewing, multipotent cells that can migrate to pathological sites and thereby provide a new treatment in diabetic animals. Superparamagnetic iron oxide/4′,6-diamidino-2phenylindole (DAPI) double-labeled BMSCs were transplanted into the pancreatic artery of macaques to treat type 2 diabetes mellitus (T2DM). The treatment efficiency of BMSCs was also evaluated. After successful induction of the T2DM model, the treatment group received double-labeled BMSCs via the pancreatic artery. Six weeks after BMSC transplantation, the fasting blood glucose and blood lipid levels measured in the treatment group were significantly lower (p < 0.05) than in the model group, although they were not reduced to normal levels (p < 0.05). Additionally, the serum Cpeptide levels were significantly increased (p < 0.05). An intravenous glucose tolerance test and C-peptide release test had significant changes to the area under the curve. Within 14 days of the transplantation of labeled cells, the pancre-
© 2014 S. Karger AG, Basel 1422–6405/14/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/cto
atic and kidney tissue of the treatment group emitted a negative signal that was visible on magnetic resonance imaging (MRI). Six weeks after transplantation, DAPI signals appeared in the pancreatic and kidney tissue, which indicates that the BMSCs were mainly distributed in damaged tissue. Labeled stem cells can be used to track migration and distribution in vivo by MRI. In conclusion, the transplantation of BMSCs for the treatment of T2DM is safe and effective. © 2014 S. Karger AG, Basel
Introduction
Type 2 diabetes mellitus (T2DM) is a complex and heterogeneous metabolic disorder that is characterized by multiple etiologies, including high blood sugar levels, insulin resistance and impaired insulin secretion. With improved standards of living and an aging population, the incidence of diabetes is increasing. However, current
Dr. Xiao-mei Sun The Institute of Medical Biology, The Chinese Academy of Medical Science Kunming 650118 (China) E-Mail sxm @ imbcams.com.cn Co-corresponding author: Dr. Guang-ping Ruan Stem Cell Engineering Laboratory of Yunnan Province Kunming General Hospital of Chengdu Military Command Kunming 650032 (China) E-Mail ruangp @ 126.com
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Key Words Bone marrow mesenchymal stem cells · Superparamagnetic iron oxide · Type 2 diabetes · Macaque
ACR AUC BMSCs DAPI FBG FBS HE IVGTT MR MRI PAS PLL SPIO STZ TEM T2DM
acute C-peptide response area under the curve bone marrow mesenchymal stem cells 4′,6-diamidino-2-phenylindole fasting blood glucose fetal bovine serum hematoxylin and eosin intravenous glucose tolerance test magnetic resonance magnetic resonance imaging periodic acid-Schiff poly-L-lysine superparamagnetic iron oxide streptozotocin transmission electron microscopy type 2 diabetes mellitus
drug treatments are only used to manage blood sugar levels and do not prevent the occurrence of complications or provide a cure for diabetes. Bone marrow mesenchymal stem cells (BMSCs) are self-renewing, multipotent cells that can migrate to pathological sites and thereby provide a new treatment in regenerative medicine [Takemiya et al., 2010; Pourrajab et al., 2013]. BMSCs have been used to treat diabetes in many animal models [Kwon et al., 2008; Urban et al., 2008]. However, the distribution and migration of BMSCs in the injured pancreatic area as well as the outcome have not been effectively identified and tracked. The development of strategies to track BMSCs and visualize their distribution is an emerging field of research [Dong et al., 2008]. A noncytotoxic fluorescent dye, 4′,6-diamidino-2-phenylindole (DAPI), does not affect cell growth. The distribution and migration of DAPIlabeled BMSCs in lesions or injured tissues can be tracked with a fluorescence microscope. Superparamagnetic iron oxide (SPIO) particles are magnetic resonance (MR) contrast agents approved by the FDA in the USA. BMSCs can be labeled with SPIO particles in vitro, which can be tracked by MR in vivo. In our experiments, we used a macaque, which is biologically, genetically and behaviorally similar to a human, to improve the visualization of the distribution, migration and outcome of transplanted BMSCs in vivo. We induced T2DM in a macaque model. BMSCs were double-labeled with SPIO and DAPI in vitro and were then transplanted to treat T2DM in the macaque model. Our results provide evidence and an experimental basis for the application of transplanted BMSCs to treat T2DM. 2
Cells Tissues Organs DOI: 10.1159/000358383
Materials and Methods Materials and Animals The following materials and equipment were used in our study: SPIO particles (Sigma, USA), DAPI (Invitrogen Corp., USA), DMEM/F12 medium, fetal bovine serum (FBS), 0.25% trypsin (Gibco, USA), streptozotocin (STZ; Sigma, USA), egg-yolk powder (Dalian Greensnow Egg Products Development Co. Ltd., China), a Heal Force CO2 incubator (Shanghai Li Shen Scientific Instruments Ltd., China), an inverted phase-contrast optical microscope, a fluorescence microscope (Olympus, Japan) and a trace blood glucose meter (Johnson, USA). Healthy 5-year-old macaques were provided by the Animal Center of Kunming Medical Biology Institute. All the experimental protocols were approved by the Experimental Animal Ethics Committee of Kunming General Hospital of Chengdu Military Command. BMSC Culture and SPIO/DAPI Double Label BMSC Isolation, Culture and Expansion Healthy 5-year-old macaques were anesthetized with an intravenous injection of 3% sodium pentobarbital (1 mg/kg). Under sterile conditions, a bone marrow biopsy needle was used to puncture the posterior superior iliac spine and extract 15–20 ml bone marrow into a 50-ml syringe containing 1 ml of heparin saline (1,200 U/ml). Collected bone marrow was diluted with 30 ml physiological saline, mixed by pipetting and filtered through a 200mesh sieve. Cells were centrifuged for 5 min at 400 g. The supernatant was removed and 10 ml of 0.38% NH4Cl was added and thoroughly mixed for 10 min. The solution was centrifuged at 400 g for 4 min and the supernatant was removed. This was repeated 3 times. Cells were resuspended and counted. The collected cells were cultured in DMEM/F12 medium (from Hyclone) containing 20% FBS (from Biological Industries) at a cell concentration of 3 × 105/ml. Cells were seeded into 175-cm2 culture flasks and cultured in an incubator at 37 ° C with 5% CO2 and saturated humidity. The medium was changed every 3–4 days. Cells were 80% confluent after 8–12 days. Cells were detached with 0.25% trypsin and passaged 1: 2 for further subculture. Cell dynamics were observed under an inverted phase-contrast microscope.
Detection of Cell Surface Markers Passage-3 cells were cultured to 80% confluence. Cells were collected by digestion with 0.25% trypsin and 5 × 105 cells were resuspended in 100 μl FB (PBS + 2% FBS + 0.1% NaN3) solution. The appropriate amount (10 μl) of fluorescein isothiocyanate-labeled CD29, CD31, CD34, CD44, CD90 or phycoerythrin-labeled CD105 antibody was added to the cell suspension (100 μl) and the samples were stained in the dark for 30 min. The appropriate amount of sheath fluid was added and the stained cell suspension was analyzed by flow cytometry. BMSCs Labeled with DAPI Passage-3 BMSCs were digested with 0.25% trypsin and centrifuged at 400 g for 4 min. The supernatant was removed and BMSCs were resuspended in DMEM/F12 medium containing 20% FBS. Single-cell suspensions were diluted to a concentration of 5 × 105/ ml and 100 μl were seeded in each well of a 6-well plate. Once cells were 80% confluent, the culture medium was removed and 10 μl DAPI working solution (100 μg/ml) was added per milliliter of culture medium for a final concentration of 1 μg/ml. Cell morphol-
Pan /Song /Dai /Yao /Wang /Pang /He /Li / Sun /Ruan
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Abbreviations used in this paper
ogy, proliferation and labeling efficiency were observed for 12, 24, 48 and 72 h. Labeled cells were washed 6 times with PBS and the intensity of DAPI-labeled cells was examined under a fluorescence microscope. Every 2–3 days, the medium was changed and the fluorescence intensity was evaluated. The DAPI labeling efficiency was determined by evaluating at least 500 cells with fluorescence microscopy: the number of positively labeled cells/the total number of cells × 100%. SPIO-Labeled BMSCs Following a previously established protocol [Kraitchman et al., 2011], 500 μl SPIO particles (1 mg/ml) and 500 μl poly-L-lysine (PLL; 0.06 mg) were mixed at room temperature and incubated for 60 min. DMEM/F12 medium (20 ml) containing 20% FBS was then added for a final concentration of 25 μg/ml SPIO and 1.5 μg/ ml PLL. The SPIO solution was added to 80%-confluent, passage-3 BMSCs, which were then cultured at 37 ° C and 5% CO2. Cell morphology, proliferation and labeling efficiency were observed at 12, 24, 48 and 72 h.
DAPI/SPIO-Double-Labeled BMSCs According to the methods described above, passage-3 BMSCs were double-labeled by SPIO and DAPI. BMSCs were incubated for 24 h at 37 ° C in an incubator with 5% CO2. Cell morphology and proliferation were observed. The DAPI labeling efficiency was observed under a fluorescence microscope. Prussian blue was used to determine the SPIO labeling efficiency of the BMSCs. At least 500 cells from each group of BMSCs were evaluated, and the labeling efficiency was determined by: the number of positively stained cells/the total number of cells × 100%.
Establishment of the Diabetic Macaque Model In this study, a high-sugar, high-fat diet combined with a low dose of STZ was used to induce T2DM in a macaque model. The following conditions were used as model standards: a fasting blood glucose (FBG) level ≥11.1 mmol/l that continued for 4 weeks and a C-peptide level 0.05). Labeling BMSCs with SPIO and DAPI did not significantly affect the proliferative activity (data not shown). Pan /Song /Dai /Yao /Wang /Pang /He /Li / Sun /Ruan
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id and C-peptide, were detected prior to diabetes induction and then at 3 days and 4, 8 and 12 weeks after induction also by radioimmunoassay.
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Fig. 1. Cultivation of macaque BMSCs. a The morphology of MSCs after 8 days of culture. ×200. b The MSCs approached 80% confluence by passage 3. ×200. c Phe-
notype of passage-3 BMSCs analyzed by flow cytometry (with isotype). The cells expressed CD29 (95.1%), CD44 (97.1%), CD90 (100.0%) and CD105 (99.9%), but did not express CD31 (0.0%) or CD34 (1.0%). FITC = Fluorescein isothiocyanate; PE = phycoerythrin.
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Evaluation of the Diabetic Model In this experiment, diabetes was successfully induced in the model group. No animals died during the experiment. The levels of FBG (fig. 3a, b) and serum C-peptide (fig. 3d) in the model group were significantly different from the levels in the control group (p < 0.05). The area under the curve (AUC) of the IVGTT (fig. 3c) and ACR experiments (fig. 3d) show that the model group reacted to glucose stimulation; the insulin secretion of islet β-cells
Glucose Levels A high-sugar, high-fat diet combined with a low dose of STZ induced diabetes and significantly increased the FBG of macaques in the model group. This hyperglycemic state was maintained for 12 weeks (fig. 3a). Seven days after BMSC transplantation, the FBG of the treatment group
BMSCs for Treatment of T2DM in a Macaque Model
Cells Tissues Organs DOI: 10.1159/000358383
in the model group was significantly different from that in the control group.
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Fig. 2. Labeled BMSCs. a BMSCs visualized in bright field. b Nuclei of BMSCs labeled
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Cells Tissues Organs DOI: 10.1159/000358383
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with DAPI appeared blue with a fluorescence microscope in dark field. Bluestained nucleoli were observed in nearly all cells with a labeling efficiency of approximately 98%. c Unlabeled BMSCs. d SPIO/ DAPI-labeled BMSCs showed normal cell growth and the distribution of brown iron particles in their cytoplasm. An inverted phase-contrast microscope was used to visualize BMSCs. e Prussian blue staining of unlabeled BMSCs. f Prussian blue staining of labeled BMSCs shows efficient intracellular uptake of particles into endosomes. Cells were labeled for 24 h with 25 μg Fe/ml Feridex and 1.5 μg/ml PLL, which resulted in a 95% labeling efficiency. a–f ×200. g TEM image illustrates the cellular uptake and intracellular distribution of iron particles. ×20,000. Arrows indicate Fe particles in cells.
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began to decrease, but that of the model control group did not change significantly (fig. 3b). At 6 weeks after transplantation, the FBG of the treatment group decreased from 25.9 ± 2.7 to 12.2 ± 2.1 mmol/l. The FBG of the treatment group was significantly different from that of the model group (p < 0.05). However, the FBG levels did not decrease to normal levels when compared to the control group. The difference was statistically significant (p < 0.05).
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Intravenous Glucose Tolerance Test At 6 weeks after BMSC transplantation, IVGTT were performed on 3 macaques each from the control group, the model control group and the treatment group (fig. 3c).
The FBG levels of the model group were significantly higher at all the time points. The AUC of the IVGTT graph for the model group increased significantly compared to the control group (p < 0.05). The FBG levels of the treatment group were significantly lower at each time point than those of the model control group (p < 0.05), as measured by IVGTT. These levels did not decrease to normal levels compared to the control group, and the difference is statistically significant (p < 0.05; fig. 3c). As observed in the figure, the AUC for the treatment group was significantly less than that of the model control group. This indicates that the impaired glucose tolerance was restored and that the insulin resistance was gradually reduced.
BMSCs for Treatment of T2DM in a Macaque Model
Cells Tissues Organs DOI: 10.1159/000358383
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the control group (n = 3) and in the model group (n = 9). Changes were induced by a high-glucose, high-fat diet combined with a low dose of STZ. FBG levels remained high in the model group for 12 weeks. b After transplantation, the FBG levels in the BMSC treatment group (n = 6) had significantly decreased when compared to the model group. c IVGTT of each group 6 weeks after BMSC transplantation (mean n = 3). The serum glucose levels and the AUC in response to intravenous glucose stimulation were significantly different for each group. d ACR of each group at 6 weeks after BMSC transplantation (mean n = 3). The serum C-peptide levels and the AUC in response to intravenous glucose stimulation were significantly different for each group.
C-peptide (ng/ml)
Fig. 3. Changes in the FBG, IVGTT and ACR of the study groups. a FBG levels in
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Table 1. Blood lipid levels (mean ± SD)
Triglycerides
Control Model
Total cholesterol
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0.50 ± 0.07 0.54 ± 0.22
0.60 ± 0.30a 1.83 ± 0.74a, b
0.58 ± 0.13 1.15 ± 0.30b
2.46 ± 0.22 2.56 ± 0.33
2.49 ± 0.08a 3.71 ± 0.50a, b
2.59 ± 0.18 3.18 ± 0.29b
a
p < 0.05. The difference between the model group and the control group was statistically significant. p < 0.05. After treatment with BMSCs for 6 weeks, the levels had significantly decreased compared to pretreatment levels. b
Lipid Levels Model group animals were fed the high-sugar, high-fat diet for 4 weeks. The blood lipid levels (triglycerides and total cholesterol) increased, and a state of high cholesterol was maintained for 12 weeks after STZ injection (a total of 16 weeks after starting the high-sugar, high-fat diet). The difference between the model group and the control group was statistically significant (p < 0.05; table 1). After treatment with BMSCs, the lipid levels significantly decreased (p < 0.05) compared to the levels before treatment. In vivo Tracking of Cells by MR Imaging No change in image signal (i.e. reduction in signal area) was observed in the pancreas, kidney, liver or other organs after intravenous injection of unlabeled cells or saline solution. However, 3 days after the infusion of SPIO-labeled BMSCs, a regional area with a low-intensity signal was observed in the pancreas and kidney of T2DM monkeys in T2W1 sequence (fig. 4). At 7 days, the 8
Cells Tissues Organs DOI: 10.1159/000358383
signal was significantly reduced. At 14 days, a scattered, low-intensity signal with spotty distribution was observed in the kidney. At 21 days, the low-intensity signal had disappeared. The change in the MR scan signal 3 days after the transplantation of labeled cells showed that SPIO-labeled BMSCs had accumulated in the injured pancreas, kidney and other injured areas. Therefore, cells can be traced by MR with the optimal concentration of SPIO particles. The weakening of the MR signal indicates that the number of intercellular iron particles decreases with time; this can be attributed to cell proliferation and a sign of cells disappearing. The negative signal from the MR scan began to decline and at 21 days, it was so weak that it was difficult to scan. Pathological Examination Six weeks after BMSC transplantation in the treatment group, HE staining of the pancreas from the model group revealed a decreased number of islet cells and an increased number of atrophied islet cells. The morphology of the islet cells was damaged; they displayed irregular and jagged edges that lacked a boundary (fig. 5d). PAS staining exposed a large amount of glycogen deposition (fig. 5g). Masson staining showed a few residual islet cells. Hyperplastic islets were surrounded by fibrous tissue that was stained with a green, transparent-like substance, which is indicative of a typical diabetic pancreas (fig. 5j). The renal biopsy of the model group revealed a significantly thicker mesangium and glomerular hypertrophy with HE staining. A glass-like substance was deposited on the glomerular wall and eosinophilic material was deposited in the glomerular capillaries. We observed glomerular deposits and intracapsular fibrosis, labeled with a red dye (fig. 5e). PAS staining showed significant deposition of sugars and proteins in the renal glomerular matrix and under the capillary endothelium (fig. 5h). Masson staining revealed glomerular hyaline degeneration and wall thickening of Pan /Song /Dai /Yao /Wang /Pang /He /Li / Sun /Ruan
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C-peptide Release Test At 6 weeks after BMSC transplantation, an ACR test was completed on 3 macaques from the control group, the model control group and the treatment group. At each time point, the serum C-peptide levels of the model group were significantly lower than those of the control group with intravenous glucose stimulation. The AUC was significantly reduced. After BMSC transplantation, the serum C-peptide levels were significantly greater at each time point in the treatment group than in the model control group with intravenous glucose stimulation. The AUC increased significantly compared to the model group. However, serum C-peptide levels were not at normal levels compared to the control group; the difference was statistically significant (fig. 3d). This demonstrates the gradual reduction in insulin resistance.
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the afferent, efferent and small arteries (fig. 5k). In addition, blue nodular substances were found in the glomerular capillary and mesangial cell zone (fig. 5k). Unstructured, red, cellulose-like substances were found deposited in the glomerular capillary lumen (fig. 5k). The glomerular cells were hypertrophic and showed typical diabetic nephropathy and mesangial thickening (fig. 5k). HE (fig. 5f) and PAS (fig. 5i) staining revealed lobular structural damage, a disordered hepatic cord, narrowing of the sinus space and increased liver cell volume in the model group. Cell necrosis, fat droplets and an increased amount of glycogen deposition and fatty liver cells were observed. Six weeks after the transplantation of double-labeled BMSCs in treatment group A, fluorescence microscopy showed a large number of fluorescent signals in the frozen sections of the pancreas (fig. 5l) and the kidney (fig. 5o), mainly in the seriously damaged areas. Prussian blue staining of the pancreas (fig. 5n) and kidney (fig. 5q) la-
beled the cells with blue dye and indicated the oriented migration of BMSCs to more severely injured diabetic tissues.
BMSCs for Treatment of T2DM in a Macaque Model
Cells Tissues Organs DOI: 10.1159/000358383
Discussion
Advantages of BMSCs as Seed Cells for Diabetes Treatment In 2011, according to the National Institutes of Health (USA) public database of clinical trial sites (http://clinical trials.gov), Trounson et al. [2011] analyzed recent clinical trials of stem cell therapy. The results revealed that there are already 123 applications of MSCs being tested in clinical studies. BMSCs are derived from the mesoderm and have the ability to self-renew and self-differentiate into MSC lineages. They are abundant and relatively easy to obtain and culture in vitro, and there are no ethical issues 9
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Fig. 4. MR images of a T2DM model in macaques injected with 2 × 106/kg SPIO-labeled BMSCs. Labeled BMSCs were tracked in horizontal slices of T2-weighted MR images in vivo. After injection, regions with a hypointense signal indicated the location of SPIO-labeled cells. The hypointense signal was still present after 14 days and spread towards the injury site. Furthermore, MRI showed a real-time reduction in the signal of labeled BMSCs after 21 days. Arrows indicate cell location. Horizontal position of the kidney 3 days after injection (a), 7 days after injection (b), 14 days after injection (c) and 21 days after injection (d). e Horizontal position of the pancreas 7 days after injection.
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concerning the use of MSCs. In addition, they can migrate to pathological sites to repair or reconstruct damaged tissues. They are ideal seed cells for tissue engineering applications and provide a new direction for clinical regenerative medicine. In recent years, improved living standards have caused and increased the incidence of diabetes in the world. The statistics show that in the USA in 10
Cells Tissues Organs DOI: 10.1159/000358383
(For legend see next page.)
2011 [Herman, 2011], 6% of the total population were diabetic patients, and the treatment of diabetes accounted for one fifth of medical and health care costs. According to Yang et al. [2010], 9.7% of China’s population aged ≥20 years have diabetes; this is approximately 92 million people. Current methods of treatment include managing diabetes with hypoglycemic medications, diet control, rehaPan /Song /Dai /Yao /Wang /Pang /He /Li / Sun /Ruan
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5
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Fig. 5. Histopathological examination of pancreatic, kidney and liver tissue of macaques 6 weeks after BMSC transplantation. a– c HE staining of the control group confirmed normal pancreatic (a), kidney (b) and liver (c) sections. Arrows show the normal islet (a), glomerular (b) and liver (c) cells. ×400. d–k Pathological sections of the model group showed lesioned pancreatic (d, g, j), kidney (e, h, k) and liver (f, i) tissue. ×400. d–f HE staining of pancreatic (d), kidney (e) and liver (f) tissue from the model group, respectively. Arrows show the abnormal islet (d), glomerular (e) and liver (f) cells. g–i PAS staining of pancreatic (g), kidney (h) and liver (i) tissue from the model group, respectively. Arrows show glycogen deposition. Masson staining of pancreatic (j) and kidney (k) tissue from the model group, respectively. Arrows show the
BMSCs were tracked in the pancreas (l, m) and kidney (o, p) with a fluorescence microscope. ×100. Blue dots represent DAPI-labeled BMSCs. l After injection with double-labeled BMSCs, a high distribution of DAPI-labeled cells was observed in the pancreas. m After injection with unlabeled BMSCs, few blue dots were observed in the pancreas. o After injection, many DAPI-labeled BMSCs were distributed in the kidney. p After injection of unlabeled BMSCs, few blue dots were observed in the kidney. Prussian blue staining of pancreatic (n) and kidney (q) tissue sections. ×200. Arrows indicate the location of cells positively labeled with SPIO. Histology of the BMSC-treated diabetic macaques: kidney (r) and pancreatic (s) tissue. ×400. After BMSC treatment, the glomerular and islet cells appear normal.
bilitation exercises and symptomatic treatments; however, no radical measures exist. There is an urgent need for improved diabetes treatments, such as a new method that can treat or cure diabetes and its complications. BMSCs
can repair cell damage, adjust immune responses and promote angiogenesis and the secretion of cell growth factors. Milanesi et al. [2012] reported that stem cells in the microenvironment of pancreatic tissue induced the
BMSCs for Treatment of T2DM in a Macaque Model
Cells Tissues Organs DOI: 10.1159/000358383
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tissue fibrosis. Six weeks after transplantation, double-labeled
Selection and Induction of the Diabetic Animal Model The establishment of an animal model is the prerequisite and basis for preventive research on T2DM. Rat and mouse models, which are established animal models, are commonly used for experimental studies on T2DM. Diabetes can be spontaneously induced in animal models by a variety of methods, such as a high-fat diet, chemical factors or genetic modifications [Kitamura et al., 2003; Kim et al., 2010; Singh et al., 2010; Kitada et al., 2011; Nakamaki et al., 2011]. These animal models are developed to evaluate T2DM treatments and to screen potential drugs. However, in order to be more relevant, treatment evaluation studies need to be performed in animals that are more closely related to humans, such as primate models. Few such studies have been conducted because of a lack of standardized protocols and models [Qiao et al., 2009; Graham et al., 2011]. Monkeys were the first cloned nonhuman primates, and their sequenced genome has up to 93% similarity to the human genome [Pennisi, 2007]. In terms of biology and the genetic and behavioral aspects, monkeys are highly similar to humans. Monkeys and humans are also immunologically, physiologically and metabolically similar. The pharmacological efficacy of treatment in preclinical evaluations performed on monkeys has more relevance for human health than any other species of experimental animals. Monkeys also spontaneously develop T2DM [Najafian et al., 2011]. Therefore, the macaque is an ideal disease model for human T2DM. Macaques are critical for the development of new diabetes drugs and in preclinical experiments. Research has shown that insulin resistance and dyslipidemia are closely related [Cooper and Jandeleit-Dahm, 2005]. Diabetic patients have abnormal lipid metabolism. A high-calorie diet is an 12
Cells Tissues Organs DOI: 10.1159/000358383
important incentive. Studies have found that a healthy, high-calorie diet [Shamekh et al., 2011] will cause dyslipidemia, reduce insulin sensitivity and induce metabolic syndromes of insulin resistance. The experimental study of a high-sugar, high-fat diet was chosen to imitate the modern lifestyle choice of high-calorie diets. STZ is a fermentation product of colorless Streptomyces. It selectively destroys the pancreatic β-cells of some species of animals and, therefore, can be used to induce diabetes. A large intravenous dose of STZ will cause a stable level of high blood sugar in many animals after 24 h. Most of the damage occurs to islet β-cells, similar to what occurs in human type 1 diabetes. However, there are some differences in the manifestation of T2DM with insulin resistance [Jin et al., 2010]. We believe that in our study, the high-sugar, high-fat diet and low dose of STZ successfully induced T2DM in a macaque model. Stem Cell Marker Selection and Safety Evaluation Conventional cell transplantation tracking methods include using fluorescent dyes, transgenic methods and labeling chromosomes or nucleic acids. These techniques may require in vitro conditions for histological analysis and identification. Therefore, it is difficult for stem cell transplantation to meet the criteria of clinical applications. In recent years, with the development of molecular imaging, SPIO particles have been used as negative MR imaging (MRI) contrast agents for a variety of in vivo cell labeling applications and tracker studies following transplantation [Huang et al., 2007; Zhou et al., 2010]. This approach has given rise to a growing and wide range of interests. MRI has high spatial resolution and enables the noninvasive visualization of SPIO-labeled cells in vitro as well as the observation of cell migration, survival and proliferation in vivo. Presently, there are many studies using magnetically labeled stem cells and in vivo tracers [Wang et al., 2011; Xie et al., 2011]. SPIO particles are nanoscale iron particles that cause changes in MR signals at very low concentrations. Cells labeled with SPIO in vitro are transplanted in vivo and used as MRI contrast agents to explicitly capture images via signals. SPIO particles modified by the positive charges of PLL [Cromer Berman et al., 2013] are endocytosed to the cell cytoplasm through electrostatic interactions with negatively charged cell membrane ligands, thereby effectively labeling the BMSCs. This experiment effectively labeled BMSCs with 25 μg/ml SPIO. TEM confirmed that cytoplasmic vesicles contained a high density of SPIO particles; the labeling efficiency was greater than 95%. An MTT assay confirmed that cell viability and proliferation were not affected, indicating that Pan /Song /Dai /Yao /Wang /Pang /He /Li / Sun /Ruan
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proliferation and differentiation of islet-like cells, which replaced damaged islet β-cells and secreted insulin to restore pancreatic endocrine function. However, stem cells can also increase the production of intracellular sugars and proteins, which can promote the binding of the insulin receptor to islet cells and decrease insulin resistance. The stem cells can repair the islet cells to produce new islet β-cells with restored insulin secretion. Stem cell repair and regeneration of these cellular functions can improve insulin secretion and play a role in hypoglycemic control. In our study, the plasmatic lipid levels decreased significantly after treatment with BMSCs compared to the levels before treatment; this suggests that treatment with BMSCs plays a role in this decrease. This demonstrates the potential of stem cells to control T2DM and its associated complications.
SPIO particles were safely and effectively endocytosed into the cell cytoplasm. DAPI is a fluorescent dye for DNA-specific binding with no significant cytotoxicity because it can penetrate the cell membrane. It quickly penetrates living cells and binds to DNA. DAPI-DNA complexes have excitation and emission wavelengths of 360 and 460 nm, respectively, and under excitation with UV light, they are visualized by blue fluorescence [Ocarino et al., 2008]. In addition, the high spatial resolution of MR permitted in vitro observations and the in vivo visualization of cell migration, survival and proliferation using noninvasive SPIO-labeled cells. The double-labeling method was both complementary and mutually authentic.
secretion of attractive chemokines. As reported in the literature [Liu and Velazquez, 2008], following tissue damage, SDF-1 and other chemokines become highly expressed, which allows damaged tissues to recruit BMSCs. In this experiment, a high-sugar, high-fat diet combined with a low dose of STZ induced pathological changes to the pancreas and kidney indicative of T2DM. Reinfusion of SPIO/DAPI-double-labeled cells in the model animal pancreas and kidney was confirmed by histopathological examination, which revealed the distribution of BMSCs. This verified that BMSCs home primarily to these damaged tissues and organs and repair the damage.
Conclusions
BMSCs for Treatment of T2DM in a Macaque Model
In summary, BMSCs were transplanted into the pancreatic artery of macaques to treat T2DM. We demonstrated that this transplantation is a safe and effective treatment for T2DM in a macaque model. It reduced the level of FBG, lipids and other metabolic parameters, increased the level of serum C-peptide and reduced insulin resistance. The transplanted BMSCs also improved numerous bodily and cellular functions and repaired damaged tissue. In addition, SPIO particles provide a safe and effective method for tracking stem cells in vivo. Tracking with MR will become a future method for the effective detection and tracking of the migration and survival of stem cells in vivo.
Acknowledgments This work was supported by the National Natural Science Foundation of China (31172170), 973 Projects (2012CB518106) and the Special Project of High-New Technology Industrial Development in Yunnan Province (201204). We thank American Journal Experts for assisting in the preparation of the manuscript.
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