Mesenchymal Stem Cell and Islet Co-Transplantation Promotes ... : Transplantation (original) (raw)
A significant loss of islets immediately after transplantation has remained one of the major obstacles to the wide-spread application of islet transplantation in type 1 diabetic patients. Davalli et al. (1) showed a decline in islet cell survival, insulin content, and β-cell mass during the first 3 days after transplantation. Using magnetic resonance imaging of mice, Jirak et al. (2) showed a significant loss of signal soon after infusion of syngeneic islets into the liver. The posttransplantation signal loss continued during the first week and slowed during the second week. Major causes of islet cell loss include a lack of blood supply and inflammation associated with transplantation (3, 4). Pancreatic islets are micro- organs, which contain a dense capillary network, approximately 10 times higher than that of the surrounding exocrine tissue (5, 6). Although the islet cell mass comprises only 1% to 2% of the total pancreatic mass, islets receive 5% to 10% of the pancreatic blood flow (7). The blood vessels within the islets are lined with fenestrated endothelial cells (ECs), indicating that islets receive a greater partial pressure of oxygen (8, 9). Increased blood supplies are necessary not only for islet cell survival but also for normal islet cell function (10).
Islet isolation and culture processes not only destroy external vasculature but may also compromise the internal islet vascular network. Angiogenesis and revascularization involving complex processes starts 2 to 4 days after islet transplantation and are generally completed by 10 to 14 days (11–14). Both donor and recipient ECs contribute to revascularization of transplanted islets (15). However, the vascular density has been shown to be less and oxygen tension is lower in revascularized, transplanted islets than islets in the native pancreas (16, 17). The development of strategies that promote islet graft revascularization would significantly reduce islet cell loss and improve the outcome of islet transplantations.
As suggested by Brissova and Powers (18), revascularization of transplanted islets involves intra-islet ECs and regulatory factors, particularly vascular endothelial growth factor (VEGF)-A (10), to recruit ECs from recipient vasculature to form a new network. Furthermore, damage and hypoxia of the islets and surrounding liver cells may release signals for mesenchymal progenitor cells in the bone marrow (BM) to migrate and assist in wound healing and angiogenesis. BM-derived mesenchymal stem cells (MSCs), especially those immunoselected for STRO-1 and vascular-cell adhesion molecule 1 (VCAM1/CD106), produce VEGF-A, and are effective in supporting neovascularization in vivo (19). Brissova et al. (10) demonstrated that VEGF-A is essential for islet vascularization, revascularization, and function after transplantation. It has also been reported that MSCs can differentiate into ECs in vitro (20) and in vivo (21).
To test the effect of MSCs, we transplanted islets together with MSCs into the liver through the portal vein of syngeneic, diabetic Lewis (LEW) rats. Our results have shown that co-infusion of MSCs significantly improves islet graft function, reduces the islet number required for reversal of diabetes, markedly increases capillary formation, and preserves normal islet structure.
MATERIALS AND METHODS
Experimental Animals
Male LEW rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA) and male nonobese diabetic severe combined immunodeficiency (NOD SCID) mice were obtained from the City of Hope Animal Resource Center colony. Male LEW rats weighing 200 to 250 g were used as recipients and those weighing 300 to 350 g were used as donors. All animals were maintained in specific pathogen-free conditions at the City of Hope Animal Resource Center. The use of animals and animal procedures in this study was approved by the City of Hope/Beckman Research Institute Research Animal Care Committee.
MSC Isolation and Culture
Preparation of rat MSC followed the method previously described (22). Briefly, BM cells were obtained from femur and tibia of LEW rats by flushing with phosphate buffered saline (PBS) and treated with ACK lysis buffer (Invitrogen Corp., Carlsbad, CA) to remove red blood cells. BM cells were plated in a six-well plate (Nunc, Roskilde, Denmark) at a concentration of 107 cells per well in a MSC-specific medium composed of Iscove's modified Dulbecco medium, l-glutamine, antibiotics, insulin-transferrin-selenium (Invitrogen), linoleic acid-albumin, 10−4 M ascorbic acid, 10−9 M dexamethasone (Sigma-Aldrich, St. Louis, MO), 10 ng/mL of epidermal growth factor, 10 ng/mL of platelet-derived growth factor-BB (R&D Systems, Inc., Minneapolis, MN), 10 ng/mL leukemia inhibitory factor (Millipore Corp., Billerica, MA), and 20% fetal bovine serum (Omega Scientific, Inc., Tarzana, CA). After 48 hr, the medium was changed, unattached cells were discarded, and adherent cells were cultured for an additional week. Cells expanded in monolayers were subcultured when they reached 70% to 80% confluency. Most of the floating hematopoietic cells disappeared by the first passage. Cells were repeatedly subcultured or used for experiments between 3rd and 10th passages. MSCs were identified by morphology, phenotypic analysis by fluorescence-activated cell sorting, and the capability to differentiate into adipocytes or osteoblasts under specific conditions previously described (23). Supplemental Figure 1 shows representative results obtained from MSCs used in this study (see Supplemental Digital Content 1, https://links.lww.com/TP/A194). Tests were performed every 5 to 10 passages, with consistent results.
Labeling of MSCs
MSCs were labeled with either carboxyfluorescein succinimidyl ester (CFSE; Invitrogen) or fluorescent Qdot nanocrystals (Qtracker 605 cell labeling kit; Invitrogen) for identification in histologic sections. For CFSE labeling, MSCs grown in monolayer were harvested by trypsinization and resuspended in PBS. Approximately 107 MSCs were suspended in 1 mL of 5-μM CFSE in PBS and incubated at 37°C in the dark for 15 min. After incubation, cells were washed twice with culture medium and cultured overnight before use. For Qdot nanocrystal labeling, adherent cells were incubated in a 6-nM Qtracker labeling solution for 1 hr at 37°C, washed twice with medium, and then incubated in fresh medium overnight.
Preparation of Islets and MSCs for Transplantation
Islet Preparation
Islets were isolated from LEW pancreata using our standard procedure. Briefly, the pancreas was distended by infusing Hanks' balanced salt solution (HBSS; Sigma-Aldrich) supplemented with 0.1% bovine serum albumin (BSA; Sigma-Aldrich; HBSS/BSA) containing 1 mg/mL of Liberase (Roche Diagnostics GmbH, Mannheim, Germany), dissected out from the surrounding tissues, and incubated at 37°C for 30 min. After incubation, ice-cold HBSS/BSA was added to stop the enzymatic digestion, and the tissue was further dissociated by repeated shaking and washing cycles. Islets were purified by gradient centrifugation on Histopaque-1077 (Sigma-Aldrich), followed by handpicking to ensure high purity. For transplantation, islets were aspirated into a sterile PE50 tube, placed in a 15-mL tube, and centrifuged at 1150_g_ for 1 min. An islet number was determined by measuring the length of the packed islet mass by referring the standard curve that indicated the correlation between the length of packed rat islets in PE50 tube versus islet number. For transplantation, the tube was cut at the length containing a desired islet number by referring the standard curve. Islets were released from the tube into a sterile Eppendorf tube (Sigma-Aldrich), suspended in HBSS/BSA, and kept on ice until the time of transplantation. Before this study, the standard curve for rat islets was constructed by plotting the length of PE50 tube by packing handpicked, known number of islets ranging from 300 to 1500. The standard curve was further validated by counting the islet number eluted out from a known length of PE50 tube.
Mesenchymal Stem Cells
MSCs grown in monolayers were trypsinized, harvested from culture flasks, washed, resuspended in HBSS/BSA, and kept at room temperature until transplantation.
Co-Infusion of Islets and MSCs Into the Liver of Syngeneic, Diabetic LEW Rats and Monitoring Islet Graft Function
Islets were infused into the liver of syngeneic LEW recipients with or without MSCs. Recipients were made diabetic by intravenous injection of streptozotocin (70 mg/kg; Sigma-Aldrich) 7 days before transplantation. Diabetes was confirmed by blood glucose levels more than 350 mg/dL for two consecutive measurements. Just before transplantation, a desired number of islets and MSCs were suspended together in 0.2 mL HBSS and immediately infused into the portal vein of a diabetic recipient, under isoflurane anesthesia, using a 25-gauge winged needle connected to a 1-mL syringe. After transplantation, blood glucose was measured daily to twice weekly to monitor islet graft function. Blood glucose levels that decreased to less than 200 mg/dL indicated reversal of diabetes. Samples of the liver were obtained at specific time points after transplantation for immunohistochemistry.
Co-Transplantation of LEW Islets and MSCs Under the Renal Capsule of NOD SCID Mice
For immunohistologic study, 100 islets and 1×106 MSCs from LEW donors were mixed together and immediately placed under the kidney capsule of male NOD SCID mice. MSCs were labeled either with CFSE or Qdots before transplantation to locate cells in histologic examinations. After 6 days, the graft-bearing kidney was removed from the killed mouse, and slides were prepared for histology.
Immunohistochemical Examination
Antibodies
Guinea pig anti-insulin antibody was used as the primary antibody for insulin staining, and Cy2-labeled anti-guinea pig antibody was used as the secondary antibody. Rabbit anti-von Willebrand factor (vWF) antibody was used as the primary antibody to determine vascular ECs, followed by Cy2-conjugated anti-rabbit secondary antibody. Both primary antibodies were from DAKO (Carpinteria, CA), and both secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Antibody directed against VEGF was purchased from R&D Systems.
Immunohistochemistry
Five-micrometer-thick paraffin sections of rat livers and NOD SCID kidneys transplanted with islets and MSCs, either unlabeled or labeled with Qdots, were prepared by the City of Hope Anatomical Pathology Core facility. Kidneys bearing islets and CFSE-labeled MSCs were embedded in OCT compound (Ted Pella, Inc., Redding, CA) and snap-frozen in liquid nitrogen for frozen sections prepared by the core facility. Paraffin sections were stained for vWF and insulin. To obtain a ratio of capillary segments per β-cell, the largest area of the graft was selected, and the number of insulin-positive β-cells and vWF-positive capillary segments were counted in the entire field. Each strand of capillary segment was counted as one, regardless of length. Frozen sections containing CFSE-labeled MSCs were stained with anti-VEGF and anti-vWF antibodies. Sections containing Qdot-labeled MSCs were stained for insulin and vWF. Slides were visualized using a BX51 fluorescent microscope equipped with a Pixera 600 CCD camera. The pictures were processed using Adobe Photoshop.
Statistical Analysis
Values were shown by mean±standard error of the mean. Differences between groups were analyzed by two-tailed unpaired Student's t test and log-rank test. P values less than 0.05 were considered statistically significant.
RESULTS
Co-Infusion of MSCs Reduces the Islet Number Required for Reversal of Diabetes
Transplantation of 600 of syngeneic islets through the portal vein achieved reversal of diabetes in 83% (five of six) of LEW rats (Fig. 1A), whereas the success rate decreased to 30% (3 of 10) when transplanted with 500 islets (Fig. 1B). Therefore, the marginal syngeneic islet number that reverses diabetes in 50% of the recipients was approximately 550 islets in our hand. Ten million MSCs were mixed with 500 islets and infused into the liver through the portal vein. Islets co-transplanted with MSCs (islet-MSCs) reversed diabetes in all eight recipients compared with 3 of 10 in the islet-alone group (Fig. 1B). Diabetes was reversed only in 10% (1 of 10) of the recipients transplanted with 300 islets alone. In contrast, the same number of islets co-infused with MSCs reversed diabetes in 56% (five of nine) of the recipients (Fig. 1C). The difference in islet requirement between islet-MSCs and islet-alone transplant was clearly shown by the cumulative diabetes reversal curves (Fig. 2), demonstrating a significant improvement of islet graft function in the liver mediated by MSC co-transplantation.
Co-infusion of mesenchymal stem cells (MSCs) and islets reduces the number of islets required for reversal of diabetes. Approximately, 300 to 600 islets alone or with MSCs were infused in the liver of streptozotocin- diabetic, syngeneic Lewis rats, and blood glucose was monitored thereafter. (A) Approximately, 600 islets were sufficient to reverse diabetes in five of the six recipients receiving islets alone. (B) Approximately, 500 islets reversed diabetes in 3 of the 10 islets-alone and eight of the eight islet-MSCs co-transplant recipients. (C) Approximately, 300 islets reversed diabetes in 1 of the 10 islet-alone and five of the nine islet-MSCs recipients.
Cumulative diabetes reversal curves (% recipients reversed) demonstrate increased islet graft function promoted by mesenchymal stem cell co-transplantation (*P<0.05 and **P<0.01 by log-rank test).
Islet-MSCs Co-Transplant Recipients Exhibit Better Glucose Tolerance Than Islet-Alone Recipients
Intravenous glucose tolerance tests were performed on all recipients 56 days after intrahepatic transplantation of 300 or 500 islets with or without MSCs. Blood glucose levels after overnight fasting were less than or equal to 180 mg/dL even in those animals which had more than 200 mg/dL without fasting. The glucose disappearance curves were significantly better in the islet-MSCs recipients compared with those exhibited by islet-alone recipients (Fig. 3).
Co-infusion of mesenchymal stem cells (MSCs) with islets into the liver improves islet graft function. Intravenous glucose tolerance test was performed on day 56 in all recipients receiving 500 or 300 islets alone, or islet-MSCs, and six age-matched normal Lewis rats as controls (n=10 and 8 for 500 islets and n=10 and 9 for 300 islets). Fasting blood glucose levels were less than or equal to 180 mg/dL in all rats, although some recipients showed more than 200 mg/dL nonfasting blood glucose. Glucose disappearance was significantly faster in islet-MSCs recipients than islet-alone recipients (values were shown by mean±standard error of the mean; *P<0.05 and **P<0.01).
Co-Transplantation of MSCs Promotes Revascularization of Transplanted Islets
One possible mechanism involved in the improved islet graft function would be that MSCs promote revascularization of transplanted islets, resulting in reduced cell death. MSCs are also known to contribute to healing damaged tissue by migrating from BM to the wound site. To evaluate vascularization of islet grafts, the liver was resected 6 days after islet transplantation and processed for histology. Sections were stained for insulin and vWF, a marker for ECs. Figure 4 shows an example of each transplant, islet-MSCs (Fig. 4A) and islet-alone (Fig. 4B). In the islet-MSCs co-transplant, the islet structure was well preserved and surrounded by capillaries, whereas the islet in the control graft was fragmented, with few cells staining positive for vWF.
Co-transplantation of mesenchymal stem cells (MSCs) and islets promotes the graft revascularization. Representative micrographs of insulin (red)- and von Willebrand factor (green)-stained grafts in syngeneic Lewis rats liver (day 6). (A) The islet-MSCs graft contains islets with normal intact structure surrounded by capillaries. (B) The islet-alone graft shows fragmentation and poor capillary formation. (C) The number of capillary segments and β-cells were counted in a whole section, comparing the capillary per β-cell ratio between the islet-MSCs and islet-alone grafts (n=5 and 4, respectively; *P<0.01).
The assessment of vascularity was difficult using islets transplanted into the liver because islets were scattered over a large area. To overcome this problem, 100 islets from LEW rats were transplanted with or without MSCs under the kidney capsule of NOD SCID mice. Grafts were removed along with the carrier kidney on day 7 for immunohistochemistry to evaluate graft revascularization. The number of cells positively stained for insulin or vWF was counted and determined the distribution of capillary per β-cell. As shown in Figure 4(C), the capillary number per β-cell in the islet-MSCs grafts (n=5) was more than double of that of islets-alone grafts (n=4) (0.135±0.046 vs. 0.052±0.028 capillary fragments per β-cell, P<0.01). This result clearly shows the contribution of MSCs on islet revascularization.
Co-Transplanted MSCs Produce VEGF
To reveal the mechanisms involved in the improvement of islet revascularization, MSCs were labeled with CFSE before transplantation with islets into NOD SCID mice. Grafts removed 1 week after transplantation were stained for VEGF. CFSE-positive MSCs (Fig. 5A) and the adjacent section stained for VEGF (Fig. 5B) are shown merged in Figure 5(C and D). These merged microphotographs clearly show that some CFSE-positive cells also stained positive for VEGF, indicating that VEGF was produced by CFSE-positive MSCs. Therefore, it is reasonable to think that VEGF produced by MSCs promotes revascularization of co-transplanted islets.
Mesenchymal stem cells (MSCs) co-transplanted with islets produce vascular endothelial growth factor (VEGF). Carboxyfluorescein succinimidyl ester (CFSE)- labeled MSCs and islets from Lewis rats were co- transplanted under the renal capsule of nonobese diabetic severe combined immunodeficiency mice. Cryosections of grafts removed on day 6 were examined by vascular endothelial growth factor (VEGF) staining. (A) CFSE-labeled MSCs (green). (B) An adjacent section stained for VEGF (red). (C, D) Merged photograph of A and B. Some CFSE-labeled MSCs also stained for VEGF (yellow), indicating that MSCs produce VEGF.
Co-Transplanted MSCs Differentiate Into Vascular ECs
Frozen sections of mouse kidneys containing CFSE- labeled MSCs were stained with an anti-vWF antibody. A few CFSE-positive cells located in capillary fragments also positively stained for vWF (results not shown). To further confirm this finding, MSCs were labeled with Qdots using Qtracker, which allowed for the preparation of paraffin sections. These sections revealed that many Qdot-positive cells remained around the islets 1 week after transplantation (Fig. 6). Furthermore, a few Qdot-labeled cells were found to stain positive for vWF (Fig. 6B–D). These results indicate that some MSCs differentiate into vascular ECs, but the number of these cells is low.
Mesenchymal stem cells (MSCs) co-transplanted with islets differentiate into endothelial cells. Qdot nanocrystal- labeled MSCs and islets from Lewis rats were co- transplanted. A significant number of Qdot-labeled MSCs are present around the co-transplanted islets under the renal capsule of nonobese diabetic severe combined immunodeficiency mice and some co-stained for von Willebrand factor, indicating differentiation into vascular endothelial cells. Histology sections of the grafts removed on day 6 are shown. (A) Graft stained for insulin (green)-containing Qdot (red)-labeled MSCs. (B) An adjacent section stained for the endothelial marker, von Willebrand factor (vWF; green). The areas indicated C and D in B are enlarged and shown to right. Arrows in each picture indicate corresponding Qdot-labeled MSC that positively stained for vWF, indicating that some MSCs differentiate to endothelial cells in situ.
Co-Transplanted MSCs Remain With the Islet Graft Over 3 Weeks
To examine whether MSCs remained at the islet graft site, Qdot-labeled MSCs and islets were co-transplanted in the space under the renal capsule of NOD SCID mice. Grafts were removed 0, 7, 14, and 21 days after transplantation and prepared for immunohistochemical examination. Sections were stained for insulin and vWF (Fig. 7). Immediately after transplantation, clumped MSCs appeared as large red spots close to the islet, and vWF stain was negative (Fig. 7A). On day 7, many MSCs were found in and around the islet. The vWF staining indicated the generation of intra-islet capillaries (Fig. 7B). Increased vasculatures were found outside and inside of the islets together with many MSCs on day 14 samples (Fig. 7C). Even on day 21, many Qdot-labeled MSCs were spread out in the area adjacent to the islet (Fig. 7D). In contrast, the sections of control, islet-alone grafts removed on day 28 contained far fewer number of vWF-positive cells (Fig. 7E).
Mesenchymal stem cells (MSCs) are present with co-transplanted islets for over 3 weeks after transplantation and associated with good revascularization. Lewis (LEW) MSCs were labeled with Qdot nanocrystals and co-transplanted with LEW islets under the renal capsule of nonobese diabetic severe combined immunodeficiency mice. Sections were prepared from islet-MSC grafts as well as islets alone grafts and stained for insulin (blue) and von Willebrand factor (green). Qdot nanocrystals appear in red. Representative results are shown in (A–E). (A) A graft removed immediately after transplantation shows clusters of MSCs (large red spots or dots) and negative von Willebrand factor staining. (B) A graft removed on day 7 contains many MSCs in and around the islet and displays clear intra-islet revascularization. (C) A graft removed on day 14 shows MSCs closely to the islets and good vascularity. (D) A graft removed on day 21 also presents MSCs in the area adjacent to the islets. (E) Control islets-alone graft removed on day 28 contains many β-cells but vascularization is limited.
DISCUSSION
A significant loss in donor islet mass occurs in three areas before islet engraftment: (1) innate immune reaction in brain-dead donors, (2) cold storage and warm collagenase digestion of pancreas with mechanisms similar to that of cold ischemia/reperfusion injury, and (3) inflammation and slow revascularization after transplantation. Prevention of islet loss in these processes will greatly contribute to successful islet transplantation. Data have been accumulated demonstrating the beneficial effect of MSCs in islet transplantation and controlling autoimmune diabetes (24–26). This study in vivo has also shown the MSC-mediated improvement of islet engraftment and function. Five hundred syngeneic islets reversed diabetes in all rats when co-infused with MSCs into the portal vein, whereas 30% recipients became euglycemic with islet infusion alone. Islet-MSCs transplants also reversed diabetes in 50% of the recipients receiving 300 islets (vs. 10% with islet transplantation alone). If MSCs are similarly effective in clinical islet transplantation, the islet number can significantly be reduced to achieve insulin independence.
Recognizing the unique characteristics of BM-derived MSCs, we have explored potential uses of MSCs in islet transplantation using rodent models. As reported by Itakura et al. (23), MSCs were successfully used for induction of hematopoietic mixed chimerism and islet allograft tolerance without a lethal preconditioning treatment of the recipient and without incidence of graft-versus-host disease. Because MSCs are known to produce VEGF and promote healing of damaged cells, we examined whether MSCs will promote revascularization and engraftment of islets after transplantation. It is interesting that many Qdot-labeled MSCs remain in the vicinity of the islet graft under the renal capsule of NOD SCID mice, over 3 weeks after transplantation (Fig. 7). Co-transplantation of MSCs promoted capillary formation in and around the islets, which was shown by an increased vWF-positive cell per β-cell ratio (Fig. 4).
It was previously reported that the density of the capillary network in the islets is approximately 10 times higher than that of the surrounding tissue (5, 6), indicating the need for higher oxygen levels for normal islet cell function and survival. Disruption of the external (and perhaps internal) vascular network during the isolation process requires construction of new capillaries after transplantation, involving both donor- and recipient-derived ECs, BM-derived cells, as well as angiogenic factors (18). Islets overexpressing VEGF by gene transfection have showed better engraftment than normal islets (27). ECs produce platelet-derived growth factor that attracts MSCs, which can then differentiate into pericytes (28). MSC-based regenerative therapy has been widely investigated, especially as a treatment for infarcted hearts (22, 29). Johansson et al. (30) constructed composite EC-islets to promote revascularization. Subsequently, MSCs were included to this construct based on the finding that they contribute to an increase in EC migration (31) and an up-regulation of EC growth factor expression. They conducted extensive in vitro studies on constructed composite human islets containing human MSCs and ECs (25). These in vitro studies showed that the addition of MSCs to EC-islet composites enhanced EC proliferation, sprout formation, and growth of ECs into the islets. Our increased capillaries per β-cell ratio results in vivo support such MSC-mediated EC proliferation and demonstrate significantly improved islet cell survival and function. We detected some MSCs expressing VEGF, which undoubtedly contributed to new capillary formation. MSCs can also be transfected with growth factors, such as VEGF gene in vitro, which may allow us to enhance the effect. MSCs overexpressing VEGF by lipofection release VEGF in culture medium for more than 7 days (Ito, unpublished results). Additionally, a few MSCs, both CFSE- and Qdot-labeled, were found to co-stain with anti-vWF antibody, indicating MSCs differentiated into ECs. Differentiation of MSCs into ECs has been reported both in vitro (20) and in vivo in a heart ischemia model (21).
The islets transplanted with MSCs maintained a normal, undisturbed structure, whereas islet-alone transplants appeared fragmented. This indicates that MSCs prevent cell loss during the early posttransplantation period. Islet cell loss may also be caused by proinflammatory cells and cytokines. MSCs are also known to have an immunomodulatory capacity and suppress various immune cell functions (32), which may contribute to the prevention of islet cell loss. The recent study by Solari et al. (26) reported findings similar to ours in islet allografts. Allogeneic islets transplanted into an omental pouch of LEW rats achieved long-term reversal of diabetes in three of four recipients transplanted with 1200 islets, whereas 600 islets were sufficient when co-transplanted with MSCs (9 of 10 with islet-MSCs vs. one of eight islets alone). Interestingly, they also reported that islet allograft survival was significantly prolonged with islet-MSCs co-transplantation in combination with short-period immunosuppression, indicating the involvement of an immunomodulatory effect of MSCs.
Our results and those reported by others (26, 30) indicate that MSCs co-transplanted with islets in type 1 diabetic recipients would facilitate islet revascularization, engraftment, and improved function and that islet-MSCs co-transplantation may be applicable to clinical transplantation. However, several issues have remained unclear and need to be investigated before its clinical application. These include (1) selection of an appropriate MSC donor; either the recipient, an islet donor, or a third-party donor; (2) tissue source to isolate and expand MSCs because in addition to BM, they are isolated and cultured from variety of human adult tissues, including adipose tissue, dermis, hair follicles, heart, liver, spleen, and dental pulp (33); and (3) the optimal ratio between islet and MSC numbers. Studies in rodents would provide useful information on advantages versus disadvantages of the MSC donor source and characteristics and function of MSCs derived from different tissues. Furthermore, the safety and efficacy of combined islets and MSCs infusion into the portal vein need to be proven using a large animal model.
In summary, co-transplantation of islets and MSCs significantly reduces the islet number required for reversal of diabetes by promoting graft revascularization. The production of VEGF by MSCs and the differentiation of MSCs into vascular ECs contribute, at least in part, to improved vascularization. Co-transplantation of MSCs with islets would facilitate islet engraftment and significantly improve graft function in clinical transplantation.
ACKNOWLEDGMENTS
The authors acknowledge Kohei Ishiyama, M.D., Ph.D., for critically evaluating the FACS results and the excellent technical services provided by the City of Hope Anatomical Pathology Core for preparation of histology slides.
REFERENCES
1. Davalli AM, Ogawa Y, Ricordi C, et al. A selective decrease in the beta cell mass of human islets transplanted into diabetic nude mice. Transplantation 1995; 59: 817.
2. Jirak D, Kriz J, Strzelecki M, et al. Monitoring the survival of islet transplants by MRI using a novel technique for their automated detection and quantification. MAGMA 2009; 22: 257.
3. Korsgren O, Lundgren T, Felldin M, et al. Optimising islet engraftment is critical for successful clinical islet transplantation. Diabetologia 2008; 51: 227.
4. Ryan EA, Lakey JR, Rajotte RV, et al. Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. Diabetes 2001; 50: 710.
5. Henderson JR, Moss MC. A morphometric study of the endocrine and exocrine capillaries of the pancreas. Q J Exp Physiol 1985; 70: 347.
6. Kuroda M, Oka T, Oka Y, et al. Colocalization of vascular endothelial growth factor (vascular permeability factor) and insulin in pancreatic islet cells. J Clin Endocrinol Metab 1995; 80: 3196.
7. Lifson N, Lassa CV, Dixit PK. Relation between blood flow and morphology in islet organ of rat pancreas. Am J Physiol 1985; 249(1 pt 1): E43.
8. Bearer EL, Orci L. Endothelial fenestral diaphragms: A quick-freeze, deep-etch study. J Cell Biol 1985; 100: 418.
9. Carlsson PO, Liss P, Andersson A, et al. Measurements of oxygen tension in native and transplanted rat pancreatic islets. Diabetes 1998; 47: 1027.
10. Brissova M, Shostak A, Shiota M, et al. Pancreatic islet production of vascular endothelial growth factor-a is essential for islet vascularization, revascularization, and function. Diabetes 2006; 55: 2974.
11. Jansson L, Carlsson PO. Graft vascular function after transplantation of pancreatic islets. Diabetologia 2002; 45: 749.
12. Carlsson PO, Andersson A, Carlsson C, et al. Engraftment and growth of transplanted pancreatic islets. Ups J Med Sci 2000; 105: 107.
13. Vajkoczy P, Olofsson AM, Lehr HA, et al. Histogenesis and ultrastructure of pancreatic islet graft microvasculature. Evidence for graft revascularization by endothelial cells of host origin. Am J Pathol 1995; 146: 1397.
14. Menger MD, Jaeger S, Walter P, et al. Angiogenesis and hemodynamics of microvasculature of transplanted islets of Langerhans. Diabetes 1989; 38(suppl 1): 199.
15. Brissova M, Fowler M, Wiebe P, et al. Intraislet endothelial cells contribute to revascularization of transplanted pancreatic islets. Diabetes 2004; 53: 1318.
16. Carlsson PO, Mattsson G. Oxygen tension and blood flow in relation to revascularization in transplanted adult and fetal rat pancreatic islets. Cell Transplant 2002; 11: 813.
17. Mattsson G, Carlsson PO, Olausson K, et al. Histological markers for endothelial cells in endogenous and transplanted rodent pancreatic islets. Pancreatology 2002; 2: 155.
18. Brissova M, Powers AC. Revascularization of transplanted islets: Can it be improved? Diabetes 2008; 57: 2269.
19. Martens TP, See F, Schuster MD, et al. Mesenchymal lineage precursor cells induce vascular network formation in ischemic myocardium. Nat Clin Pract Cardiovasc Med 2006; 3(suppl 1): S18.
20. Oswald J, Boxberger S, Jorgensen B, et al. Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells 2004; 22: 377.
21. Silva GV, Litovsky S, Assad JA, et al. Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model. Circulation 2005; 111: 150.
22. Shake JG, Gruber PJ, Baumgartner WA, et al. Mesenchymal stem cell implantation in a swine myocardial infarct model: Engraftment and functional effects. Ann Thorac Surg 2002; 73: 1919.
23. Itakura S, Asari S, Rawson J, et al. Mesenchymal stem cells facilitate the induction of mixed hematopoietic chimerism and islet allograft tolerance without GVHD in the rat. Am J Transplant 2007; 7: 336.
24. Ding Y, Xu D, Feng G, et al. Mesenchymal stem cells prevent the rejection of fully allogenic islet grafts by the immunosuppressive activity of matrix metalloproteinase-2 and -9. Diabetes 2009; 58: 1797.
25. Johansson U, Rasmusson I, Niclou SP, et al. Formation of composite endothelial cell-mesenchymal stem cell islets: A novel approach to promote islet revascularization. Diabetes 2008; 57: 2393.
26. Solari MG, Srinivasan S, Boumaza I, et al. Marginal mass islet transplantation with autologous mesenchymal stem cells promotes long-term islet allograft survival and sustained normoglycemia. J Autoimmun 2009; 32: 116.
27. Cross SE, Richards SK, Clark A, et al. Vascular endothelial growth factor as a survival factor for human islets: Effect of immunosuppressive drugs. Diabetologia 2007; 50: 1423.
28. Ball SG, Shuttleworth CA, Kielty CM. Mesenchymal stem cells and neovascularization: Role of platelet-derived growth factor receptors. J Cell Mol Med 2007; 11: 1012.
29. Toma C, Pittenger MF, Cahill KS, et al. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation 2002; 105: 93.
30. Johansson U, Elgue G, Nilsson B, et al. Composite islet-endothelial cell grafts: A novel approach to counteract innate immunity in islet transplantation. Am J Transplant 2005; 5: 2632.
31. Ghajar CM, Blevins KS, Hughes CC, et al. Mesenchymal stem cells enhance angiogenesis in mechanically viable prevascularized tissues via early matrix metalloproteinase upregulation. Tissue Eng 2006; 12: 2875.
32. Nauta AJ, Fibbe WE. Immunomodulatory properties of mesenchymal stromal cells. Blood 2007; 110: 3499.
33. Riekstina U, Carkstina I, Parfejevs V, et al. Embryonic stem cell marker expression pattern in human mesenchymal stem cells derived from bone marrow, adipose tissue, heart and dermis. Stem Cell Rev 2009; 5: 378.
Keywords:
Mesenchymal stem cell; Islet transplantation; Islet graft revascularization
Supplemental Digital Content
© 2010 Lippincott Williams & Wilkins, Inc.