Integration of nano- and biotechnology for beta-cell and islet transplantation in type-1 diabetes treatment.

Abstract Regenerative medicine using human or porcine β‐cells or islets has an excellent potential to become a clinically relevant method for the treatment of type‐1 diabetes. High‐resolution imaging of the function and faith of transplanted porcine pancreatic islets and human stem cell–derived beta cells in large animals and patients for testing advanced therapy medicinal products (ATMPs) is a currently unmet need for pre‐clinical/clinical trials. The iNanoBIT EU H2020 project is developing novel highly sensitive nanotechnology‐based imaging approaches allowing for monitoring of survival, engraftment, proliferation, function and whole‐body distribution of the cellular transplants in a porcine diabetes model with excellent translational potential to humans. We develop and validate the application of single‐photon emission computed tomography (SPECT) and optoacoustic imaging technologies in a transgenic insulin‐deficient pig model to observe transplanted porcine xeno‐islets and in vitro differentiated human beta cells. We are progressing in generating new transgenic reporter pigs and human‐induced pluripotent cell (iPSC) lines for optoacoustic imaging and testing them in transplantable bioartificial islet devices. Novel multifunctional nanoparticles have been generated and are being tested for nuclear imaging of islets and beta cells using a new, high‐resolution SPECT imaging device. Overall, the combined multidisciplinary expertise of the project partners allows progress towards creating much needed technological toolboxes for the xenotransplantation and ATMP field, and thus reinforces the European healthcare supply chain for regenerative medicinal products.


| INTRODUC TI ON
Diabetes is one of the most challenging and economically important areas in medicine. Current data from the International Diabetes Federation indicate that 425 million people worldwide suffer from diabetes, and this is predicted to increase to 629 million people by 2045 (https://www.idf.org/diabe tesatlas). About 10% of these subjects suffer from type-1 diabetes (T1D), which involves immune-mediated destruction of the insulin-producing pancreatic beta cells and thus requires exogenous insulin replacement.
Despite remarkable advances in recent years in treatment of diabetes, with technical developments, 1 T1D patients develop frequent hypoglycaemic episodes and extreme glycolic lability. For patients with recurrent life-threatening hypoglycaemia, pancreas or islet transplantation is considered as ultimate therapeutic option, 2 which are able to normalize glucose homeostasis and prevent macro-and microvascular complications of diabetes. However, requirement of lifelong immunosuppression and the critical lack of human donor organs are major limitations that urgently call for alternative options including stem cell-derived insulin-producing cells and xenogeneic islets. [3][4][5] Xenotransplantation of cells, tissues and organs is a rapidly developing field. [6][7][8] The pig is the favourite donor species for several reasons, including similarity with humans in the size and physiology of many organs, high fecundity and the possibility of genetic modification. However, its major limitation is the need for immunosuppressive treatment to prevent graft rejection which have significant side effect such as diminishing islet function or the induction of islet death. Immune isolation of cells by encapsulation with a permselective membrane, which protects cells against the recipient's immune system, but allows nutrient and oxygen supply, 9,10  Different strategies to generate new beta cells are currently being discussed. 12 Clinical trials with products from pluripotent stem cells have begun (eg Clinicaltrials.gov: NCT02239354, NCT03163511) but clinical efficacy still needs to be proven. Detection and monitoring testing them in transplantable bioartificial islet devices. Novel multifunctional nanoparticles have been generated and are being tested for nuclear imaging of islets and beta cells using a new, high-resolution SPECT imaging device. Overall, the combined multidisciplinary expertise of the project partners allows progress towards creating much needed technological toolboxes for the xenotransplantation and ATMP field, and thus reinforces the European healthcare supply chain for regenerative medicinal products.

F I G U R E 1
The structure and toolboxes of the iNanoBIT project. iRFP expressing xeno-islets and hiPSC-derived beta cells will be transplanted in genetically modified humanized pre-clinical diabetic pig model. Highly sensitive nanotechnology-based imaging approaches (Optoacoustic imaging, SPECT/CT) will be designed to monitor of survival, engraftment, proliferation, function and whole-body distribution of the cellular transplants

| G ENER ATI ON OF TR AN S PL ANTAB LE P ORCINE PAN CRE ATI C IS LE TS AND H UMAN B E TA CELL S WITH REP ORTER G ENE S FOR DIFFERENT IMAG ING MODALITIE S
Cell labelling with reporter genes such as fluorescent proteins (eGFP, dsRed, mCherry) allows tracking of transplanted cells fate. [14][15][16] Unlike traditional indirect methods of cell labelling, such as superparamagnetic iron oxide (SPIO) and radionuclide labelling, reporter genes are inherited genetically (direct labelling) and can be used to monitor cell proliferation and survival for the lifetime of transplanted and their progeny cells. Current challenges and comparison of methods for stem cell tracking are reviewed. [17][18][19][20] In case of fluorescent reporters, changes in fluorescent signal indicate cell death or proliferation. These reporter genes cause the coloration of the cells expressing them and thus also allow the discrimination from histological sections as well as permit non-invasive, real-time tracking in vivo, but they are limited due to the penetration depths of visible light in the body. 21 Near-infrared fluorescent protein (iRFP) has been developed from the DrBphP bacterial phytochrome of Deinococcus radiodurans partially overcome this limitation, thus became a new prospect in the application of IFPs for protein labelling and in vivo tracking. 22,23 After careful consideration, for subsequent transplantation and imaging purposes we decided to develop the iRFP reporter driven by a pCAG promoter, both for porcine xenoislet and the hiPSC-derived beta-cell transplants. On the one hand, pCAGdriven iRFP has already been reported to express reliably both in transgenic mice 24 and in human cell lines implanted into mice. 25 Tran et al (2014) measured carefully several physiological parameters of wild-type control and pCAG-iRFP transgenic mice and found no difference between them in body size, blood indices, reproductive performance or organ growth and morphology. Both studies reported a strong expression with well-balanced intensity.
On the other hand, the CAG promoter has been used successfully to drive the expression of several reporter genes in hiPSCs. [26][27][28] These studies report a constitutive, stable and scalable reporter expression of the transgenes, both in pluripotent and differen-

| IN VIVO S TUD IE S IN PI G S -THE B I OARTIFI CIAL PAN CRE A S (BAP)
Cell encapsulation currently represents the most promising method of avoiding autoimmune rejection of transplanted pluripotent stem cell-derived β cells, and therefore, it is an active area of worldwide research. This technique was introduced by Lim and Sun in 1980 38 and has been continuously developed and adapted since then.
Recent reviews summarized advances in the encapsulation methods. 36,37 This strategy has the potential to overcome graft rejection without the need for long-term immunosuppressive medication, thus avoiding related side effects. 38 Primordial for the success of such cellular transplantation therapy, the safety and functionality post-implantation are key features of the bioartificial pancreas (BAP). Therefore, the BAP should be resistant enough to protect the encapsulated islets/cells from the host's immune system, but also to protect the receiving organism from the cells that could be of animal origin or stem cell-derived. Moreover, the BAP should allow optimal exchanges between the encapsulated cells and the receiving organism. Inadequate oxygen supply causes the gradual loss of cell mass and function, and this effect can be aggravated with encapsulation, thus pose one of the challenges in BAP development. 39 To overcome this problem, several different experimental approaches have been tested such as the stimulation of vascularization growth prior to cell transplantation, 10,40-43 the use of a hypoxia-resistant cell line from Tilapia, 44 microencapsulation, 45 increased oxygen permeability of the encapsulating material. 46 Incorporated refillable oxygen reservoir in the βAir device has already been tested in small and large animal models, [47][48][49] and recently, in a clinical phase I study (Clinicaltrials. gov: NCT02064309) has been conducted to evaluate its safety. 34 However, the βAir device requires daily refilling of the oxygen chamber thus patient needs to carry the equipment to refill the device which is inconvenient. 34 The novel MailPan® (MAcroencapsulation of PANcreatic IsLets, see Figure 4) device 50 uses of a permselective membrane allowing the passage of glucose and insulin, but impermeable to the immune system. Thanks to this property, no immunosuppressive treatment shall be needed. Innovative feature of MailPan ® is its "In" and "Out" implantable access ports that allow to fill or empty MailPan ® , without the need of a surgery, whenever the cells might become non-functional. Thanks to the in and out ports, the device can be implanted weeks before loading the cells which allow pre-vascularizing the device prior the islet filling, which should improve their survival where media and cells can be loaded and removed from the device without surgically removing it from the patient will allow repeated application of imaging agents during prolonged observations of several months, while keeping the benefits of encapsulation technology. This is a novel use of the device and would provide added value.
We implanted two MailPan ® devices for a 2-month period, in one pig (Sus scrofa domesticus) to determine whether a subcutaneous implantation of MailPan ® device was feasible since MailPan ® devices were previously implanted in pre-peritoneal site, which is not ideal for the MSOT imaging method, due to limitation of infrared laser light penetration into tissues, in the range of a few centimetres. Therefore, one MailPan ® device was implanted in subcutaneous site and the other one in pre-peritoneal site to compare biointegration at both sites. The recipient pig was followed up during 2 months post-implantation and was sacrificed. During the follow-up, the implanted pig was in good conditions. Both recovered MailPan ® devices were intact at explantation after 2-month follow-up. In summary, the results showed the feasibility to implant MailPan ® device in subcutaneous as well as the integrity of the device. Depending on the maximum depth allowing accurate optoacoustic imaging, the MailPan ® device could be implanted subcutaneously which is a less deep site than pre-peritoneal site.

| DE VELOPMENT AND TE S TING OF NANOMATERIAL S FOR MULTIMODALIT Y ENHAN CED IMAG ING
In order to overcome the current sensitivity limitations of imaging technology, we take advantage of the unique characteristics of the nanoparticles, already established as innovative therapeutic and diagnostic agents. 51,52 Nanoparticles have the capacity to circulate in the body, as carriers loaded with small molecules such as drugs, diagnostic and imaging agents, minimizing the dispersion and degradation of such molecules and allowing them to reach more easily the target cells/ organs in the body. [53][54][55] The safe journey of the nanoparticles in the body requires prevention of their elimination by macrophages, which is possible by making them "stealth" using PEGylation or glycosylation at their surface. 56 The dimension and shape 57

| DE VELOPMENT OF NOVEL CLINI C ALLY APPLI C AB LE IMAG ING APPROACHE S
High-resolution and sensitive whole-body imaging in large animals and humans are areas where development is fast and promising, but the currently available imaging agents and clinical equipment are not sufficient to satisfy all of the needs of cell and tissue transplantation.
Clinical imaging in humans allows larger size objects to be imaged, including head, limbs, arms or full body, on the expense of resolution and sensitivity. We are currently developing higher sensitivity equipment for pre-clinical large animal models and humans, as well as developing automated or semi-automated data analysis tools with new mathematical algorithms to enhance the information gained from the data acquired.
The design of collimators has fundamental effect on the final performance of the SPECT imaging system. Multi-pinhole collimators provide enough flexibility to effectively optimize the parameters of the camera for specific applications. We designed a new multi-pinhole collimator, for imaging of transplanted islets inside certain regions of various sized pigs (AnyScan TRIO system, Figure 6

| IN VIVO IMAG ING WITH N UCLE AR MED I CINE TECHNOLOGY PE T AND S PEC T IN COMB INATI ON WITH C T AND WITH M SOT
Finally, imaging of the transplanted pigs will be performed, applying our novel nanotools, reporter pig and cell lines. In order to establish PET/CT and SPECT/CT imaging of pigs, there are several prerequisites, which need to be fulfilled. These include approvals in the context of animal welfare and radiation safety. Furthermore, the installation of our new SPECT/CT device is currently being developed near to the pig facility.
In order to create the imaging protocols for pigs, we prepared PET/CT measurements on pigs using fluorodeoxyglucose. Using this well-established tracer for measuring glucose metabolism before labelled, exendin derivatives would become available in iNa-noBIT, will yield quantitative measurements of glucose metabolism.
Importantly, basic information on animal welfare during PET/CT scanning can be gained as a prerequisite for optimization of the planned exendin scans and subsequent detailed description for the application to achieve approval by the authorities. This study is also essential to establish logistics and radiation safety procedures in animal transport, housing, anaesthesia, as well as monitoring during PET/CT scanning and wake-up phase after the imaging session. In addition, these scans provide specific information on PET sensitivity and organ sizes in the pig.

| CON CLUS IONS
The potential socio-economic impact of the regenerative medicine solutions for T1D is high. According to the IDF Diabetes Atlas globally,

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm that the data supporting the findings of this study are available within the article.