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Co-combination of islets with bone marrow mesenchymal stem cells promotes angiogenesis
Biomedicine & Pharmacotherapy 78 (2016) 156–164
Available online at
ScienceDirect www.sciencedirect.com
Original article
Co-combination of islets with bone marrow mesenchymal stem cells promotes angiogenesis Xian-kui Caoa , Rui Lib , Wei Suna , Yang Gea , Bao-lin Liua,* a b
The sixth Department of General Surgery, Shengjing Hospital of China Medical University, Shenyang, 110004, China Department of General Surgery, People’s Hospital of Liaoning Province, Shenyang, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 August 2015 Received in revised form 22 December 2015 Accepted 13 January 2016
Background: Islet transplantation is a commonly therapeutic strategy for diabetes mellitus. However, avascular phase and the poor formation of blood vessels in the late period lead to islet allograft loss which contributed to inefficiency and short-acting of islet transplantation. Recently, to speed up new angiogenesis and increase the density of blood vessels around transplanted islets became the hotspot in research of islet transplantation. Methods: In this study, we undergone co-combination transplantation of allogeneic islet and bone marrow mesenchymal stem cells (BM-MSCs) into non-obese diabetic (NOD) mice and investigated the influence of BM-MSCs in transplanted islet function and neovascularization. Results: In mice of co-combination transplantation of islet with BM-MSCs, level of blood glucose was improved compared with only BM-MSCs transplanted mice; proliferation of islet cell was enhanced while apoptosis of islet cell was reduced; 2, 4, and 8 weeks post transplantation, peripheral vascular density of islet grafts were significantly more than the islet transplantation group alone; donor lymphocytic chimerism in graft was increased. In result of immunofluorescence analysis, we observed that BM-MSCs can migrate to transplanted islet, differentiate into vascular smooth muscle cells (VSMC) and vascular endothelial cells (VEC), and also secrete vascular endothelial growth factor (VEGF). Conclusion: BM-MSCs can migrate to transplanted islet and promote neovascularization. Also, it enhanced allograft immune tolerance of islet grafts via increasing donor lymphocytic chimerism. ã 2016 Published by Elsevier Masson SAS.
Keywords: Type 1 diabetes mellitus Mesenchymal stem cell Islet transplantation Islet graft revascularization
1. Introduction Islet transplantation is one of the ideal methods for the treatment of type 1 diabetes [1]. However, the development of this was restricted by inefficiency and short-term of islet transplantation[2]. Islet cells with high metabolic activity, the ATP and insulin secretion requires large amounts of oxygen supply to maintain. That was the reason why only the pancreas islet volume 1–2% occupies 5–10% of blood supply of pancreas [3]. While, during transplantation, islet was separated from pancreatic tissue and blood supply was damaged. Initial transplanted islet survives depending on environmental oxygen diffusion that resulted in very low oxygen partial pressure in the center of the islets. The lack of oxygen causes islet cell necrosis and apoptosis. After islet transplantation, the vascularization was taken about 14 days [4].
* Corresponding author at: The sixth Department of General Surgery, Shengjing Hospital of China Medical University, 36 Sanhao Street, Shenyang 110004, People’s Republic of China. E-mail address: [email protected] (B.-l. Liu). http://dx.doi.org/10.1016/j.biopha.2016.01.007 0753-3322/ ã 2016 Published by Elsevier Masson SAS.
However, even complete revascularization, blood supply and oxygen partial pressure in graft is obviously lower than normal pancreas islet [5]. So to speed up islets around new angiogenesis and increase the density of blood vessels after transplanted has become the hotspot in research of islet transplantation in the near future. Mesenchymal stem cells (MSCs) are multipotent stem cells which are characterized of self-renewal and multi-directional differentiation potential and derived from a variety of organs and tissues, such as bone marrow, fat and umbilical cord [6]. In addition, MSCs also play a pivotal role in immune adjustment, tissue repair regeneration and blood vessel formation [7–9]. Its application into the treatment of ischemic diseases such as ischemic cardiomyopathy has been obtained the good curative effect [10,11]. Accumulated evidences showed that MSCs promoted the formation of new blood vessels around the transplanted islet. Taihei et al. transplanted MSCs companied with islet of Lewis rat into renal capsule of non-obese diabetic-severe combined immunodeficiency disease (NOD-SCID) mice and observed the new VSMCs generated from MSCs differentiation [12]. Ito and his colleagues found that co-transplantation of islet with MSCs into
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NOD-SCID mice not only reduced insulin administration dose but also facilitated the formation of new blood vessels on account of VGEF secretion in MSCs [13]. This was also proved in researches of Figliuzzi and Rackham C [14]. In present study, NOD mice were induced immune tolerance through receiving bone marrow transplantation and immunosuppressant, and subsequently experienced co-treatment with BMMSCs and allogeneic islet, aiming to evaluate the assist-function of BM-MSCs on islet transplantation and neovascularization.
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2. Material and methods 2.1. Animals and reagents All mice were obtained from Department of medical experimental animals of China Medical University. Female BALB/c mice (aged 8 weeks) were used for donor of bone marrow cells and islet; female BALB/c mice (aged 4 weeks) were used for donor of BMMSCs; NOD/Lt mice (aged 12 week) were used for transplantation
Fig. 1. Graft function. (A) Blood glucose was monitored in islet + MSCs, islet, MSCs and PBS transplanted mice for 56 days. (B) Survival curve was to evaluate viability of transplanted mice of four groups. Data were presented as mean s.d. *p < 0.05 and #p < 0.05 compared with mice transplanted with PBS.
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recipients. All mice are specific-pathogen free mice and maintained in a controlled environment with standard conditions of temperature and humidity. After 12 weeks, the blood glucose of NOD/Lt mice were monitored every week and successful diabetic mice was with a standard of non-fasting mice with greater than 400 mg/dl blood glucose in both consecutive examinations. The NOD/Lt mice were randomly divided into four groups: (1) islet + MSCs group (n = 12), mice were co-transplanted with islet and MSCs; (2) islet group (n = 12), mice were transplanted with islet only; (3) MSCs group, mice were injected with MSCs (n = 6); (4) sham-operated group (n = 3), mice were undergone surgery but without islet and/or MSCs transplantation. Fluorescein-conjugated monoclonal antibodies against CD105, CD29, Sca-1 and CD45 were purchased from purchased from Abcam (Cambridge, MA, USA). FITC labelled-H-2Dd, PE labelled-H2Db and PerCP labelled-CD3 monoclonal antibodies were obtained
from BD PharMingen (SanDiego, CA, USA). Antibodies of goat antimice-vWF antibody, rabbit anti-mice-a-actin antibody, rabbit anti-mice-VEGF antibody, rabbit anti-mice-HGF antibody and guinea pig anti-mice insulin were purchased from Abcam (Cambridge, MA, USA). 2.2. Mesenchymal stem cell isolation The tibia and fibula were removed from mice (aged 4 weeks) and used for generate of MSCs. In brief, the marrow was washed using phosphate buffer (PBS, pH 7.4) and the scrubbing solution was transferred to the culture bottle that containing DMEM/F12 medium (GIBCO, USA) supplemental with 10% FBS. The cells were incubated at condition of 37 C and 5% CO2 in incubator. The medium was changed every 24 hours, with removal of nonadherent cells. When cultures reached confluence of 80–90%, the
Fig. 2. Effect of graft on islet cell growth. Comparison of (A) proliferation rate and (B) apoptosis rate of islet cell in transplanted mice between islet only group and cotransplantation of islet with MSCs group. Comparison of proliferation rate and apoptosis rate between mice transplanted with (C) islet and (D) islet plus MSCs. Data were presented as mean s.d. *p < 0.05 compared with 1 week post transplantation with islet; #p < 0.05 compared with 1 week post islet + MSCs transplantation, ¥ p < 0.05 compared with proliferation.
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cells were digested and were sub-cultured. The cells were cultured for 3 generation and collected for identification or transplantation. 2.3. Immunophenotyping of MSCs The presence of surface marker of MSCs was analyzed using flow cytometry. In brief, MSCs concentration was adjusted to 107/ ml and added with FC block. The mixture was incubated at room temperature for 10 min to block non-specific combination between cells and antibodies. 100 ml samples were equally divided into microcentrifuge tubes and incubated with PE labelled antibodies to CD105, CD29, Sca-1 and CD45 for 30 min in dark and cool (0–4 C) environment. The reaction solution was washed twice and suspended with 400 ml PBS. Cells were analyzed in a FACS calibur cytometer equipped with 488 nm argon laser (BD Pharmingen). 2.4. Immunologic tolerance induction Three days before bone marrow transplantation, 1 108/ml spleen cells of BALB/c mice were infused into NOD/Lt mice via tail intravenous injection while one day before bone marrow transplantation, NOD/Lt mice were treated with a single dose of cyclophosphamide (200 mg/Kg) via abdominal subcutaneous. On day of bone marrow transplantation, NOD/Lt mice were received
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6 107/ml bone marrow stem cell and then were cured with antibody treatment. MR1 (0.5 mg/day), the monoclonal antibody against CD154, was treated via intraperitoneal injection for 5 days, followed by injected on day 7, 10 and 14; 0.5 mg anti-BTLA monoclonal antibody was treated via intraperitoneal injection on the day of transplantation followed by 0.25 mg treated dose on day 2, 4, 6, 8 and 10 post transplantation. 2.5. Bone marrow cell extraction and transplantation The tibia and fibula were removed from mice (aged 8 weeks) and used for generate of bone marrow cell. In brief, 20 ml syringe with no. 26 needle absorbed PBS and used for washing the marrow. The washing fluid was collected and filtered with 70 um mesh filter to remove bone fragments, muscle and cell mass. In the filtered liquid, erythrocyte was lyzed and total cells were counted. The cell concentration was adjusted to 2 108/ml and 0.3 ml of it was injected into NOD/Lt mice via intraperitoneal injection. 2.6. Islet isolation Pancreas of mice was injected with 3 ml collagenase (2 mg/ml; type XI; Sigma–Aldrich) through choledoch to fully expanded pancreas. The entire pancreas was completely removed and digested at 37 C thermostatic water bath for 10 min. Islets were
Fig. 3. Growth of islet and blood vessels in transplant. (A) Generation of new vessels in islet of mice transplanted with islet only or islet plus MSCs post 2 and 4 weeks transplantation was displayed using light microscopy (magnification is 100). (B) four weeks post islets plus MSCs transplantation, islet was visualized using HE staining (left) and insulin immunohistochemistry (right) and was labeled by black frames; Arrow was pointed to lymphocytes infiltration. (C) Islet and vessels were visualized in islet or islet plus MSCs transplanted groups using immunohistochemistry. (D) Blood vessels were normalized to b-cell. Data were presented as mean s.d. *p < 0.05 compared with 2 week post transplantation with islet; #p < 0.05 compared with 2 week post transplantation with islet + MSCs (For interpretation of the references to colour in text, the reader is referred to the web version of this article.).
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isolated by density gradient separation (Histopaque-1077; Sigma– Aldrich) at 1750 rpm for 5 min. The islet was carefully extracted and washed with M199 medium containing 5% FBS. 2.7. Transplantation The purified islets were selected under the microscope and 300 IEQ of them were transferred into PE50 tube followed by centrifugal sedimentation. During transplantation, the mice were anaesthetized and were exposed kidney via oblique incision of the left rib. The PE50 polyethylene tube (Becton Dickinson, Sparks, MD, USA) were inserted into renal capsule and slowly injected into islet under the subcapsular space. The sham-operated mice were injected with 30 ml PBS. Meanwhile, the CFDA-SE labelled MSCs was adjusted into 1.67 107/ml and 0.3 ml of it was injected into mice via tail intravenous injection. The blood glucose was monitored every day after transplantation. At week 1, 2 and 4 post transplantation, kidneys on the transplanted side were removed for following proliferation, apoptosis, immunofluorescence and immunohistochemistry analysis.
Immunohistochemical double dye was used to detect new blood vessels around the islet grafts. The fixed kidney was sliced by araffin method. The section was performed with dimethyl benzene dewaxing, rehydration by gradient alcohol, antigen repair, the nonspecific serum closed, endogenous biotin, endogenous peroxidase and block by endogenous alkaline phosphatase, and then incubated with goat anti-mice-vWF antibody and guinea pig antimice insulin at 4 C overnight. The next day, the section was incubated with biotin labelled ass anti-goat second antibody or peroxidase labelled goat anti-guinea pig second antibody. After incubated, the section was stained by DAB and StayRed followed by hematoxylin staining. 2.11. Statistical analysis All data were presented as mean standard deviation (s.d.). Individual differences were analyzed by SPSS13.0 statistical software. Comparison between the two groups was used student’s t test; survival analysis was performed with Kaplan Meier method; survival curve contrast was compared using the log-rank test. p < 0.05 was considered as statistically significant.
2.8. Proliferation and apoptosis 3. Results Analysis of islet cell proliferation was performed with EdU assay. The mice were received EdU (100 mg/kg) via intraperitoneal injection one day before nephrectomy. The kidney was quickly put into liquid nitrogen. The frozen samples were embedded by OCT and were carried on the frozen sections followed by fixture, semipermeable film with 1% Triton-100 and staining with Apollo. After 30 min staining, the section was washed by PBS containing 0.5% TritonX-100. Cell apoptosis was examined using TUNEL assay according to manufacturers’ protocols. 2.9. Donor lymphocytic chimerism Induction of tolerance in T cells is an important for long-term survival of organ allografts. Thus we detected donor lymphocytic chimerism to evaluate organ transplantation efficiency. After bone marrow transplantation, 100 ml bloods was collected form mice angula vein with blood of non-treated NOD and BALB/c mice as control. The blood was added with heparin used as anticoagulant. The sample was incubated with FITC labelled anti-H-2Dd, PE labelled anti-H-2Db and PerCP-labelled anti-CD3 monoclonal antibodies (BD PharMingen, SanDiego, CA, USA) for 30 min. Cells were washed using PBS that containing 2% FBS and then were added with red blood cell lysis solution. The sample was mixed with 500 ml PBS followed by detection of chimeric level using flow cytometry.
3.1. Blood glucose in co-transplanted mice As shown in Fig. 1A, both co-transplantation of islet with MSCs and islet transplantation alone improved mice blood glucose comparing with PBS treatment and the cure rate was up to 100%. MSCs individually treatment had no curative effect on blood glucose improvement. The Fig. 1B shows the survival curve of four groups. Survival rate of mice in islet group is the same as that in cotransplanted group. Both of these two treatments substantially increased mice survival rate compared with MSCs group. MSCs alone transplantation also increase survival of mice compared with sham-operated mice while with no statistical significance. Additionally, mice in these two groups died on day 21–25 post transplantation.
2.10. Immunofluorescence and immunohistochemistry Kidneys form the side of transplantation were removed and divided into half. One portion was quickly put into liquid nitrogen for following immunofluorescence (IF) and the other portion was fixed with 4% paraformaldehyde for following immunohistochemistry (IHC). The frozen samples were carried on the frozen sections and fixed with 4 C acetone for 10 min and dried at room temperature for 30 minutes. Then the section was re-hydrated with PBS. The hydrated section was blocked with non-specific serum and then incubated with goat anti-mice-vWF antibody, rabbit anti-mice-a-actin antibody, rabbit anti-mice-VEGF antibody and rabbit anti-mice-HGF antibody at 4 C overnight. The next day, the section were incubated with DyLight1 650 labelled second antibody for 30 min. The image was finally observed via fluorescence microscope.
Fig. 4. Donor lymphocytic chimaeras in transplant were detected in week 1, 2, 3, 4, 6, 8, 10 post transplantation with flow cytometry method. Data were presented as mean s.d. *p < 0.05 compared with 1 week post transplantation with islet.
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3.2. Islet cell proliferation in transplant Islet cell proliferation and apoptosis in transplant was examined at 1, 2 and 4 weeks post graft. We observed that proliferation rate of islet was increasing (Fig. 2A) while apoptosis rate was gradually declining (Fig. 2B) by weeks post transplantation in both islet only and islet co-transplanted with MSCs mice. MSCs combined with islet were significantly promoted proliferation and reduced apoptosis of transplanted islet cell in comparison with islet alone. In Fig. 2C, the data showed that cell apoptosis rate was apparently higher than proliferation rate at 1 week after islet transplantation, and was decreasing to the same level as proliferation rate at week 2 and 4 post transplantation. In islet plus MSCs co-transplanted group, proliferation of islet cell was greater than apoptosis rate at week 1 and 4 post transplantation (Fig. 2D). 3.3. MSCs promotes neovascularization To evaluate the role of MSCs in neovascularization, the kidney was removed to examine the new vessels around transplanted islet. Images in Fig. 3A showed that new vessels in MSCs co-
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transplantation group were greater than islet alone group at 2 and 4 weeks post transplantation. In image of insular staining (left of Fig. 3B) and insulin immunohistochemistry (right of Fig. 3B), we observed that islet grew well and infiltrated with a few lymphocytes (indicated as Black arrow). In addition, the transplant was immune to antibodies against both insulin and vessels-related protein and the staining image showed that new blood vessels in MSCs co-transplantation group were greater than that in islet alone group (indicated as red) (Fig. 3C). The insulin was represented by brown. The vessels were then normalized to b-cell. As shown in Fig. 3D, MSCs co-transplantation with islet increased vessels density compared with islet alone. 3.4. MSCs enhanced lymphocytic chimaera Lymphocyte chimeric level in transplant recipients is one of the important factors that influencing the graft immune tolerance. Two weeks after NOD mice transplanted with the donor bone marrow, the islet and MSCs were injected. Levels of lymphocytic chimaera were monitored every week after transplantation. As shown in Fig. 4, lymphocytic chimaera was reduced by bone marrow transplantation and was improved by islet co-
Fig. 5. MSCs in transplant. Four weeks post transplantation, (A) CFDA-SE labelled MSCs was presented using fluorescence microscope in islet transplanted sites (100); (B) MSCs, (C) vascular smooth muscle cell and (D) cell nucleus were detected using immunofluorescent assay. (E) Superposition of three pictures of (B), (C) and (D). Magnifications of picture in B, C, D and E are 200 (For interpretation of the references to colour in text, the reader is referred to the web version of this article.).
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Fig. 6. MSCs differentiate into vascular endothelial cells in co-transplantation of islet with MSCs mice. Four weeks post transplantation; (A) MSCs, (B) vascular endothelial cell and (C) cell nucleus were detected using immunofluorescent assay. (D) Superposition of three pictures of (A), (B) and (C). All magnifications of picture are 200 (For interpretation of the references to colour in text, the reader is referred to the web version of this article.).
transplantation with MSCs, while dropped continuously in islet group. These data suggested that MSCs co-transplantation apparently enhanced NOD mice’s donor lymphocytic chimaera. 3.5. Migration and differentiation of MSCs Chemotaxis and homing ability are one of the important characteristics of MSCs. We injected COD mice with fluorescentlylabeled MSCs and explored the fluorescence around isletat 4 weeks post transplantation. As shown in Fig. 5A, we observed green
fluorescence as well as intricate network of blood vessels (represented as black). This data suggested that MSCs can migrate to islet transplantation site. The potential important role of MSCs is realized by its differentiation into various cells. We next investigated whether MSCs can transform into VSMC via immunofluorescent assay. The data showed that MSCs was visible by green fluorescence (Fig. 5B), VSMC was represented by red fluorescence which labelled by a-actin antibody (Fig. 5C) and cell nuclear was visualized by blue fluorescence (Fig. 5D). We overlapped three pictures of B, C and D and obtained a composite
Fig. 7. Expression of VGEF in co-transplantation of islet with MSCs mice. Four weeks post transplantation, (A) MSCs, (B) VEGF and (D) cell nucleus were detected using immunofluorescent assay. (E) Superposition of three pictures of (B), (C) and (D); the image in box is VEGF expression in MSCs. All magnifications of picture are 200 (For interpretation of the references to colour in text, the reader is referred to the web version of this article.).
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image (Fig. 5E) in which a-actin was expressed in some MSCs cell. These data suggested that MSCs can transform to VSMC. 3.6. MSCs differentiate into VSMCs Next, we examined whether MSCs can transform to VSMCs as method of Fig. 5. As shown in Fig. 6A, MSCs was green fluorescence labelled points. VSMCs were represented by red fluorescence which labelled by vWF antibody (Fig. 6B) and cell nuclear was visualized by blue fluorescence (Fig. 6C). Three pictures of B, C and D were composed into one image (Fig. 6D) in which vWF was expressed in some MSCs cell. These data suggested that MSCs can transform to VSMC. 3.7. VEGF expression in MSCs The VEGF in transplant of islet plus MSCs transplanted mice were also determined using immunofluorescent assay. CFDA-SE labelled MSCs was given green fluorescence under fluorescent excitation (Fig. 7A). VEGF antibody labelled expression of VEGF was represented as red in Fig. 7B. Cell nuclear cell nuclear was visualized by blue fluorescence (Fig. 7C). In composite image of Fig. 7A, 7B and 7C, we observed VEGF was expressed in MSCs cell (labelled by box) (Fig. 7D). 4. Discussion Clinical studies have shown that allogeneic islet transplantation has good short-term curative effect, can make the patient’s blood glucose and glycosylated hemoglobin value return to normal, as well as avoids the occurrence of hypoglycemia shock at the same time [15]. This treatment significantly improves the quality of life of patients and reduces the diabetes complications. However, islet transplantation has deficient, such as inefficiency and short-term. Studies have pointed out that patients who have received enough islets from many donors only possess 20-40% of normal level of insulin secretion [16]. Recently, studies demonstrated that pancreatic ischemia and lack of revascularization after transplantation are the fatal factors limited survival ability of islet [17]. Given to this, a strategy that can promote neovascularization as well as ensure islet survival is quite essential. Multipotent mesenchymal stromal cells (MSCs) are a kind of heterogeneous cell that can be separated from a variety of organs and tissues, and is characteristic of self-renewal and multidirectional differentiation potency. Based on their differentiation and the properties of the formation of blood vessels, MSCs have been used for treatment of various diseases, including ischemic cardiovascular diseases [18], hypoxia-ischemia induced movement disorders [19], liver ischemia-reperfusion injury [20]. Recently, researchers apply MSCs in islet transplantation and found that cotransplantation of islet with MSCs enhanced islet transplantation efficiency [21]. This was also observed in our present study which showed an improved blood glucose level and an elevated survival rate (shown in Fig. 1) in co-transplantation mice. MSCs cotransplantation also partially promoted islet proliferation and suppressed islet apoptosis. As to the pivotal role of new blood vessels in islet transplantation efficiency, we next evaluated the effect of MSCs on development of new blood vessels by immunohistochemistry. By results, we observed that islet in MSCs co-transplanted mice grew well and possessed higher vessels density around than in islet alone group. These data showed protective action of MSCs on islet survival suggested MSCs assisted transplantation might be a better strategy for islet treatment. New blood vessels formation is a complex process and included two important aspects: formation of an endothelial cell tube
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followed by recruitment of smooth muscle cells [22]. That is to say that the increasing of VSMCs and VECs is first and foremost condition for angiogenesis. Previous research shows that MSCs have potential to migrate and differentiate toward VSMC [23] and VEC [24]. MSCs transforming into VECs has been observed in mice co-transplanted of MSCs and islet [25]. In present study, we injected NOD mice with CFDA-SE labelled MSCs and detected production of VSMC and VEC via vessels-specific protein depended immunofluorescent assay. The results are accordance with previous evidence and showed that MSCs can migrate to islet transplantation place and differentiated into VSMC (Fig. 5E) and VEC (Fig. 6D) indicating the vascular differentiation trend of MSCs. This was further supported by expression of vascular endothelial growth factor (VEGF), being essential for angiogenesis, in fluorescence labeled MSCs. Additionally, we found level of donor lymphocytic chimaera in NOD mice was elevated. This might be benefit for islet tolerance and protect translated islet against immune interference. 5. Conclusion All these experimental protocols were performed in diabetic NOD mice that are similar to human’s diabetes pathogenesis. MSCs co-transplantation with islet improved islet transplantation efficiency via prompting neovascularization mediated by production of VSMC and VSMC as well as enhanced VEGF expression. In addition, MSCs also can heighten the donor lymphocyte chimaera of NOD mice. Our data suggested that MSCs induced neovascularization and immunologic tolerance of allograft transplant might contributed islet transplantation efficiency and further provide theoretical support for co-transplantation of islet with MSCs in diabetic treatment. Conflict of interest The authors have no actual or potential conflicts of interest to declare. References [1] P. Srinivasan, G.C. Huang, S.A. Amiel, N.D. Heaton, Islet cell transplantation, Postgraduate Med. J. 83 (2007) 224–229. [2] D. Mineo, A. Pileggi, R. Alejandro, C. Ricordi, Point: steady progress and current challenges in clinical islet transplantation, Diabetes Care 32 (2009) 1563– 1569. [3] A. Barbu, L. Jansson, M. Sandberg, M. Quach, F. Palm, The use of hydrogen gas clearance for blood flow measurements in single endogenous and transplanted pancreatic islets, Microvasc. Res. 97 (2015) 124–129. [4] A.R. Pepper, B. Gala-Lopez, O. Ziff, A.M. Shapiro, Revascularization of transplanted pancreatic islets and role of the transplantation site, Clin. Dev. Immunol. 2013 (2013) 352315. [5] L. Jansson, P.O. Carlsson, Graft vascular function after transplantation of pancreatic islets, Diabetologia 45 (2002) 749–763. [6] X. Gao, L. Song, K. Shen, H. Wang, M. Qian, W. Niu, et al., Bone marrow mesenchymal stem cells promote the repair of islets from diabetic mice through paracrine actions, Mol. Cell. Endocrinol. 388 (2014) 41–50. [7] S. Bruno, M.C. Deregibus, G. Camussi, The secretome of mesenchymal stromal cells: role of extracellular vesicles in immunomodulation, Immunol. Lett. (2015). [8] A.I. Caplan, R. Hariri, Body management: mesenchymal stem cells control the internal regenerator, Stem Cells Transl. Med. (2015). [9] D.M. Smadja, M. Levy, L. Huang, E. Rossi, A. Blandinieres, D. Israel-Biet, et al., Treprostinil indirectly regulates endothelial colony forming cell angiogenic properties by increasing VEGF-A produced by mesenchymal stem cells, Thromb. Haemost. (2015) 114. [10] C.C. Sheng, L. Zhou, J. Hao, Current stem cell delivery methods for myocardial repair, Biomed Res. Int. 2013 (2013) 547902. [11] L. Bockeria, V. Bogin, O. Bockeria, T. Le, B. Alekyan, E.J. Woods, et al., Endometrial regenerative cells for treatment of heart failure: a new stem cell enters the clinic, J. Transl. Med. 11 (2013) 56. [12] T. Ito, S. Itakura, I. Todorov, J. Rawson, S. Asari, J. Shintaku, et al., Mesenchymal stem cell and islet co-transplantation promotes graft revascularization and function, Transplantation 89 (2010) 1438–1445.
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Original article
Co-combination of islets with bone marrow mesenchymal stem cells promotes angiogenesis Xian-kui Caoa , Rui Lib , Wei Suna , Yang Gea , Bao-lin Liua,* a b
The sixth Department of General Surgery, Shengjing Hospital of China Medical University, Shenyang, 110004, China Department of General Surgery, People’s Hospital of Liaoning Province, Shenyang, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 11 August 2015 Received in revised form 22 December 2015 Accepted 13 January 2016
Background: Islet transplantation is a commonly therapeutic strategy for diabetes mellitus. However, avascular phase and the poor formation of blood vessels in the late period lead to islet allograft loss which contributed to inefficiency and short-acting of islet transplantation. Recently, to speed up new angiogenesis and increase the density of blood vessels around transplanted islets became the hotspot in research of islet transplantation. Methods: In this study, we undergone co-combination transplantation of allogeneic islet and bone marrow mesenchymal stem cells (BM-MSCs) into non-obese diabetic (NOD) mice and investigated the influence of BM-MSCs in transplanted islet function and neovascularization. Results: In mice of co-combination transplantation of islet with BM-MSCs, level of blood glucose was improved compared with only BM-MSCs transplanted mice; proliferation of islet cell was enhanced while apoptosis of islet cell was reduced; 2, 4, and 8 weeks post transplantation, peripheral vascular density of islet grafts were significantly more than the islet transplantation group alone; donor lymphocytic chimerism in graft was increased. In result of immunofluorescence analysis, we observed that BM-MSCs can migrate to transplanted islet, differentiate into vascular smooth muscle cells (VSMC) and vascular endothelial cells (VEC), and also secrete vascular endothelial growth factor (VEGF). Conclusion: BM-MSCs can migrate to transplanted islet and promote neovascularization. Also, it enhanced allograft immune tolerance of islet grafts via increasing donor lymphocytic chimerism. ã 2016 Published by Elsevier Masson SAS.
Keywords: Type 1 diabetes mellitus Mesenchymal stem cell Islet transplantation Islet graft revascularization
1. Introduction Islet transplantation is one of the ideal methods for the treatment of type 1 diabetes [1]. However, the development of this was restricted by inefficiency and short-term of islet transplantation[2]. Islet cells with high metabolic activity, the ATP and insulin secretion requires large amounts of oxygen supply to maintain. That was the reason why only the pancreas islet volume 1–2% occupies 5–10% of blood supply of pancreas [3]. While, during transplantation, islet was separated from pancreatic tissue and blood supply was damaged. Initial transplanted islet survives depending on environmental oxygen diffusion that resulted in very low oxygen partial pressure in the center of the islets. The lack of oxygen causes islet cell necrosis and apoptosis. After islet transplantation, the vascularization was taken about 14 days [4].
* Corresponding author at: The sixth Department of General Surgery, Shengjing Hospital of China Medical University, 36 Sanhao Street, Shenyang 110004, People’s Republic of China. E-mail address: [email protected] (B.-l. Liu). http://dx.doi.org/10.1016/j.biopha.2016.01.007 0753-3322/ ã 2016 Published by Elsevier Masson SAS.
However, even complete revascularization, blood supply and oxygen partial pressure in graft is obviously lower than normal pancreas islet [5]. So to speed up islets around new angiogenesis and increase the density of blood vessels after transplanted has become the hotspot in research of islet transplantation in the near future. Mesenchymal stem cells (MSCs) are multipotent stem cells which are characterized of self-renewal and multi-directional differentiation potential and derived from a variety of organs and tissues, such as bone marrow, fat and umbilical cord [6]. In addition, MSCs also play a pivotal role in immune adjustment, tissue repair regeneration and blood vessel formation [7–9]. Its application into the treatment of ischemic diseases such as ischemic cardiomyopathy has been obtained the good curative effect [10,11]. Accumulated evidences showed that MSCs promoted the formation of new blood vessels around the transplanted islet. Taihei et al. transplanted MSCs companied with islet of Lewis rat into renal capsule of non-obese diabetic-severe combined immunodeficiency disease (NOD-SCID) mice and observed the new VSMCs generated from MSCs differentiation [12]. Ito and his colleagues found that co-transplantation of islet with MSCs into
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NOD-SCID mice not only reduced insulin administration dose but also facilitated the formation of new blood vessels on account of VGEF secretion in MSCs [13]. This was also proved in researches of Figliuzzi and Rackham C [14]. In present study, NOD mice were induced immune tolerance through receiving bone marrow transplantation and immunosuppressant, and subsequently experienced co-treatment with BMMSCs and allogeneic islet, aiming to evaluate the assist-function of BM-MSCs on islet transplantation and neovascularization.
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2. Material and methods 2.1. Animals and reagents All mice were obtained from Department of medical experimental animals of China Medical University. Female BALB/c mice (aged 8 weeks) were used for donor of bone marrow cells and islet; female BALB/c mice (aged 4 weeks) were used for donor of BMMSCs; NOD/Lt mice (aged 12 week) were used for transplantation
Fig. 1. Graft function. (A) Blood glucose was monitored in islet + MSCs, islet, MSCs and PBS transplanted mice for 56 days. (B) Survival curve was to evaluate viability of transplanted mice of four groups. Data were presented as mean s.d. *p < 0.05 and #p < 0.05 compared with mice transplanted with PBS.
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recipients. All mice are specific-pathogen free mice and maintained in a controlled environment with standard conditions of temperature and humidity. After 12 weeks, the blood glucose of NOD/Lt mice were monitored every week and successful diabetic mice was with a standard of non-fasting mice with greater than 400 mg/dl blood glucose in both consecutive examinations. The NOD/Lt mice were randomly divided into four groups: (1) islet + MSCs group (n = 12), mice were co-transplanted with islet and MSCs; (2) islet group (n = 12), mice were transplanted with islet only; (3) MSCs group, mice were injected with MSCs (n = 6); (4) sham-operated group (n = 3), mice were undergone surgery but without islet and/or MSCs transplantation. Fluorescein-conjugated monoclonal antibodies against CD105, CD29, Sca-1 and CD45 were purchased from purchased from Abcam (Cambridge, MA, USA). FITC labelled-H-2Dd, PE labelled-H2Db and PerCP labelled-CD3 monoclonal antibodies were obtained
from BD PharMingen (SanDiego, CA, USA). Antibodies of goat antimice-vWF antibody, rabbit anti-mice-a-actin antibody, rabbit anti-mice-VEGF antibody, rabbit anti-mice-HGF antibody and guinea pig anti-mice insulin were purchased from Abcam (Cambridge, MA, USA). 2.2. Mesenchymal stem cell isolation The tibia and fibula were removed from mice (aged 4 weeks) and used for generate of MSCs. In brief, the marrow was washed using phosphate buffer (PBS, pH 7.4) and the scrubbing solution was transferred to the culture bottle that containing DMEM/F12 medium (GIBCO, USA) supplemental with 10% FBS. The cells were incubated at condition of 37 C and 5% CO2 in incubator. The medium was changed every 24 hours, with removal of nonadherent cells. When cultures reached confluence of 80–90%, the
Fig. 2. Effect of graft on islet cell growth. Comparison of (A) proliferation rate and (B) apoptosis rate of islet cell in transplanted mice between islet only group and cotransplantation of islet with MSCs group. Comparison of proliferation rate and apoptosis rate between mice transplanted with (C) islet and (D) islet plus MSCs. Data were presented as mean s.d. *p < 0.05 compared with 1 week post transplantation with islet; #p < 0.05 compared with 1 week post islet + MSCs transplantation, ¥ p < 0.05 compared with proliferation.
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cells were digested and were sub-cultured. The cells were cultured for 3 generation and collected for identification or transplantation. 2.3. Immunophenotyping of MSCs The presence of surface marker of MSCs was analyzed using flow cytometry. In brief, MSCs concentration was adjusted to 107/ ml and added with FC block. The mixture was incubated at room temperature for 10 min to block non-specific combination between cells and antibodies. 100 ml samples were equally divided into microcentrifuge tubes and incubated with PE labelled antibodies to CD105, CD29, Sca-1 and CD45 for 30 min in dark and cool (0–4 C) environment. The reaction solution was washed twice and suspended with 400 ml PBS. Cells were analyzed in a FACS calibur cytometer equipped with 488 nm argon laser (BD Pharmingen). 2.4. Immunologic tolerance induction Three days before bone marrow transplantation, 1 108/ml spleen cells of BALB/c mice were infused into NOD/Lt mice via tail intravenous injection while one day before bone marrow transplantation, NOD/Lt mice were treated with a single dose of cyclophosphamide (200 mg/Kg) via abdominal subcutaneous. On day of bone marrow transplantation, NOD/Lt mice were received
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6 107/ml bone marrow stem cell and then were cured with antibody treatment. MR1 (0.5 mg/day), the monoclonal antibody against CD154, was treated via intraperitoneal injection for 5 days, followed by injected on day 7, 10 and 14; 0.5 mg anti-BTLA monoclonal antibody was treated via intraperitoneal injection on the day of transplantation followed by 0.25 mg treated dose on day 2, 4, 6, 8 and 10 post transplantation. 2.5. Bone marrow cell extraction and transplantation The tibia and fibula were removed from mice (aged 8 weeks) and used for generate of bone marrow cell. In brief, 20 ml syringe with no. 26 needle absorbed PBS and used for washing the marrow. The washing fluid was collected and filtered with 70 um mesh filter to remove bone fragments, muscle and cell mass. In the filtered liquid, erythrocyte was lyzed and total cells were counted. The cell concentration was adjusted to 2 108/ml and 0.3 ml of it was injected into NOD/Lt mice via intraperitoneal injection. 2.6. Islet isolation Pancreas of mice was injected with 3 ml collagenase (2 mg/ml; type XI; Sigma–Aldrich) through choledoch to fully expanded pancreas. The entire pancreas was completely removed and digested at 37 C thermostatic water bath for 10 min. Islets were
Fig. 3. Growth of islet and blood vessels in transplant. (A) Generation of new vessels in islet of mice transplanted with islet only or islet plus MSCs post 2 and 4 weeks transplantation was displayed using light microscopy (magnification is 100). (B) four weeks post islets plus MSCs transplantation, islet was visualized using HE staining (left) and insulin immunohistochemistry (right) and was labeled by black frames; Arrow was pointed to lymphocytes infiltration. (C) Islet and vessels were visualized in islet or islet plus MSCs transplanted groups using immunohistochemistry. (D) Blood vessels were normalized to b-cell. Data were presented as mean s.d. *p < 0.05 compared with 2 week post transplantation with islet; #p < 0.05 compared with 2 week post transplantation with islet + MSCs (For interpretation of the references to colour in text, the reader is referred to the web version of this article.).
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isolated by density gradient separation (Histopaque-1077; Sigma– Aldrich) at 1750 rpm for 5 min. The islet was carefully extracted and washed with M199 medium containing 5% FBS. 2.7. Transplantation The purified islets were selected under the microscope and 300 IEQ of them were transferred into PE50 tube followed by centrifugal sedimentation. During transplantation, the mice were anaesthetized and were exposed kidney via oblique incision of the left rib. The PE50 polyethylene tube (Becton Dickinson, Sparks, MD, USA) were inserted into renal capsule and slowly injected into islet under the subcapsular space. The sham-operated mice were injected with 30 ml PBS. Meanwhile, the CFDA-SE labelled MSCs was adjusted into 1.67 107/ml and 0.3 ml of it was injected into mice via tail intravenous injection. The blood glucose was monitored every day after transplantation. At week 1, 2 and 4 post transplantation, kidneys on the transplanted side were removed for following proliferation, apoptosis, immunofluorescence and immunohistochemistry analysis.
Immunohistochemical double dye was used to detect new blood vessels around the islet grafts. The fixed kidney was sliced by araffin method. The section was performed with dimethyl benzene dewaxing, rehydration by gradient alcohol, antigen repair, the nonspecific serum closed, endogenous biotin, endogenous peroxidase and block by endogenous alkaline phosphatase, and then incubated with goat anti-mice-vWF antibody and guinea pig antimice insulin at 4 C overnight. The next day, the section was incubated with biotin labelled ass anti-goat second antibody or peroxidase labelled goat anti-guinea pig second antibody. After incubated, the section was stained by DAB and StayRed followed by hematoxylin staining. 2.11. Statistical analysis All data were presented as mean standard deviation (s.d.). Individual differences were analyzed by SPSS13.0 statistical software. Comparison between the two groups was used student’s t test; survival analysis was performed with Kaplan Meier method; survival curve contrast was compared using the log-rank test. p < 0.05 was considered as statistically significant.
2.8. Proliferation and apoptosis 3. Results Analysis of islet cell proliferation was performed with EdU assay. The mice were received EdU (100 mg/kg) via intraperitoneal injection one day before nephrectomy. The kidney was quickly put into liquid nitrogen. The frozen samples were embedded by OCT and were carried on the frozen sections followed by fixture, semipermeable film with 1% Triton-100 and staining with Apollo. After 30 min staining, the section was washed by PBS containing 0.5% TritonX-100. Cell apoptosis was examined using TUNEL assay according to manufacturers’ protocols. 2.9. Donor lymphocytic chimerism Induction of tolerance in T cells is an important for long-term survival of organ allografts. Thus we detected donor lymphocytic chimerism to evaluate organ transplantation efficiency. After bone marrow transplantation, 100 ml bloods was collected form mice angula vein with blood of non-treated NOD and BALB/c mice as control. The blood was added with heparin used as anticoagulant. The sample was incubated with FITC labelled anti-H-2Dd, PE labelled anti-H-2Db and PerCP-labelled anti-CD3 monoclonal antibodies (BD PharMingen, SanDiego, CA, USA) for 30 min. Cells were washed using PBS that containing 2% FBS and then were added with red blood cell lysis solution. The sample was mixed with 500 ml PBS followed by detection of chimeric level using flow cytometry.
3.1. Blood glucose in co-transplanted mice As shown in Fig. 1A, both co-transplantation of islet with MSCs and islet transplantation alone improved mice blood glucose comparing with PBS treatment and the cure rate was up to 100%. MSCs individually treatment had no curative effect on blood glucose improvement. The Fig. 1B shows the survival curve of four groups. Survival rate of mice in islet group is the same as that in cotransplanted group. Both of these two treatments substantially increased mice survival rate compared with MSCs group. MSCs alone transplantation also increase survival of mice compared with sham-operated mice while with no statistical significance. Additionally, mice in these two groups died on day 21–25 post transplantation.
2.10. Immunofluorescence and immunohistochemistry Kidneys form the side of transplantation were removed and divided into half. One portion was quickly put into liquid nitrogen for following immunofluorescence (IF) and the other portion was fixed with 4% paraformaldehyde for following immunohistochemistry (IHC). The frozen samples were carried on the frozen sections and fixed with 4 C acetone for 10 min and dried at room temperature for 30 minutes. Then the section was re-hydrated with PBS. The hydrated section was blocked with non-specific serum and then incubated with goat anti-mice-vWF antibody, rabbit anti-mice-a-actin antibody, rabbit anti-mice-VEGF antibody and rabbit anti-mice-HGF antibody at 4 C overnight. The next day, the section were incubated with DyLight1 650 labelled second antibody for 30 min. The image was finally observed via fluorescence microscope.
Fig. 4. Donor lymphocytic chimaeras in transplant were detected in week 1, 2, 3, 4, 6, 8, 10 post transplantation with flow cytometry method. Data were presented as mean s.d. *p < 0.05 compared with 1 week post transplantation with islet.
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3.2. Islet cell proliferation in transplant Islet cell proliferation and apoptosis in transplant was examined at 1, 2 and 4 weeks post graft. We observed that proliferation rate of islet was increasing (Fig. 2A) while apoptosis rate was gradually declining (Fig. 2B) by weeks post transplantation in both islet only and islet co-transplanted with MSCs mice. MSCs combined with islet were significantly promoted proliferation and reduced apoptosis of transplanted islet cell in comparison with islet alone. In Fig. 2C, the data showed that cell apoptosis rate was apparently higher than proliferation rate at 1 week after islet transplantation, and was decreasing to the same level as proliferation rate at week 2 and 4 post transplantation. In islet plus MSCs co-transplanted group, proliferation of islet cell was greater than apoptosis rate at week 1 and 4 post transplantation (Fig. 2D). 3.3. MSCs promotes neovascularization To evaluate the role of MSCs in neovascularization, the kidney was removed to examine the new vessels around transplanted islet. Images in Fig. 3A showed that new vessels in MSCs co-
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transplantation group were greater than islet alone group at 2 and 4 weeks post transplantation. In image of insular staining (left of Fig. 3B) and insulin immunohistochemistry (right of Fig. 3B), we observed that islet grew well and infiltrated with a few lymphocytes (indicated as Black arrow). In addition, the transplant was immune to antibodies against both insulin and vessels-related protein and the staining image showed that new blood vessels in MSCs co-transplantation group were greater than that in islet alone group (indicated as red) (Fig. 3C). The insulin was represented by brown. The vessels were then normalized to b-cell. As shown in Fig. 3D, MSCs co-transplantation with islet increased vessels density compared with islet alone. 3.4. MSCs enhanced lymphocytic chimaera Lymphocyte chimeric level in transplant recipients is one of the important factors that influencing the graft immune tolerance. Two weeks after NOD mice transplanted with the donor bone marrow, the islet and MSCs were injected. Levels of lymphocytic chimaera were monitored every week after transplantation. As shown in Fig. 4, lymphocytic chimaera was reduced by bone marrow transplantation and was improved by islet co-
Fig. 5. MSCs in transplant. Four weeks post transplantation, (A) CFDA-SE labelled MSCs was presented using fluorescence microscope in islet transplanted sites (100); (B) MSCs, (C) vascular smooth muscle cell and (D) cell nucleus were detected using immunofluorescent assay. (E) Superposition of three pictures of (B), (C) and (D). Magnifications of picture in B, C, D and E are 200 (For interpretation of the references to colour in text, the reader is referred to the web version of this article.).
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Fig. 6. MSCs differentiate into vascular endothelial cells in co-transplantation of islet with MSCs mice. Four weeks post transplantation; (A) MSCs, (B) vascular endothelial cell and (C) cell nucleus were detected using immunofluorescent assay. (D) Superposition of three pictures of (A), (B) and (C). All magnifications of picture are 200 (For interpretation of the references to colour in text, the reader is referred to the web version of this article.).
transplantation with MSCs, while dropped continuously in islet group. These data suggested that MSCs co-transplantation apparently enhanced NOD mice’s donor lymphocytic chimaera. 3.5. Migration and differentiation of MSCs Chemotaxis and homing ability are one of the important characteristics of MSCs. We injected COD mice with fluorescentlylabeled MSCs and explored the fluorescence around isletat 4 weeks post transplantation. As shown in Fig. 5A, we observed green
fluorescence as well as intricate network of blood vessels (represented as black). This data suggested that MSCs can migrate to islet transplantation site. The potential important role of MSCs is realized by its differentiation into various cells. We next investigated whether MSCs can transform into VSMC via immunofluorescent assay. The data showed that MSCs was visible by green fluorescence (Fig. 5B), VSMC was represented by red fluorescence which labelled by a-actin antibody (Fig. 5C) and cell nuclear was visualized by blue fluorescence (Fig. 5D). We overlapped three pictures of B, C and D and obtained a composite
Fig. 7. Expression of VGEF in co-transplantation of islet with MSCs mice. Four weeks post transplantation, (A) MSCs, (B) VEGF and (D) cell nucleus were detected using immunofluorescent assay. (E) Superposition of three pictures of (B), (C) and (D); the image in box is VEGF expression in MSCs. All magnifications of picture are 200 (For interpretation of the references to colour in text, the reader is referred to the web version of this article.).
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image (Fig. 5E) in which a-actin was expressed in some MSCs cell. These data suggested that MSCs can transform to VSMC. 3.6. MSCs differentiate into VSMCs Next, we examined whether MSCs can transform to VSMCs as method of Fig. 5. As shown in Fig. 6A, MSCs was green fluorescence labelled points. VSMCs were represented by red fluorescence which labelled by vWF antibody (Fig. 6B) and cell nuclear was visualized by blue fluorescence (Fig. 6C). Three pictures of B, C and D were composed into one image (Fig. 6D) in which vWF was expressed in some MSCs cell. These data suggested that MSCs can transform to VSMC. 3.7. VEGF expression in MSCs The VEGF in transplant of islet plus MSCs transplanted mice were also determined using immunofluorescent assay. CFDA-SE labelled MSCs was given green fluorescence under fluorescent excitation (Fig. 7A). VEGF antibody labelled expression of VEGF was represented as red in Fig. 7B. Cell nuclear cell nuclear was visualized by blue fluorescence (Fig. 7C). In composite image of Fig. 7A, 7B and 7C, we observed VEGF was expressed in MSCs cell (labelled by box) (Fig. 7D). 4. Discussion Clinical studies have shown that allogeneic islet transplantation has good short-term curative effect, can make the patient’s blood glucose and glycosylated hemoglobin value return to normal, as well as avoids the occurrence of hypoglycemia shock at the same time [15]. This treatment significantly improves the quality of life of patients and reduces the diabetes complications. However, islet transplantation has deficient, such as inefficiency and short-term. Studies have pointed out that patients who have received enough islets from many donors only possess 20-40% of normal level of insulin secretion [16]. Recently, studies demonstrated that pancreatic ischemia and lack of revascularization after transplantation are the fatal factors limited survival ability of islet [17]. Given to this, a strategy that can promote neovascularization as well as ensure islet survival is quite essential. Multipotent mesenchymal stromal cells (MSCs) are a kind of heterogeneous cell that can be separated from a variety of organs and tissues, and is characteristic of self-renewal and multidirectional differentiation potency. Based on their differentiation and the properties of the formation of blood vessels, MSCs have been used for treatment of various diseases, including ischemic cardiovascular diseases [18], hypoxia-ischemia induced movement disorders [19], liver ischemia-reperfusion injury [20]. Recently, researchers apply MSCs in islet transplantation and found that cotransplantation of islet with MSCs enhanced islet transplantation efficiency [21]. This was also observed in our present study which showed an improved blood glucose level and an elevated survival rate (shown in Fig. 1) in co-transplantation mice. MSCs cotransplantation also partially promoted islet proliferation and suppressed islet apoptosis. As to the pivotal role of new blood vessels in islet transplantation efficiency, we next evaluated the effect of MSCs on development of new blood vessels by immunohistochemistry. By results, we observed that islet in MSCs co-transplanted mice grew well and possessed higher vessels density around than in islet alone group. These data showed protective action of MSCs on islet survival suggested MSCs assisted transplantation might be a better strategy for islet treatment. New blood vessels formation is a complex process and included two important aspects: formation of an endothelial cell tube
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followed by recruitment of smooth muscle cells [22]. That is to say that the increasing of VSMCs and VECs is first and foremost condition for angiogenesis. Previous research shows that MSCs have potential to migrate and differentiate toward VSMC [23] and VEC [24]. MSCs transforming into VECs has been observed in mice co-transplanted of MSCs and islet [25]. In present study, we injected NOD mice with CFDA-SE labelled MSCs and detected production of VSMC and VEC via vessels-specific protein depended immunofluorescent assay. The results are accordance with previous evidence and showed that MSCs can migrate to islet transplantation place and differentiated into VSMC (Fig. 5E) and VEC (Fig. 6D) indicating the vascular differentiation trend of MSCs. This was further supported by expression of vascular endothelial growth factor (VEGF), being essential for angiogenesis, in fluorescence labeled MSCs. Additionally, we found level of donor lymphocytic chimaera in NOD mice was elevated. This might be benefit for islet tolerance and protect translated islet against immune interference. 5. Conclusion All these experimental protocols were performed in diabetic NOD mice that are similar to human’s diabetes pathogenesis. MSCs co-transplantation with islet improved islet transplantation efficiency via prompting neovascularization mediated by production of VSMC and VSMC as well as enhanced VEGF expression. In addition, MSCs also can heighten the donor lymphocyte chimaera of NOD mice. Our data suggested that MSCs induced neovascularization and immunologic tolerance of allograft transplant might contributed islet transplantation efficiency and further provide theoretical support for co-transplantation of islet with MSCs in diabetic treatment. Conflict of interest The authors have no actual or potential conflicts of interest to declare. References [1] P. Srinivasan, G.C. Huang, S.A. Amiel, N.D. Heaton, Islet cell transplantation, Postgraduate Med. J. 83 (2007) 224–229. [2] D. Mineo, A. Pileggi, R. Alejandro, C. Ricordi, Point: steady progress and current challenges in clinical islet transplantation, Diabetes Care 32 (2009) 1563– 1569. [3] A. Barbu, L. Jansson, M. Sandberg, M. Quach, F. Palm, The use of hydrogen gas clearance for blood flow measurements in single endogenous and transplanted pancreatic islets, Microvasc. Res. 97 (2015) 124–129. [4] A.R. Pepper, B. Gala-Lopez, O. Ziff, A.M. Shapiro, Revascularization of transplanted pancreatic islets and role of the transplantation site, Clin. Dev. Immunol. 2013 (2013) 352315. [5] L. Jansson, P.O. Carlsson, Graft vascular function after transplantation of pancreatic islets, Diabetologia 45 (2002) 749–763. [6] X. Gao, L. Song, K. Shen, H. Wang, M. Qian, W. Niu, et al., Bone marrow mesenchymal stem cells promote the repair of islets from diabetic mice through paracrine actions, Mol. Cell. Endocrinol. 388 (2014) 41–50. [7] S. Bruno, M.C. Deregibus, G. Camussi, The secretome of mesenchymal stromal cells: role of extracellular vesicles in immunomodulation, Immunol. Lett. (2015). [8] A.I. Caplan, R. Hariri, Body management: mesenchymal stem cells control the internal regenerator, Stem Cells Transl. Med. (2015). [9] D.M. Smadja, M. Levy, L. Huang, E. Rossi, A. Blandinieres, D. Israel-Biet, et al., Treprostinil indirectly regulates endothelial colony forming cell angiogenic properties by increasing VEGF-A produced by mesenchymal stem cells, Thromb. Haemost. (2015) 114. [10] C.C. Sheng, L. Zhou, J. Hao, Current stem cell delivery methods for myocardial repair, Biomed Res. Int. 2013 (2013) 547902. [11] L. Bockeria, V. Bogin, O. Bockeria, T. Le, B. Alekyan, E.J. Woods, et al., Endometrial regenerative cells for treatment of heart failure: a new stem cell enters the clinic, J. Transl. Med. 11 (2013) 56. [12] T. Ito, S. Itakura, I. Todorov, J. Rawson, S. Asari, J. Shintaku, et al., Mesenchymal stem cell and islet co-transplantation promotes graft revascularization and function, Transplantation 89 (2010) 1438–1445.
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