The aim of this study was to investigate whether tolerance-induced protection of islets in the renal subcapsular space can also prevent subcutaneous allogeneic islets from being rejected. We used bone marrow stem cells from C57BL/6 (H2b) mice to construct donor chimerism in conditioned diabetic BALB/c (H2d) mice and investigated the effect of donor chimerism on engraftment and survival of subcutaneously transplanted allogeneic islets in streptozotocin-induced diabetic mice. We also studied the anti-inflammatory effect of mesenchymal stem cell on islet engraftment. Full but not low-grade or no donor chimerism was associated with successful engraftment of allogeneic islets and restoration of normoglycemia in the treated diabetic mice. The temporary hyperglycemia was 11 ± 1 versus 19 ± 5 days (
Transplantation of islets into the subcutaneous space to treat diabetes has been studied for decades; however, it has yet to be applied clinically [
All chemicals, including streptozotocin, histopaque-1077, and type XI collagenase, were obtained from Sigma Chemical Company (St. Louis, MO, USA). RPMI-1640 medium was purchased from GIBCO Invitrogen, Life Technologies, Inc. (Grand Island, NY, USA). Intravenous busulfan (Busulfex) was sourced from P.D.L. Bio Pharma, Inc. (Incline Village, NV, USA), and cyclophosphamide was supplied by Baxter (Deerfield, IL, USA). Anti-insulin antibodies were obtained from Abcam Inc. (Cambridge, MA, USA). Whole blood glucose was determined using a One Touch II portable glucometer (Lifescan Inc., Milpitas, CA, USA) using dry reagent technology based on the glucose oxidase method via tail snipping. Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC) were gifted by Dr. Ing-Kae Wang from the Industrial Technology Research Institute of Taiwan.
Inbred, 8 to 12 weeks old male BALB/c (H2d) and C57BL/6 (H2b) mice were supplied by local breeders. Three to five mice were housed in each cage and fed with a standard pelleted food and tap water ad libitum. The animal room had an automatic lighting cycle with 12 hours of light and 12 hours of darkness. To induce diabetes, the BALB/c (H2d) mice received a single intraperitoneal injection of streptozotocin 200 mg/kg body weight. The mice with a whole blood glucose level exceeding 360 mg/dL for more than 2 weeks were used as the diabetic recipients. The body weight and nonfasting whole blood glucose levels of the mice were determined twice a week between 8:00 AM and 10:00 AM. After islet transplantation, the cure of diabetes in the recipients was defined as a whole blood glucose levels maintained below 200 mg/dL for two consecutive tests and thereafter. The days of temporary hyperglycemia were calculated as the duration before diabetes was cured. All of the animals were treated humanely in accordance with the laboratory animal guidelines of Chang Gung Memorial Hospital.
Pancreatic islets were isolated from the C57BL/6 (H2b) mice by collagenase digestion, enriched on a histopaque density gradient, and finally hand-picked. Briefly, under anesthesia, the pancreases of the nonfasted healthy mice were distended with 2.5 mL RPMI-1640 medium containing 1.5 mg/mL collagenase and then excised and incubated in a 37°C water bath. The islets were purified using a density gradient and were hand-picked under a dissecting microscope. Isolated islets with diameters of 125–150
The islets of C57BL/6 (H2b) mice were centrifuged in PE-50 tubing connected to a 200
The islets of C57BL/6 (H2b) mice were mixed with 30
With the donor C57BL/6 (H2b) mice under anesthesia, both femurs and tibiae were removed using a sterile procedure and bone marrow cells (BMCs) were flushed out of the bone into phosphate buffer solution (PBS). After being gently dispersed by pipetting cells in and out, whole bone marrow cells were washed twice in PBS and recovered by centrifugation at 1150 ×
The induction of donor chimerism in the BALB/c mice was modified from a previously described protocol [
Mice in group A (
A diagrammatic timetable illustrating the experimental design and grouping. The day of islet transplantation is day 0. BMCs: bone marrow cells; WJ-MSC: Wharton’s jelly-derived mesenchymal stem cells.
Kaplan-Meier plot of cumulative cure rate of diabetes. Diabetic mice in both groups A and B received conditioning and BMC transplantation. The mice in group A received 300 allogeneic islets under the left kidney subcapsular space, and the mice in group B received 900 allogeneic islets under the subcutaneous space of the back. After islet transplantation, nonfasting whole blood glucose levels of the recipients were determined twice weekly, and the cure of diabetes was defined as a whole blood glucose level below 200 mg/dL for two consecutive tests and thereafter. The difference in cumulative cure rate of diabetes between two the groups was analyzed with the log-rank test, and the
Immunohistochemical examination of the retrieved islet grafts. The graft-bearing kidneys and the subcutaneous grafts from the mice in groups A, B, C, and D were retrieved at 20 to 30 weeks posttransplantation. The immunohistochemical examinations were performed using anti-insulin antibodies. The secondary antibody was prepared from rabbits and conjugated with horseradish peroxidase, and diaminobenzidine was used as the chromogenic substance. The dark-brownish precipitates indicate insulin-containing cells. (a), (b), (c), and (d) show the graft histology of mice in groups A, B, C, and D, respectively. Photos are 40 × 10 magnification. Scale bars indicate 0.1 mm.
The effect of donor chimerism on blood glucose and body weight changes in the mice. All mice in groups A and C received 300 allogeneic islets under the left kidney subcapsular space. The mice in group A received conditioning and BMC transplantation, whereas the mice in group C received conditioning but no BMC infusion. The nonfasting whole blood glucose level (a) and body weight (b) of the mice in groups A and C were determined twice weekly. All mice in groups B and D received 900 allogeneic islets under the subcutaneous space of the back. The mice in group B received conditioning and BMC transplantation, whereas the mice in group D received conditioning and transplantation of both BMCs and MSCs. The nonfasting whole blood glucose level (c) and body weight (d) of the mice in groups B and D were determined twice weekly.
Chimerism in the BALB/c mice was detected by staining for H2-Db-expressing (C57BL/6 donor) cells that were distinguishable from BALB/c cells expressing H2-Dd. Other antibodies used for chimerism analysis including CD45, CD4, CD8, CD11b, CD11c, CD19, Ly6G/6C (Gr1), and Foxp3 were purchased from BD Biosciences (San Jose, CA, USA), eBioscience Inc., (San Diego, CA, USA), and BioLegend Inc., (San Diego, CA, USA). Peripheral blood was sampled to determine the degree of chimerism in the recipient mice. A volume of 0.15 mL of whole blood was collected in 0.5 mL of PBS containing 3 mg/mL of EDTA and 5 U/mL of heparin. After red blood cells had been lysed, approximately 2-3 × 105 cells were incubated with different antibodies, and the cells were then washed and analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA).
We identified T cell subsets by flow cytometry analysis as naive T cells (TN, defined as CD44loCD62L+), central memory T cells (TCM, defined as CD44hiCD62L+), and effector memory T cells (TEM, defined as CD44hiCD62L−). Antibodies were purchased from BD Biosciences (San Jose, CA), eBioscience (San Diego, CA), and BioLegend (San Diego, CA) and included anti-CD44 (IM7), anti-CD62L (MEL-14), anti-CD4 (RM4-5), and anti-CD8 (53.6.7). After primary staining, the cells were washed three times and then run on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) and analyzed using FlowJo software (TreeStar, Ashland, OR).
Subcutaneous graft and graft-bearing left kidneys of the recipient mice were removed for histological examination at sacrifice. Immunohistochemical examinations of tissue sections were performed using anti-insulin antibodies. The secondary antibody was prepared from rabbits and conjugated with horseradish peroxidase, and 3,3′-diaminobenzidine was used as the chromogenic substrate.
Insulin content in the pancreas remnant was extracted using the acid-ethanol method. Briefly, the pancreases from nonfasted mice were removed randomly between 8:00 AM and 10:00 AM, homogenized in an acid-ethanol solution, and stored at 4°C overnight. After centrifugation at 2400 rpm for 30 minutes at 4°C, the supernatant was collected and stored at −20°C. The pancreas remnants were then homogenized again in a new aliquot of acid-ethanol solution, and insulin was reextracted overnight. Following centrifugation, the supernatant was collected and pooled with the first extracted sample. Finally, the insulin concentration was measured using a mouse-specific ELISA kit (Ultrasensitive Mouse Insulin ELISA, Mercodia, Uppsala, Sweden).
Data are expressed as mean ± standard error. Statistical differences between means were analyzed using a paired or unpaired Student’s
A state of high-grade (>60%) multilineage donor chimerism of the hematopoietic cells of C57BL/6 (H2b) origin was rapidly established in the conditioned diabetic BALB/c (H2d) mice in both group A (renal site) and group B (subcutaneous site) at 2 weeks post-BMC transplantation (Table
Donor chimerism achieved in the peripheral blood of mice that received conditioning and bone marrow cell and/or mesenchymal stem cell transplantation.
Donor chimerism (%) | Group A |
Group B |
Group D |
|||
---|---|---|---|---|---|---|
Time | 2 weeks | 8 weeks | 2 weeks | 8 weeks | 2 weeks | 8 weeks |
CD4 | 61.6 ± 8.9 | 98.8 ± 0.5 | 69.4 ± 12.9 | 98.7 ± 1.2 | 0.3 ± 0.1 | 1.6 ± 0.5 |
CD8 | 94.0 ± 0.9 | 97.2 ± 0.4 | 91.2 ± 3.4 | 98.3 ± 0.9 | 4.8 ± 3.0 | 4.1 ± 1.7 |
CD45 | 94.3 ± 2.1 | 99.0 ± 0.4 | 94.8 ± 5.2 | 99.7 ± 0.1 | 3.3 ± 3.1 | 0.1 ± 0.0 |
CD11b | 94.2 ± 0.8 | 96.6 ± 2.6 | 94.4 ± 4.4 | 98.8 ± 0.2 | 3.8 ± 3.6 | 0.8 ± 0.2 |
CD11c | 56.6 ± 8.3 | 81.5 ± 1.5 | 62.3 ± 4.4 | 74.1 ± 3.0 | 2.1 ± 1.1 | 1.9 ± 0.7 |
CD19 | 90.5 ± 2.6 | 98.6 ± 0.1 | 86.8 ± 9.1 | 98.2 ± 0.7 | 2.4 ± 2.0 | 2.1 ± 0.7 |
Gr1 | 90.5 ± 1.4 | 96.2 ± 1.2 | 92.6 ± 5.0 | 97.8 ± 0.3 | 2.3 ± 2.0 | 1.8 ± 0.4 |
Foxp3 | 30.8 ± 9.3 | 58.1 ± 1.6 | 38.1 ± 1.4 | 50.8 ± 1.8 | 9.1 ± 3.4 | 9.4 ± 0.4 |
Chimerism in the BALB/c mice was detected by staining for H-2Db-expressing (C57BL/6 donor) cells that were distinguishable from BALB/c cells expressing H-2Dd at 2 and 8 weeks posttransplantation. Other antibodies used for chimerism analysis included CD45, CD4, CD8, CD11b, CD11c, CD19, Ly6G/6C (Gr1), and Foxp3. All comparisons between group A and group B did not differ. All comparisons between group B and group D were statistically significant and had a
The time frame of myeloid cell development during the establishment of chimerism revealed that more than 80% of the peripheral myeloid cells were CD11b+ and Gr1+ cells at 2 weeks after BMC transplantation in the mice in groups A and B which had established high-grade donor chimerism (Table
Population of different cells developed in the peripheral blood of mice that received conditioning and bone marrow cell and/or mesenchymal stem cell transplantation.
Cell population (%) | Group A |
Group B |
Group D |
|||
---|---|---|---|---|---|---|
Time | 2 weeks | 8 weeks | 2 weeks | 8 weeks | 2 weeks | 8 weeks |
CD4 | 2.8 ± 1.0 | 9.7 ± 1.9 | 2.3 ± 1.3i | 7.1 ± 2.8o | 15.2 ± 2.8i | 17.9 ± 1.6o |
CD8 | 8.5 ± 1.9 | 3.6 ± 0.5 | 5.9 ± 1.0 | 4.5 ± 0.7 | 6.8 ± 1.8 | 6.3 ± 0.8 |
CD45 | 93.1 ± 1.5 | 92.8 ± 1.8 | 91.9 ± 1.0 | 89.8 ± 1.9 | 88.9 ± 1.3 | 98.2 ± 0.3 |
CD11b | 83.4 ± 1.5 | 48.3 ± 6.6 | 85.7 ± 2.8j | 53.3 ± 14.7 | 57.4 ± 2.7j | 44.6 ± 4.9 |
CD11c | 9.0 ± 2.2a,b | 6.2 ± 1.1e,f | 3.9 ± 0.6a,k | 1.1 ± 0.2e,p | 11.8 ± 4.3b,k | 9.6 ± 2.5f,p |
CD19 | 1.1 ± 0.2c | 15.7 ± 2.3g | 5.8 ± 1.7l | 17.7 ± 5.5 | 15.7 ± 4.7c,l | 19.9 ± 4.9g |
Gr1 | 81.2 ± 1.5 | 45.1 ± 6.8 | 81.1 ± 3.3m | 49.9 ± 15.4 | 61.6 ± 5.1m | 54.0 ± 5.0 |
Foxp3 | 4.9 ± 0.8d | 6.5 ± .0.5h | 5.3 ± 1.2n | 5.9 ± 1.2q | 10.4 ± 1.9d,n | 10.7 ± 0.6h,q |
The mean frequency of different cells in the venous blood was measured by flow cytometry at 2 and 8 weeks posttransplantation. a,b,f,g,l,n
T cell subsets were identified by flow cytometry analysis.
T subset (%) | Group A |
Group B |
Group D |
|||
---|---|---|---|---|---|---|
Time | 2 weeks | 8 weeks | 2 weeks | 8 weeks | 2 weeks | 8 weeks |
CD4-TN | 31.1 ± 5.4 | 66.5 ± 9.2 | 38.4 ± 11.3 | 59.6 ± 15.9 | 60.8 ± 7.9 | 31.2 ± 9.5 |
CD4-TEM | 37.7 ± 5.5 | 14.6 ± 8.9 | 35.8 ± 8.6a | 24.6 ± 13.8 | 12.0 ± 3.8a | 15.3 ± 1.9 |
CD4-TCM | 5.5 ± 1.0 | 9.9 ± 1.5 | 5.6 ± 1.0 | 9.1 ± 1.6 | 8.7 ± 1.0 | 9.2 ± 2.5 |
CD8-TN | 1.4 ± 0.3 | 47.4 ± 9.5 | 3.4 ± 1.5b | 34.4 ± 15.9 | 15.7 ± 2.8b | 12.4 ± 4.3 |
CD8-TEM | 90.7 ± 0.9 | 29.6 ± 9.4 | 86.0 ± 1.9c | 49.9 ± 17.2 | 41.9 ± 6.1c | 31.8 ± 3.8 |
CD8-TCM | 4.3 ± 0.4 | 16.0 ± 1.5 | 7.9 ± 1.2d | 13.9 ± 2.4 | 21.7 ± 3.5d | 22.2 ± 3.3 |
Naive T cells (TN), central memory T cells (TCM), and effector memory T cells (TEM) were defined as CD44loCD62L+, CD44hiCD62L+, and CD44hiCD62L−, respectively. a
The duration of temporary hyperglycemia in the mice in group A that received 300 islets under the renal capsule was significantly shorter than that in the mice in group B that received 900 islets subcutaneously (10.8 ± 1.2,
Full donor chimerism of hematopoietic cells in group A enhanced engraftment of renal subcapsular donor islets and restored normoglycemia at 2 weeks posttransplantation (basal: 387 ± 18 mg/dL versus 2 weeks: 131 ± 15 mg/dL,
Bethge et al. previously analyzed the outcomes in patients that received donor lymphocyte infusion (DLI) for low or falling chimerism after nonmyeloablative conditioning for allogeneic hematopoietic cell transplantation. They found that patients who remained low donor CD3 chimerism (<5%) eventually rejected their grafts, suggesting that low donor CD3 chimerism appeared to predict rejection [
In this study, full multilineage donor chimerism was achieved rapidly after two infusions of BMCs into the conditioned diabetic BALB/c mice regardless whether that islets were implanted into the subcutaneous or renal subcapsular space. The achievement of full donor chimerism was associated with a higher transient expansion of CD11b+ and Gr-1+ myeloid progenitor cells and effector memory CD4 and CD8 T cells in both the subcutaneous and renal subcapsular islet transplantation groups. Our results suggest that the site of islet transplantation did not affect the establishment of donor chimerism using nonmyeloablative conditioning and BMC transplantation. Previous study has shown that induction of bone marrow chimerism in irradiated mice is associated with a transient expansion of CD11b+ Gr-1+ cells, defined as myeloid-derived suppressor cells (MDSC) in mice, with in vitro T cell suppressive activity [
Graft-versus-host disease (GvHD) is a pathologic attack by donor T cells on normal host tissues including the skin, liver, and gastrointestinal tract. Mohty et al. found that those who have full donor T cell chimerism at day 30 had a higher incidence of grades 2–4 acute GvHD compared with mixed T cell chimerism cases [
In conclusion, full donor chimerism protected both renal subcapsular and subcutaneous allogeneic islets. Using the subcutaneous site for islet transplantation, many more islets and rapidly achieving full donor chimerism were required to restore normoglycemia. Recipient mortality due to graft-versus-host disease and/or loss of body weight offsets the prolonged survival of allogeneic islets from donor chimerism without immunosuppressive treatment.
The authors declare no conflicts of interest.
This work was supported by Chang Gung Memorial Hospital Research Grants CMRPG3E1571, CMRPG3E1572, CMRPG3E1573, and CMRPG3F1451.