The authors have declared that no competing interests exist.
The bone marrow cavity (BMC) has recently been identified as an alternative site to the liver for islet transplantation. This study aimed to compare the BMC with the liver as an islet allotransplantation site in diabetic monkeys. Diabetes was induced in Rhesus monkeys using streptozocin, and the monkeys were then divided into the following three groups: Group1 (islets transplanted in the liver with immunosuppressant), Group 2 (islets transplanted in the tibial BMC), and Group 3 (islets transplanted in the tibial BMC with immunosuppressant). The C-peptide and blood glucose levels were preoperatively measured. An intravenous glucose tolerance test (IVGTT) was conducted to assess graft function, and complete blood cell counts were performed to assess cell population changes. Cytokine expression was measured using an enzyme-linked immune sorbent assay (ELISA) and MILLIPLEX. Five monkeys in Group 3 exhibited a significantly increased insulin-independent time compared with the other groups (Group 1: 78.2 ± 19.0 days; Group 2: 58.8 ± 17.0 days; Group 3: 189.6 ± 26.2 days) and demonstrated increases in plasma C-peptide 4 months after transplantation. The infusion procedure was not associated with adverse effects. Functional islets in the BMC were observed 225 days after transplantation using the dithizone (DTZ) and insulin/glucagon stains. Our results showed that allogeneic islets transplanted in the BMC of diabetic Rhesus monkeys remained alive and functional for a longer time than those transplanted in the liver. This study was the first successful demonstration of allogeneic islet engraftment in the BMC of non-human primates (NHPs).
Allogeneic islet transplantation has been considered a potential treatment for type 1 diabetes mellitus (T1DM) [
The instant blood-mediated inflammatory reaction is an innate immune response that causes significant islet graft loss[
Therefore, many studies have focused on developing alternative sites for islet transplantation. Perez et al. reported the long-term survival and function of allogeneic islet transplantation in the anterior chamber of the eye in diabetic baboons [
Recently, the bone marrow cavity (BMC) has been suggested as an alternative site for islet transplantation[
Non-human primates (NHPs) are an outstanding preclinical animal model because of their high genetic and immunological similarities to humans[
Rhesus monkeys (3–5 years old, 5.8–6.9 kg in weight) were purchased from the Chengdu Ping'an Experimental Animal Reproduction Center (License No. SCXK (CHUAN) 2014–013, Chengdu, China). Diabetes was induced in the monkeys (fasting blood glucose (FBG) levels > 11.1 mmol/L on two consecutive days) with a single intravenous injection of streptozocin (STZ, 80 mg/kg, Sigma-Aldrich, CA, USA) 30 days prior to transplantation[
The animal protocols used in this work were approved by the Institutional Animal Care and Use Committee of the Traditional Chinese Medicine National Center (Chengdu, China) (Protocol:IACUC-2014001C), and the protocols strictly abided by the Laboratory Protocol of Animals created by the IACUC. The animals were housed at the Traditional Chinese Medicine National Center(Chengdu, China)in separate stainless steel cages (800×900×2060 mm) at a constant temperature (16–26°C) in HEPA-filtered air under a 12-h light and dark cycle from 7:30am-7:30pm and received insulin injections twice daily before feeding. The monkeys were provided opportunities to play with toys (polyvinyl chloride (PVC) toys and mirrors) while in their cages, and they could perch on the crossbars in the cages. The animal care staff and technicians talked softly to animals to provide animals with auditory stimulation and made good visual contact with the animals. The animals were fed a diet of monkey chow (Ke’ao Xieli Feed Co. Ltd., Beijing, China) twice daily and received sterilized water adlibium. None of the monkeys died during the experiment prior to euthanasia.
The islets were purified and isolated as previously described [
Super paramagnetic iron oxide (SPIO) was kindly provided by the College of Materials Science and Engineering of Sichuan University. A 5-μl dose of SPIO (200 μg/ml) was mixed with the islets in 5 ml of 1640 medium, and the mixture was cultured at 37°C for 2 hours. The islets were then washed and collected. For control purposes, the SPIO alone and islets alone were transplanted into the tibiae of monkeys.
After 16 hours of fasting, the recipient monkeys received an intramuscular (IM) injection of ketamine (10 mg/kg, Jiangsu Hengrui Medicine Co. Ltd., Jiangsu, China) and 20 m/kg IV propofol and fentanyl (Jiangsu Hengrui Medicine Co. Ltd., Jiangsu, China) to induce anesthesia. The animals were administered isoflurane and monitored using a breathing machine during islet transplantation. After surgery, the animals were injected with tramadol hydrochloride (100 mg i.m. every 12 hours) for analgesia for a total of 24 hours. The intrahepatic group(Group 1, n = 5) received an islet transplant into the liver as previously described [
Low-molecular-weight heparin was administered for anticoagulation; methylprednisolone served as an anti-inflammatory agent; anti-human thymocyte globulin (ATG), tacrolimus and sirolimus were administered to maintain immunosuppression (Sigma-Aldrich, CA, USA) (
Treatment | Dose | Time Point | Route |
---|---|---|---|
300 IU/Day | Days -2,-1, and 0 | i.m. | |
250 mg | Day -1 | i.m. | |
25 mg | Day 0 | i.m. | |
4 mg/kg | Days -1, 0, 1, and 2 | i.v. | |
0.2 mg/kg/d | From day of Tx until graft failure | p.o. | |
0.2 mg/kg/d | From day of Tx until graft failure | p.o. |
i.m., intramuscular; i.v., intravenous; p.o., per os (by mouth); Tx, transplantation
The graft function was evaluated by assessing the fasting blood glucose (FBG, every morning before feeding), fasting C-peptide (every 2 months), fasting insulin (every 2 months), exogenous insulin(every day depending on the FBG), and glycated hemoglobin (HbA1c, every 2 months) levels. The C-peptide and insulin levels in the bone marrow were measured every 2 months following islet transplantation. IVGTT was performed prior to STZ injection (normal group), after STZ injection(hyperglycemia group), and 1, 2, 4, and 6 months after transplantation. Briefly, after 16 hours of fasting, the monkeys were intravenously administered glucose (0.5 g/kg). Blood specimens were collected 0, 1, 3, 5, 10, 30, 60, 90 and 120 minutes after glucose injection. The glucose levels were measured in all blood specimens obtained. The serum was collected and analyzed for insulin using an electrochemiluminescence immunoassay (Roche Diagnostics, Basel, Switzerland). One month after transplantation, the glucose levels (0, 1, 3, 5, and 10 minutes)were measured in the bone marrow when IVGTT was performed.
MRI scans of the tibia and the abdomen were performed 1, 30, and 225 days after transplantation on a clinical 3.0 T system (Sigma, General Electric, USA).The resultant images were quantitatively analyzed by manually counting the area of hypo-intense spots visible in the bone marrow tissue in each slice.
To assess the effect of islet infusion in the BM on the hematopoietic activity, aperipheral whole blood cell count was performed every 2 months after transplantation(Department of Experimental Medicine, West China Hospital).Kidney function and liver function were assessed using blood biochemical assays every 2 months after transplantation(Department of Experimental Medicine, West China Hospital).
Blood was collected from all monkeys on the day of the transplantation and 10 days, 1 month, and 3 months after transplantation. The serum was immediately separated and maintained at -80°Cfor future use.
The levels of high-sensitivity C-reactive protein (Hs-CRP), thrombin-antithrombin complex(TAT)and fibrinogen (Fbg) in the serum were measured using a monkey enzyme-linked immune sorbent assay (ELISA) kit (Cusabio Biotech Co.,LTD, Hubei, China). The human soluble intercellular adhesion molecule-1 (sICAM) level was measured using an ELISA kit (Invitrogen, CA, USA) following the manufacturer’s instructions. All data were measured using a plate reader (Molecular Devices).
The MILLIPLEX MAP NHP cytokine kit was purchased from Merck Millipore (Merck Millipore, BOSTON, USA). The kit contained specific components for the assessment of NHP IFN-γ, IL-2,IL-10 and TGF-β. The assays were performed according to the manufacturer’s instructions, and the samples were measured on a Luminex 200 with the Milliplex Analyst Software.
Monkeys in Group 3 (at the first sign of a loss of graft function) were euthanized with an overdose of pentobarbitone sodium (iv, 300mg/kg) 225 days after transplantation. The bone marrow was collected and stained with DTZ and Prussian blue. The tibia containing islet grafts was removed and fixed in 10% formalin, decalcified in 10% EDTA for 30 days at room temperature and then embedded in paraffin. For the immunofluorescent staining, 5-μm sections were stained with rabbit-anti-human insulin polyclonal antibody (1:100; LifeSpan BioSciences, USA) and goat-anti-human glucagon polyclonal antibody (1:500; Santa Cruz Biotechnology Inc., USA). The secondary antibodies included goat-anti-rabbit FITC (1:500) and rabbit anti-goat Cy3 (1:500)(Golden Bridge Biological Technology, Beijing, China). The nuclei were stained with DAPI(Golden Bridge Biological Technology, Beijing, China).
Peripheral blood and bone marrow from monkeys were collected 10 days after islet transplantation. The mononuclear fractions of the peripheral blood and bone marrow were isolated using LSM. The cells were stained with monoclonal antibodies (mAbs), including FITC-conjugatedanti-CD4,PE-conjugatedanti-CD25, APC-conjugated anti-Foxp3 (eBioscience, San Diego, USA),PE-Cy5-conjugated anti-CD3 and PE-conjugated anti-CD8 (Beckman-Coulter, Miami, FL)according to the manufacturer’s instructions.
All data are represented as the means ± standard deviations. One- or two-way ANOVAs followed by the t-test were used to assess the significance of differences using the SPSS software. For all analyses, a 2-tailed P value of 0.05 was considered significant.
All blood glucose levels in the recipient monkeys decreased after transplantation (
(A) Average FBG of diabetic monkeys following islet transplantation in each group. (B) Average exogenous insulin requirement expressed as IU/day. (C) Days of normal blood glucose levels without exogenous insulin. (D) Fasting serum C-peptide levels following islet transplantation. (E) HbA1c levels following transplantation. The data are expressed as the mean±SD. **,
The blood glucose changes in the IVGTT of the transplanted groups (Groups 1, 2 and 3) were similar to that of the normal group (
The IVGTT was measured 1, 2, 4, and 6 months after islet transplantation into the BM or liver. Normal and hyperglycemia were used as controls. (A-D) Blood glucose levels during IVGTT. (E-H) AUC for glucose during IVGTT. The data were expressed as the mean±SD. **,
The glucose level in the peripheral blood of Group3 was higher than that of the bone marrow 3 and10 minutes after glucose injection (
Comparison of blood glucose levels (Fig 3A) and AUC for glucose (Fig 3B) in the peripheral blood (PB) and bone marrow (BM) during IVGTT following islet transplantation in Group 3. The C-peptide level (Fig 3C) was measured in the bone marrow and peripheral blood. Tibial bone was harvested 225 days after transplantation and stained for insulin and glucagon to confirm islet survival. The bone marrow collected from the tibia 225 days after islet transplantation was stained with DTZ(Fig 3E) and Prussian blue (Fig 3F). Tibial bone was stained for insulin and Prussian blue to confirm SPIO (Fig 3G).**,
The immunofluorescence staining of tibiae revealed the presence of insulin and glucagon-positive cells at the site of transplantation (
Animals underwent MRI scans 1, 30, and 225 days after islet, SPIO, or islet+SPIO transplantation. As shown in
MRI was performed 1, 30 and 225 days after islet(left), SPIO (left middle) and islet+SPIO (right middle) transplantation. MRIs of the liver were acquired 1and 30 days after islet+SPIO (right) transplantation. A small hypo-intense area (black circle) inside the normal hyperintense signal was evident at the site of the islet infusion at the level of the tibia. The area of hypointense spots visible in the bone marrow tissue and liver in each slice in different groups (below). **,
The peripheral blood was measured every 2 months after transplantation and did not show significant differences in the hematopoietic parameters, including the platelet count (PLT), hemoglobin(HGB),red blood cell, and white blood cell counts, among the three groups (
The hematopoietic activity of the BM (HGB, RBC, PLT, WBC; Figure 5A), Liver function (ALT, AST, GGT; Figure 5B)and the kidney function (BUN, Scr, UA; Figure 5C) were measured every 2 months following transplantation. The data are presented as the means ± SD. A statistical analysis was performed using two way ANOVAs.
The serum samples from each group were collected on 0, 10, 30, and 90 days after transplantation, and the resultant data are shown in
The serum IFN-γ, IL-2, IL-10 and TGF-β levels were measured following islet transplantation. The data are presented as the means ± SEM. **,
The expression levels of CD3+CD4+ and CD3+CD8+ were significantly lower in bone marrow cells than in the peripheral blood (both
Bone marrow and peripheral blood were collected from Group 3 10 days after transplantation. (A) CD4+ and CD8+ T cells and (B) Treg cells were analyzed. (C) Percentage of lymphocyte subsets. Data are presented as the means ± SEM. **,
Currently, the liver is the preferred receptor site for clinical islet transplantation, but this approach is associated with a number of important limitations. The purpose of our study was to investigate the BMC as an alternative site for islet allotransplantation in a preclinical model. We are the first group to demonstrate that transplanting islets in the BMCs of diabetic NHPs reversed diabetes.
Specifically, our results showed that islets could be easily and safely transplanted into the BMC, and no side effects were observed in Rhesus monkeys that had undergone BMC islet transplantation. Furthermore, none of the monkeys that had received a BMC transplant developed hypoglycemia after transplantation, and routine whole blood or blood biochemical indices of liver functionality (ALT, AST and GGT) and kidney functionality(BUN, Scr and UA)did not significantly differ between groups until one year after islet transplantation. Transplantation into the BMC is considered a less invasive and low-risk ambulatory procedure in clinical settings that provides unlimited opportunities for repeated transplantation and an easily accessible site for engraftment[
Moreover, islets engrafted in the BMC exhibited better function than those transplanted in the liver. Specifically, our results demonstrated that islets transplanted to the BMC remained insulin-independent longer than intrahepatic transplanted islets(189.6 ± 26.2 days vs. 78.2±19.0 days). The IVGTT result also confirmed that the islet graft was functioning well in the BMC. Kover et al. reported that isograft islets transplanted in the BMC of dark agouti rats lasted longer (10–14 days) than islets transplanted into the liver and kidney capsule (3 days) [
Changes in the graft were more easily monitored in the BMC than in the liver. After the islets were transplanted into the liver via the hepatic portal vein, the islets were immediately randomly scattered, which may have precluded the detection of hypo-intense spots 30 days after transplantation into the liver. Therefore, harvesting biopsy samples of the islets to monitor early graft changes was difficult in the liver transplantation group, and Toso C et al. reported that less than30%of biopsies contained the desired islets [
In this study, bone marrow samples were collected 10 days after transplantation, and flow cytometry was performed to measure the changes in T cell (CD3, CD4 and CD8) and Treg populations. CD3+CD4+ T cells are important in the initiation of the immune response because they recruit macrophages[
To better elucidate the mechanisms involved in graft survival, we also analyzed growth factors (IL-2, IL-10, TGF-β, and IFN-γ) secreted by cells in the BMC because other factors secreted in the BMC, such as osteocalcin, EGFR, and VEGFA, have been reported to enhance the survival and function of islet grafts[
In conclusion, we demonstrated that islets were easily and safely transplanted and monitored in the BMC. Thus, the tibial BMC is an alternative site for clinical islet transplantation and cellular transplantation for other diseases.
Comparison of blood glucose levels (S1A Fig) and AUC for glucose (S1B Fig) in the peripheral blood (PB) and bone marrow (BM) during IVGTT following islet transplantation in Group1, Group 3 and normal group. **,
(TIF)
The authors would like to thank Professor Hua Ai (Sichuan University, China) for providing SPIO. Careful reading of this manuscript by Dr. Xi Jin is also greatly appreciated.