Bone marrow cell cotransplantation with islets improves their vascularization and function

Abstract

BACKGROUND.: To test the angiogenesis-promoting effects of bone marrow cells when cotransplanted with islets. METHODS.: Streptozotocin-induced diabetic BALB/c mice were transplanted syngeneically under the kidney capsule: (1) 200 islets, (2) 1 to 5x10 bone marrow cells, or (3) 200 islets and 1 to 5x10 bone marrow cells. All mice were evaluated for blood glucose, serum insulin, and glucose tolerance up to postoperative day (POD) 28, and a subset was monitored for 3 months after transplantation. Histologic assessment was performed at PODs 3, 7, 14, 28, and 84 for the detection of von Willebrand factor (vWF), vascular endothelial growth factor (VEGF), insulin, cluster of differentiation-34, and pancreatic duodenal homeobox-1 (PDX-1) protein. RESULTS.: Blood glucose was the lowest and serum insulin was the highest in the islet+bone marrow group at POD 7. Blood glucose was significantly lower in the islet+bone marrow group relative to the islet only group after 63 days of transplantation (P<0.05). Significantly more new periislet vessels were detected in the islet+bone marrow group compared with the islet group (P<0.05). Vascular endothelial growth factor staining was more prominent in bone marrow than in islets (P<0.05). Pancreatic duodenal homeobox-1-positive areas were identified in bone marrow cells with an increase in staining over time. However, there were no normoglycemic mice and no insulin-positive cells in the bone marrow alone group. CONCLUSIONS.: Cotransplantation of bone marrow cells with islets is associated with enhanced islet graft vascularization and function.

Figure 1

Blood glucose (A) and serum insulin (C) after islet and / or bone marrow transplantation up to postoperative day (POD) 28. Blood glucose was lowest in the islet + bone marrow group and was decreased significantly compared to the bone marrow and sham groups. The same tendency was observed in serum insulin, which was highest in the islet + bone marrow group. There was a significant increase in serum insulin in the islet + bone marrow group relative to the islet group at POD 28. The percentage of animals reaching normoglycemia (B) was higher in the islet + bone marrow group (46.2%, 6/13) than in the islet group (25%, 3/12) (p=0.32) and there were no normoglycemic mice in the bone marrow only or sham groups. There was also no significant difference between the islet (3.7±0.5 days) and islet + bone marrow (5.8±1.7 days) groups in the days to normoglycemia (B). Glucose tolerance tests (GTT) at POD 7 (D), POD 14 (E) and POD 28 (F) revealed that there was a significant improvement in the islet + bone marrow group compared to the bone marrow and sham groups at POD 7. Statistical analysis was performed by two ways repeated measurement ANOVA and significant difference was p<0.05.

Figure 2

Blood glucose (A), serum insulin (B) and GTT (C) after transplantation up to POD 84. Mice were selected randomly for either histological assessment or for long term observation. Blood glucose decreased significantly in the islet + bone marrow group compared to the islet group about two months after transplantation (= POD 63, 70, 77 and 84). Serum insulin was also increased at POD 28. No significant difference was seen between islet + bone marrow and islet groups in GTT. Statistical analysis was performed by two ways repeated measurement ANOVA and significant difference was p<0.05.

Figure 3

Photomicrographs of fresh kidneys recovered from islet, bone marrow and islet + bone marrow group mice at POD3, 7, 14, 28 and 84. While new vessels could not be detected in any groups at POD 3, we saw newly formed vessels (arrows) in and around the transplant site (outline) in bone marrow and islet + bone marrow groups at POD 7 and more prominently at POD 14. Mature vasculature was seen at POD 28 and 84.

Figure 4

vWF staining of subcapsular grafts. A. More new vessels (positive for vWF, indicated as arrow) could be detected in the islet + bone marrow group than in the islet group. (magnification = 100x and bar = 500 μm). B. Vessel numbers in the field of view (magnification is 100x) were significantly more in bone marrow and islet + bone marrow groups compared to the islet group at POD 14, 28 and 84. C. Vessel numbers divided by graft area (mm²) were also significantly more in bone marrow and islet + bone marrow groups at POD 3 and 7. Statistical analysis was performed by Dunnet test and significant difference was p<0.05.

Figure 5

A. Fluorescent double staining for insulin and VEGF (upper) and for CD34 and VEGF (lower). VEGF staining was more prominent within CD34 positive cells (=bone marrow cells) than within insulin positive cells (=β cells) at POD 14 and 28. B. Ratio of VEGF positive area to total CD34 positive or insulin positive cells area (=%VEGF). The ratio was significantly higher in VEGF/CD34 (both bone marrow and islet bone marrow groups) than VEGF/insulin in islet group. Statistical analysis was performed by Dunnet test and significant difference was p<0.05.

Figure 6

A. Ratio of PDX-1 positive area to CD34 positive area in bone marrow and islet + bone marrow groups. Change in the ratio over time within each group was tested. Significant difference was seen in the bone marrow group between POD 3 and 14. B. Fluorescent double staining for PDX-1 (red) and CD34 (green). Double stained cells were seen at POD 14 and 28. Statistical analysis was performed by Dunnet test and significant difference was p<0.05.