Mesenchymal stromal cells support endothelial cell interactions in an intramuscular islet transplantation model
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Mesenchymal stromal cells (MSC) have been under investigation for a number of therapies and have lately been in focus as immunosuppressive actors in the field of transplantation. Herein we have extended our previously published in vitro model of MSC-islets in an experimental setting of islet transplantation to the abdominal muscle.
Human islets coated with luciferase-GFP transduced human MSC were transplanted to the abdomen muscle tissue of NOD-scid ILR2γnull mice and cellular interactions were investigated by confocal microscopy.
The MSC reduced fibrotic encapsulation and facilitated endothelial cell interactions. In particular, we show a decreased fraction of αSMA expressing fibrotic tissue surrounding the graft in presence of MSC-islets compared to islets solely distributed into the muscle tissue. Also, in the presence of MSC, human islet endothelial cells migrated from the center of the graft out into the surrounding tissue forming chimeric blood vessels with recipient endothelial cells. Further, in the graft periphery, MSC were seen interacting with infiltrating macrophages.
Here, in our experimental in vivo model of composite human islets and luciferase-GFP-transduced human MSC, we enable the visualization of close interactions between the MSC and the surrounding tissue. In this model of transplantation the MSC contribute to reduced fibrosis and increased islet endothelial cell migration. Furthermore, the MSC interact with the recipient vasculature and infiltrating macrophages.
KeywordsMesenchymal stromal cell Islets of Langerhans Transplantation Endothelial cells
alpha smooth muscle actin
Cluster of differentiation
green fluorescent protein
instant blood mediated inflammatory reaction
mesenchymal stromal cells
Mesenchymal stromal cells (MSC) are a subpopulation of multipotent cells originally identified in the bone marrow . MSC are characterized by their fibroblast-like appearance, differentiation and colony forming unit capacity including their rapid adherence to plastic surfaces . MSC have been used in both experimental models and in the clinical setting as immunosuppressive treatment [3, 4] and catalyzers of endothelial cell sprout formation . The in vitro immunosuppressive capacity combined with proven therapeutic efficacy has paved the way of MSC in the clinic. MSC in an allogeneic nonhuman primate model of islet transplantation showed increased engraftment, indicating a capacity for these cells to reduce rejection . Safety concerns and efficacy of MSC in solid organ transplantation are currently under investigation but so far they have proven to be safe and so far no detrimental effects have been reported . MSC have further been under investigation in a clinical trial as immune modulatory therapy for diabetic patients where early onset type 1 diabetic patients received autologous MSC in an attempt to halt the disease (ClinicalTrials.gov Identifier: NTC01068951 ).
MSC have also been under investigation in the transplantation setting of islets (ClinicalTrials.gov Identifier: NCT01967186). Today islets are transplanted to the portal vein of the liver. Unfortunately, due to the instant blood mediated inflammatory reaction (IBMIR) a substantial fraction of islets are destroyed and multiple infusions of islets are usually needed to acquire insulin independence . Therefore, alternative transplantation sites such as the striated muscle have been investigated . The muscle as a transplantation site has shown great potential to support islet revascularization in in vivo experimental models . MSC could facilitate the engraftment processes both as immune regulators but also as supporters for the ingrowth of recipient’s vasculature and by producers of stimulatory growth factors . In our previous in vitro studies we have shown that the presence of MSC contributed to increase sprout formation of endothelial cells into fibrin gels after being coated onto islets . One benefit of creating composite islets i.e. coating the MSC onto the islet surface instead of performing co-transplantation of MSC in suspension with islets is besides increased possibility of cellular interactions, also a greater possibility of the MSC to reside during a prolonged time at the site of transplantation.
Herein, we present an in vivo normoglycemic experimental model of islet transplantation utilizing human composite MSC-islets. MSC expressed GFP/luciferase to enable in vivo imaging studies over time and ex vivo confocal analysis post explantation. The MSC-islets were transplanted to the abdominal muscle of NOD-scid ILR2γnull mice to improve the engraftment of human cells  and analyzed three days to seven days post transplantation for revascularization, infiltration and fibrosis. Our results provide knowledge about the close interactions between the MSC, the recipient’s vasculature and the endogenous islet endothelial cells as well as the accumulation of macrophages.
Detection of luciferase/GFP-transduced MSC after transplantation
Analysis of the islet graft size
MSC act as an interactive barrier against accumulating macrophages
Fibrotic process surrounding islets in the absence of MSC
Human islet endothelial cell movement in vitro and at site of transplantation
MSC facilitate endogenous and recipient vascular interactions
In the current study we have been able to investigate the interactions between human MSC and human islets in an in vivo model of transplantation to enlighten initial cellular events by utilizing advanced imaging techniques. The findings here correlate with our previous in vitro model of MSC-islets where it was shown that the presence of MSC induced endothelial cell migration  and as an additional suggested mechanism in the in vivo setting, the MSC was shown to interact with infiltrating macrophages. Furthermore, the MSC contributed to significantly reduced fibrosis compared to control islets.
Today, clinical islet transplantation suffers from loss of islets in part caused by the IBMIR occurring upon infusion into the portal vein of the liver. Over the past decade there has been an increased interest in alternative transplantation sites due to mentioned obstacles and deterioration of islet graft function post intraportal transplantation. Further, the need of rapid revascularization processes increases the demand on the transplantation site. The striated muscle has a natural ongoing angiogenesis and a high oxygen tension and might therefore to be considered as a preferable site for transplantation . The muscle as a transplantation site has previously shown great potential to support islet revascularization in experimental models of islets transplanted to the immune privileged cremaster muscle .
Macrophages and neutrophils are known to infiltrate grafted tissues and cause degradation and tissue damage  but also to support revascularization . In our experimental model with transplanted human MSC-islets, we show that the framework of MSC surrounding the islets was interacting with accumulating macrophages. The interactions at depth showed that the MSC seemed to be wrapped around and at some locations fused with the macrophages. Image analysis verified a potential effect by the MSC on day 3 post transplantation as shown by accumulation of F4/80 at a longer distance from the islet graft in the presence of MSC compared to control islets.
One element of consideration within this study is the disappearance of MSC from the transplantation site. Upon clinical intravenous administration of MSC, the cells are trapped within the lung and are thereafter rapidly disappearing. However, as the therapy has effect in situations such as graft versus host disease, there may be a small part of the cells that survive and utilize their function, such as homing to sites of injury or releasing immune modulatory factors [18, 19]. With our data we can confirm that there is a loss of a majority of the transplanted MSC at site of transplantation. By estimation of the luciferase signal the transplanted MSC were reduced by approximately 90 % one-week post transplantation. Still, the remaining MSC were highly active and interacted with the infiltrating cells and migrated out to the surrounding muscle tissue. Others have also shown that MSC have a tendency to disappear from the site of transplantation  and that MSC migration from the injured site is dose and time dependent and before all, exclusively CXCR4 dependent . Further, MSC seem to have a systemic effect even when not present at the site  so the amount of cells and for how long time the MSC are needed at one particular site is inconclusive. Along with the continuous reduction of the luciferase signal we did not observe any specific pattern of migration. However, there is always a possibility that some MSC detach from the islets upon injection and they can probably be drained from the site of transplantation when the surrounding tissue soak up injected excess liquid. Furthermore, the loss of MSC could of course also be due to cell death. We can only speculate about this, but the luciferase dots that appeared in three of the animals (Additional file 2: Figure S2) may indicate deposit of MSC within the spleen or the liver.
Pancreatic islets have a rich vascular supply in the native pancreas and some report that islets receive 5–10 % of pancreatic blood flow despite coverage of 1–2 % in the pancreas [23, 24]. Even though we could not see a full revascularization process of the implanted grafts in our transplantation model, we could observe that when the MSC were present, the distance of CD31+ islet sprouting endothelial cells were significantly longer than in islets solely distributed into the muscle tissue. In our in vitro studies of cultured human islets we have seen that the resident endothelial cells are retracted to the center part of the islet and as the in vitro culture progressed, the CD31 expression declined as a reflection of lost CD31 expression from the islet vasculature or actual endothelial cell loss. This can be related to the work of Nyqvist and colleagues  where they investigated the expression of CD31 in cultured mouse islets. They further showed a retraction and a loss of islet endothelial cells as early as 3–4 days post isolation. To compare, with our results it was shown that human islet endothelial cells remain within the islet center after 6 days in culture. A phenomenon likely to occur due to increased expression of vascular endothelial growth factor (VEGF) within the hypoxic islet core . MSC are known to produce VEGF  and it is not unlikely that growth factors produced by the MSC is a cause of islet endothelial recruitment into the surrounding tissue. MSC have further been shown to produce several trophic molecules that trigger angiogenesis in a model of islet transplantation . If our finding of recruitment of islet endothelial cells to the muscle tissue is of benefit or not needs to be further investigated, but the interaction of human islet endothelial cells to the recipient mouse vasculature is in itself an indication of increased cellular interaction in the presence of MSC. A similar scenario was observed in the in vivo setting where CD31 expression in control islets solely could be seen close to the center of the islet mass non-connecting to the recipient vasculature while MSC facilitated the migration of the endothelial cells out into the surrounding tissue where they were interactive with the ingrowing vasculature. Further, MSC was observed at some sites to align with and wrap around the human endothelial cells as to form chimeric blood vessels in a smooth muscle cell behavior. It has been shown in vitro that co-cultures of human MSC are stimulated towards a smooth muscle phenotype through the cell-cell interactions with endothelial cells . It has further been reported that islet endothelial cells and recipient blood vessels form chimeric blood vessels in an animal model . Also, in a rat transplantation model in the presence of immunosuppressive drugs the transplanted endothelial cells contributed to chimeric vessels that were functional during 60 days . Here we show the interactions between human MSC, human islet endothelial cells and mouse vasculature. However, the current study lacks the parameters to answer inquiries of enhanced vascular functionality at the chosen site and requires further investigations.
αSMA is normally expressed by vascular smooth muscle cells but can also be expressed by stromal fibroblastic cells in pathological conditions leading to fibrosis . Herein, αSMA expression was seen in the tissue surrounding the graft. Surprisingly, analysis of apoptosis revealed no significant difference between control islets compared to MSC-islets. A possible reason for this could be of a technical matter due to actual loss of apoptotic tissues during sectioning and staining process, as indicated by the increased fibrosis. Without the supportive framework of MSC, the grafted control islets showed accumulation of F4/80 macrophages close to the islet mass, had an active fibroproliferation process that could be detected to a significantly higher extent. However, in the presence of MSC, areas in the islet grafts could be found that were not surrounded by fibrotic tissue. This finding could indicate a healing process of the transplantation area in the presence of MSC.
The MSC in this model of transplantation contribute to reduced fibrosis, islet endothelial cell migration and interaction with the recipient vasculature and infiltrating macrophages.
All work involving human tissue was conducted according to the principles expressed in the Declaration of Helsinki and in the European Council’s Convention on Human Rights and Biomedicine. The healthy volunteers donating bone marrow gave written informed consent and the Regional Ethics Committee in Stockholm, Sweden approved the study. Consent for organ donation (for clinical transplantation and for use in research) was obtained verbally from the deceased’s next of kin by the attending physician and documented in the medical records of the deceased in accordance with Swedish law and as approved by the Regional Ethics Committee. The study was approved by the Regional Ethics Committee in Uppsala, Sweden, according to the Act Concerning the Ethical Review of Research Involving Humans. MSC were isolated and expanded from bone marrow of healthy donors as previously described  following approval by the ethics committee at Huddinge University Hospital and thereafter cultured and utilized at Uppsala University (EPN Uppsala, Dnr2013/410). All laboratory animal experiments were approved by the local ethics committee (Dnr C261/12, 362/10).
Isolation and expansion of adult human MSC
The MSC were cultured in MSC medium consisting of Dulbecco’s modified Eagle’s medium-low glucose (DMEM-LG), supplemented with 10 % heat-inactivated fetal bovine serum (FBS from PAA Laboratories GmbH, Pasching, Austria). The release criteria for MSC was based on spindle shaped morphology, cell viability >95 % and flow cytometry of cells with >95 % positivity for CD73, CD90, CD105, HLA-ABC and <5 % for CD14, CD31, CD34, CD45 and HLA-DR as previously described as previously described . The cultures were negative for bacteria, fungi and polymerase chain reaction (PCR)-negative for Mycoplasma pneumoniae. In this study, MSC in passages 5–9 from three different donors were used in separate experiments.
Human islet isolation
Islets of Langerhans were isolated from human pancreas received from brain-dead donors using a semi-automated method . Islet preparations with purity of 69–85 % based upon Ditizone staining were used in separate experiments. Purity of islet preparation was estimated with digital image analysis . Freshly isolated islets and exocrine tissue were cultured free-floating in islet medium, CMRL 1066 culture medium with 10 % ABO serum and supplements, for about 48 h at 37 °C (5 % CO2) prior to experiments .
Lentiviral construction and production
A lentiviral plasmid, pBMN (G2L), with the genes encoding copepod green fluorescent protein (GFP), codon-optimized firefly luciferase separated by a sequence encoding a self-cleaving 2A peptide from Thosea asigna virus (T2A) was constructed using pGreenPuro (SBI System Biosciences, Mountain View, CA). A large-scale third generation self-inactivating (SIN) lentivirus batch was produced in HEK-293T cells (Life Technologies, Carlsbad, CA) using polyethyleneimine (Sigma-Aldrich, St Louis, MO) transfection of plasmids pBMN (G2L), pLP1, pLP2 and pVSV-G (Life Technologies) at a ratio of 2:1:1:1. The supernatant was harvested 48 and 72 h post-transfection and concentrated through ultracentrifugation at 75,000 x g for 90 min. The viral pellet was resuspended in PBS and stored at −80 °C until further use. The virus titer was determined using the lentivirus qPCR Titer Kit (Applied Biological Materials Inc, Richmond, Canada) following the provider’s instructions.
Viral transduction of MSC and creation of composite MSC-islets
Viral supernatants (20 μl) were added to 200 000 MSC cultured in 25 cm2 flasks in MSC medium supplemented with 8 μg/ml Polybrene (Sigma-Aldrich Inc, Saint Louis, MO, USA). Cells were incubated for 24 h at 37 °C, 5 % CO2 and the media was replaced the following day. Transduction efficiency was analyzed for GFP expression using FACScanto II (BD Biosciences, San Diego, CA).
Human islets of Langerhans were manually picked and used one to three days post islet isolation. Islets were incubated with blue cell tracker (1-3 uM Cell Tracker, Molecular Probes, Eugene, OR) in 5cm Sterilin dishes (Sterilin Ltd, New Port, UK) in islet culture medium (see above), 1 h, 37 °C, followed by change of medium and 1h incubation in 37 °C. For creation of composite MSC-islets, we followed earlier established protocols for coating of the islets [13, 37]. In short, approximately 200 islets were added to 5 mL polystyrene tubes (Sarstedt, Numbrecht, Germany) together with 185 000 GFP/luciferase-transduced MSC followed by careful mixing every 30 min during 2 h at room temperature (RT). Islets and composite MSC-islets were thereafter cultured in 5 cm Sterilin dishes with MSC complete medium (see above), 37 °C, overnight.
Experimental in vivo model
Female NOD-scid ILR2γnull mice were obtained from (MTA TLJ Ref No 005557, Jackson). Within 3–4 days after isolation approximately 200 islets or approximately 200 MSC-islets were upon transplantation dispersed to the abdomen muscle in isoflourane-anesthetized normoglycemic mice with end points and removal of grafts 3 (n = 4/group) and 7 (n = 5/group) days post transplantation. Islets or MSC-islets were collected and let to sediment in 20−30 ul of islet culture medium using 25G butterfly infusion needles before injection into the muscle tissue. To monitor GFP-luciferase/MSC in the MSC-islet receiving mice, luciferase activity was analyzed using the IVIS-100 Imaging system (Xenogen Corporation, Alameda, CA). An intraperitoneal injection of 10 μl/g body weight D-luciferin (Xenogen) followed by visualization was measured at 5, 10, 15 and 20 min after injection and the mean luciferase signal in each investigated animal was calculated. Luciferase activity measurements were performed each or every second day until endpoint.
Preparation of tissue for immunohistochemistry
After explantation, mouse abdominal muscles were fixed in 1 % paraformaldehyde (PFA) at 4 °C overnight followed by additional incubation in PBS 4 °C overnight and further in 20 % sucrose/PBS overnight to finally be stored at −70 °C. Longitudinal cryosections (5-7 μm) were cut of the abdominal muscle after mounting in optimal cutting temperature (O.C.T) medium (Tissue-Tech, Sakura Finetek, Zoeterwoude, Netherlands).
To enable a complete overview of the graft area within the muscle tissue, longitudinal 5–7 μm cryosections of the grafts were performed. The longitudinal sections were selected through the collected muscle tissue to analyze the tissue transversal (10–20 sections/analyzed marker combination at different section levels within the tissue) and the sections were stained for mouse endothelial cells (CD31 dilution 1:400 (Becton Dickinson (BD) Biosciences, Franklin Lakes, NJ) and human endothelial cells (Alexa-647 conjugated CD31 dilution 1:200, BD Biosciences), human insulin (dilution 1:100, Fitzgerald Industries International, MA), mouse macrophages (F4/80 dilution 1:200, AbD Serotec, Oxford, United Kingdom), fibrotic tissue (Cy5 conjugated α-smooth muscle actin (αSMA) dilution 1:1000, Sigma-Aldrich, St. Louis, MO) . Slides were treated with a blocking/permeabilizing solution; 5 % goat serum/2 % BSA/0,05 % Triton-X, 1 h at RT followed by over night, 4 °C, incubation with primary antibody. Slides were rinsed in TBS-Tween followed by 1 h incubation with secondary antibodies at RT (goat-α-rat Alexa 568, goat-α-rabbit Alexa 405, goat-α-rat Alexa 647, dilution 1:500, Molecular Probes, Eugene, OR, USA). For apoptotic events the ApopTag®Plus In situ Apoptosis Fluorescein Detection Kit (Merck Millipore, Billerica, MA, USA) was applied according to manufacturer’s protocol with addition of staining endocrine tissue using anti-chromogranin A antibody (pre-diluted from DAKO, Glostrup, Denmark) during 1 h at RT. The ethanol/acid fixation step in the ApopTag kit removed the green signal from GFP+ MSC thereby avoiding interference with ApopTag fluorescein detection. Slides were rinsed in TBS-Tween followed by 1 h incubation with secondary antibody (donkey-α-rabbit Alexa 568, diluted 1:1000 in 5 % goat serum/2 % BSA, Molecular Probes). Slides were rinsed in TBS followed by mounting with Fluoromount-G (SouthernBiotech, Birmingham, AL) and stored in 4 °C until analyzed.
In situ staining
Whole muscle were explanted and fixed in 1 % PFA, 4 °C, overnight followed by rinse in TBS and incubation with primary antibodies (mouse CD31 dilution 1:200) diluted in 20 % sucrose/0,05 % Triton-X 4 °C 3–4 days. Secondary antibodies (goat-α-rat Alexa 568 dilution 1:500, Molecular Probes, Eugene, OR) diluted in 20 % sucrose/0,05 % Triton-X were added after TBS-tween rinsing. The muscle were rinsed in TBS and kept in 20 % sucrose until image analysis.
Microscopy was performed on the cryosections (Zeiss LSM700, Carl Zeiss, Jena, Germany) and in situ stained muscle tissue (Zeiss 710 NLO two-photon and confocal microscope (Carl Zeiss) at Science for Life Lab, BioVis Platform, Uppsala). To analyze the quantity of a specific cell marker in a section, each image was analyzed for the amount of events estimated by positive fluorescent staining. Images were split from RGB into red, blue and green single images and made from grey to binary. Percentage of positive events was calculated by the analyze particle function in Fiji image processing software (http://fiji.sc/). Images stained for CD31 were further analyzed for endothelial cell length by using the skeleton plug-in in Fiji (http://fiji.sc/ Analyze Skeleton) and further for distance in relation to islet mass by using the function object neighbors, relation objects and Id primary object, measure correlation and relate objects in the CellProfiler image analysis software (www.cellprofiler.org). CD31 positive structures <40 pixels or 12.5 um were excluded in the analysis to avoid quantification of small dots not representing vascular structures. Also, the analysis of selected CD31 structures for quantification was visually defined as vessel-like structures. Distance analysis of F4/80+ cells in correlation to the islet grafts was preformed by measuring distance from all F4/80+ events to the closest and second closest islet graft area. Data is presented as mean distance in each analyzed image. Distance analysis was preformed using the functions primary objects, measure object neighbor, measure distance first closest object, measure distance second closest object in the CellProfiler image analysis software. Adobe Photoshop CS6v13.0.2 (Adobe Systems Incorporated, San Jose, CA) was used for all image processing.
Differences of analyzed positive events estimated by fluorescent signal between the groups and time points were compared using the GraphPad Prism (GraphPad Software Incorporated, La Jolla, CA) with significance set to p ≤ 0.05 using the non-parametric Mann–Whitney test. Data are presented as mean values with standard deviation (SD).
This study was supported by grants from EXODIAB (UFV-PA 2012/2330) and the Swedish Research Council (A0290401, A0290402), The Swedish Child Diabetes Foundation, the Swedish Society of Medicine and Novo Nordisk foundation.
The authors would like to thank the Nordic Network for human islet isolation, Clinical Immunology. Imaging was performed with equipment maintained by the Science for Life Lab BioVis Platform, Uppsala University. We offer special thanks to Dr. Deborah McClellan for editorial assistance and Berith Nilsson for excellent technical assistance.
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