Sequential intravital imaging reveals in vivo dynamics of pancreatic tissue transplanted under the kidney capsule in mice

Aims/hypothesis Dynamic processes in pancreatic tissue are difficult to study. We aimed to develop an intravital imaging method to longitudinally examine engraftment, vascularisation, expansion and differentiation in mature islets or embryonic pancreases transplanted under the kidney capsule. Methods Isolated pancreatic islets from adult mice and murine embryonic day (E)12.5 pancreases containing fluorescent biomarkers were transplanted under the kidney capsule of immunodeficient recipient mice. Human islet cells were dispersed, transduced with a lentivirus expressing a fluorescent label and reaggregated before transplantation. Graft-containing kidneys were positioned subcutaneously and an imaging window was fitted into the skin on top of the kidney. Intravital imaging using multiphoton microscopy was performed for up to 2 weeks. Volumes of fluorescently labelled cells were determined as a measure of development and survival. Results Transplanted islets and embryonic pancreases showed good engraftment and remained viable. Engraftment and vascularisation could be longitudinally examined in murine and human islet cells. Murine islet beta cell volume was unchanged over time. Transplanted embryonic pancreases increased to up to 6.1 times of their original volume and beta cell volume increased 90 times during 2 weeks. Conclusions/interpretation This method allows for repeated intravital imaging of grafts containing various sources of pancreatic tissue transplanted under the kidney capsule. Using fluorescent markers, dynamic information concerning engraftment or differentiation can be visualised and measured. Electronic supplementary material The online version of this article (doi:10.1007/s00125-016-4049-6) contains peer-reviewed but unedited supplementary material, which is available to authorised users.


Abstract
Aims/hypothesis Dynamic processes in pancreatic tissue are difficult to study. We aimed to develop an intravital imaging method to longitudinally examine engraftment, vascularisation, expansion and differentiation in mature islets or embryonic pancreases transplanted under the kidney capsule. Methods Isolated pancreatic islets from adult mice and murine embryonic day (E)12.5 pancreases containing fluorescent biomarkers were transplanted under the kidney capsule of immunodeficient recipient mice. Human islet cells were dispersed, transduced with a lentivirus expressing a fluorescent label and reaggregated before transplantation. Graftcontaining kidneys were positioned subcutaneously and an imaging window was fitted into the skin on top of the kidney. Intravital imaging using multiphoton microscopy was performed for up to 2 weeks. Volumes of fluorescently labelled cells were determined as a measure of development and survival. Results Transplanted islets and embryonic pancreases showed good engraftment and remained viable. Engraftment and vascularisation could be longitudinally examined in murine and human islet cells. Murine islet beta cell volume was unchanged over time. Transplanted embryonic pancreases increased to up to 6.1 times of their original volume and beta cell volume increased 90 times during 2 weeks. Conclusions/interpretation This method allows for repeated intravital imaging of grafts containing various sources of pancreatic tissue transplanted under the kidney capsule. Using fluorescent markers, dynamic information concerning engraftment or differentiation can be visualised and measured.

Introduction
Islet cell development, dynamic processes involved in islet engraftment and changes in islet composition are difficult processes to study in vivo. To obtain longitudinal information in combination with functional data, new imaging methods are required that allow sequential measurements in individual animals. Transplantation of pancreatic islets under the kidney capsule is considered the gold standard for the in vivo evaluation of graft insulin secretory capacity and survival in mice [1]. Isolated islets derived from humans and different animal species can be used for transplantation at this site [2]. After engraftment, there is good vascularisation of the transplanted islets, allowing the rapid release and action of insulin. Nephrectomy of the graftcontaining kidney followed by determination of blood glucose values validates graft function [3]. By transplanting a cell pellet that remains compact after transplantation, grafts can easily be retrieved and histologically analysed.
In this study, we adapted our previously published method [4,5] to perform intravital imaging on islets of Langerhans or embryonic pancreatic tissue transplanted under the kidney capsule using an abdominal imaging window. This method allows sequential measurements of survival, vascularisation, expansion and differentiation of these tissues for a period up to 2 weeks.

Methods
Mouse pancreatic islets and embryonic pancreas isolation All experiments on animals were carried out in accordance with the guidelines of the Animal Welfare Committee of the Royal Netherlands Academy of Arts and Sciences. Mature pancreatic islets and intact E12.5 embryonic pancreases were isolated from mouse insulin promoter-enhanced green fluorescent protein (MIP-EGFP) (Jackson Laboratory, Bar Harbor, ME, USA, stock no. 006864) and cytomegalovirusactin-globin promoter red fluorescent protein (CAG-DsRed) (Jackson Laboratory, stock no. 005441) mice. See ESM for further details.

Human pancreatic islets and lentiviral transduction
Human pancreatic islets were isolated at the Leiden University Medical Centre using standard procedures [6]. Isolated human islets could only be used with research consent and when the number and/or quality of the islets were insufficient for clinical islet transplantation, according to national laws. Islets were transduced with a human insulin promoter-GFP (HIP-GFP) virus before transplantation. See ESM for further details.
Islet transplantation and positioning of an intra-abdominal imaging window NOD severe combined immunodeficiency gamma (NSG) mice (Jackson Laboratory, stock no. 005557) were anaesthetised and pancreatic islets or embryonic pancreases were transplanted under the kidney capsule. The kidney was fixed subcutaneously and an imaging window was placed in the skin (ESM Fig. 1a-f), as published previously [4]. See ESM for further details.
Histological analysis Graft-containing kidneys were either fixed for paraffin sectioning or for cryosectioning. Paraffinembedded sections (4 μm) were cut at and stained with haematoxylin and eosin for the determination of graft size and capsule thickness. Cryosections (10 μm) were used for immunohistochemical staining for insulin and glucagon. See ESM for further details.
Microscopy data processing See ESM for further details.
Statistical analysis Data are presented as mean ± SEM. R (www.R-project.org) was used to perform statistical analyses. IPGTT data were analysed using two-way ANOVA. All other statistical analyses were performed using independent two-way Student's t-tests. Data were considered significant if the p value was <0.05.

Results
Transplanted islets function normally in mice fitted with an abdominal imaging window Mice recovered quickly after surgery and were fully active after 1 h. There was no impairment of movement and mice did not show behaviour indicating pain or discomfort. Body weight increased normally after surgery (ESM Fig. 2a). To test if islet graft functionality was affected by the procedure, an IPGTT was performed and human C-peptide concentrations were measured in streptozotocininduced hyperglycaemic NSG mice 4 weeks after transplantation of 2000 human islet equivalents. Both mice with an abdominal imaging window (AIW, n = 5) and controls (CTRL, n = 5) showed a normal response to an IPGTT (ESM Fig. 2b). Human C-peptide concentrations were similar between the two groups 4 weeks after surgery ( Fig. 2d). The imaging window did not affect the proportion of beta cells (relative to the total alpha and beta cells) in the transplanted islets (ESM Fig. 2e-g).

Discussion
Here we describe a novel intravital imaging model to study mature islet cells and developing pancreatic tissue using an abdominal imaging window. Tissues transplanted under the kidney capsule, which is considered the gold standard site for studying islet grafts, can be sequentially imaged for at least 2 weeks. Using fluorescent markers, we longitudinally measured engraftment, expansion, differentiation and vascularisation in transplanted developing pancreatic tissue and mature islets.
Alternative intravital imaging methods have been used to study islet function, survival and vascularisation over time. While the anterior chamber of the eye model allows prolonged (up to 6 months) sequential intravital imaging of transplanted pancreatic islets [7], it is limited by the tissue volume that can be transplanted. This reduces options to transplant large amounts of tissue, or tissues with expansion capacity over time.
Our model allows the dynamic characterisation of the embryonic pancreas, which undergoes further development and expansion after transplantation under the kidney capsule [8]. Fetal pancreases are by nature highly proliferative [9]. The time course of growth and differentiation of embryonic pancreases transplanted under the kidney capsule is very similar to that observed in eutopic pancreatic development [10]. Sequential intravital imaging shows the process of islet neogenesis, as indicated by the appearance of rounded clusters of fluorescent cells. In the majority of embryonic pancreatic tissue transplants no acinar tissue was observed, as reported previously [8]. This is probably the reason why the percentage of beta cells in our grafts is relatively high compared with normal pancreatic development.
We were also able to dynamically assess transplanted islet grafts. Islets were connected to the host vasculature within 3 days, which is in line with previous findings [7]. Generally, placement of an imaging window did not affect survival or function of islets transplanted under the kidney capsule.
Important processes such as engraftment, vascularisation, expansion and differentiation can be studied using this imaging method. The technique can therefore be a valuable tool in beta cell replacement therapy using progenitor cells, islet inflammation and rejection. In conclusion, we have developed a novel method to dynamically image both mature islet cells and developing pancreatic tissue.
Funding This work was supported by the stichting Diabetes Onderzoek Nederland (DON).

Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
Contribution statement LvG, CJML, JvR and EJPdK contributed to the design of the study. LvG, PPvK, GD, EJ, FCASR and EB contributed to the acquisition of the data. LvG and EJPdK contributed to the drafting of the article. All authors contributed to revision of the article and gave final approval. LvG is responsible for the integrity of the work as a whole.
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