Introduction
To date, insulin therapy is considered the gold standard for the treatment of type 1 diabetes. Nevertheless limitations persist, such as frequent episodes of hypoglycaemia, and development chronic micro- and macrovascular complications [
1,
2]. Islet transplantation offers an alternative treatment for patients with type 1 diabetes, especially for those with hypoglycaemic unawareness following insulin administration. Despite the improved outcome of islet transplantation over the last few years, drawbacks remain, such as a limited supply of cadaveric donors, the necessity of several donors for a single transplantation and (immediate) graft failure through metabolic pressure, continued autoimmunity, alloimmunity, high concentrations of immunosuppressive drugs and oxidative stress caused by hypoxia or due to cytokine exposure [
3]. Rapid revascularisation of the islets seems crucial to avoid beta cell stress by hypoxia and inflammatory cytokines. Several studies aiming to improve engraftment with cell therapy are currently ongoing (Clinical Trial.gov registration no. NCT00646724, NCT02384018).
Mesenchymal stem cells (MSCs, 30–35 μm in diameter), the major stem cells used for cell therapy, are self-renewing cells that can be isolated from the bone marrow and possibly also from many (if not all) tissues; for over 10 years MSCs have shown benefits in the treatment of several clinical diseases, mainly tissue injury and immune disorders [
4‐
6]. When co-transplanted with islets, MSCs improve islet transplantation outcome in preclinical animal models through immunomodulation, increasing islet revascularisation and/or preserving islet morphology [
7‐
10]. Despite the progress made in the understanding of MSC biology, there is little knowledge on the nature of their local microenvironment (probably hypoxic) and their functions in vivo. Moreover, long population doubling time, early senescence and DNA damage during in vitro expansion, as well as poor engraftment after transplantation, are considered to be among the key disadvantages of MSC therapy [
11]. Furthermore, with long-term culture expansion, MSCs can become karyotypical abnormal, potentially posing a risk of tumour formation.
Multipotent adult progenitor cells (MAPCs, 15–20 μm in diameter) are adult stem cells isolated from postnatal bone marrow, muscle or brain from rodents and humans [
12,
13]. These non-haematopoietic, non-endothelial stem cells most probably use similar immunosuppressive and angiogenic mechanisms to those used by MSCs, while displaying unique features (e.g. secretome, transcriptome and microRNA profiles) distinct from those of most adult stem cells [
14‐
17]. Interestingly, MAPCs have been shown to promote tissue repair and healing and induce neo-angiogenesis, possibly by delivering angiogenic growth factors that activate or recruit endogenous vascular cells and that seem to be specifically tailored to the immediate needs of the injured tissue [
18‐
21]. In vivo, these cells are short-lived as they experience only a minimally prolonged residence time. Moreover, in contrast to other cell types, MAPCs can be expanded in the long term (for > 80 population doublings) without genetic instability and can be administered without tissue matching, making them into an optimal stem cell product for routine clinical use (MultiStem; Athersys, Cleveland, OH, USA). Therefore, we aimed in this study to assess the therapeutic efficacy of co-transplantation of undifferentiated human MAPCs with mouse islets as separate or composite pellets in a syngeneic marginal mass islet transplantation model.
Discussion
Several hurdles still prevent the progression of clinical islet transplantation, including early graft failure and the loss of transplanted islet mass due to non-immunological reasons [
28]. In the first days following transplantation, islets lack an adequate vascular network, leading to severe hypoxia and cell death. It is believed that this is one of the major causes for the poor performance of islet grafts long-term. Therefore, there is a need of methods to improve the early survival, function and engraftment of transplanted islets. A variety of (stem) cell populations (i.e. endothelial progenitor cells, MSCs) have been described for enhancing transplanted beta cell survival and function after co-transplantation; however, there are still a number of problems relating to their wide-scale application in the clinic, such as inconsistent stem cell potency, poor cell engraftment and survival and age/disease-related host tissue impairment [
29].
MAPCs are a novel class of progenitor cells and can be derived from the postnatal bone marrow stroma compartment and also from muscle and brain of several species, including rodents and humans [
13]. These cells have demonstrated extensive in vitro expansion capacity, durable cytogenetic stability and higher plasticity when compared with MSCs [
30]. MAPCs are also non-immunogenic and have a strong immunomodulatory profile [
15,
27,
31‐
35], permitting safe non-HLA-matched allogeneic and even xenogeneic use without the need for immunosuppression [
36,
37]. Interestingly, MAPCs seemed to exhibit an unusual capacity to evade the immune system and can regulate homeostatic T cell proliferation, prevent the expansion of T helper (Th) 1, Th17 and Th22 effector T cells and block the development of pathogenic allogeneic responses [
31]. Moreover, MAPCs have been reported to secrete angiogenic growth factors and to improve vascular remodelling in different models of ischaemia, such as those for cardiac infarction and severe limb ischaemia [
20,
37]. Based on these phenotypic and functional characteristics, we wanted to study the localised effect of human MAPCs on islet graft function in a murine syngeneic marginal mass transplant model.
Here we show for the first time that human MAPCs co-transplanted as composite pellets with mouse islets can improve islet graft function as measured by the initial glycaemic control, diabetes reversal rate, glucose tolerance and serum C-peptide concentration when compared with transplantation of islets alone. Moreover, we found that grafts composed of islet–human MAPC composites had an improved revascularisation process. The human MAPCs actively participated in the revascularisation process mainly by producing angiogenic growth factors, soluble adhesion molecules and IL8.
Several studies have shown that MAPCs from both allogeneic and xenogeneic sources exert positive effects in models of ischaemia, mainly through the secretion of trophic factors such as VEGF-A, platelet-derived growth factor and IGF-1 [
37,
38]. Our present data corroborated the angiogenic potency of human MAPCs, as supernatant fractions of cultured cells were shown to contain high concentrations of vascular inflammation and angiogenic factors such as VEGF-A, PlGF and bFGF, all major regulators of islet vascularisation. The neo-angiogenic potential of human MAPCs was further validated in vivo in a chicken CAM assay, where implanted human MAPCs formed new blood vessels at a rate comparable with that observed with recombinant VEGF-A. VEGF-A is considered to be one of the major regulators of islet vascularisation, innervation and function, as beta cell-specific deletion of VEGF-A leads to diminished and abnormal islet vascularity, impaired postnatal nerve fibre ingrowth and dysregulated glucose-stimulated insulin secretion [
39‐
41]. Moreover, VEGF-A seems to be required for revascularisation of transplanted islets. Based on these data, several investigators have tried to enhance VEGF production locally in the pancreatic islets, although not continuously as this might trigger vascular tumour formation [
42‐
44]. It has been reported that islets co-transplanted with human embryonic stem cell-derived MSCs that conditionally overexpress VEGF allow a 50% reduction in the islet mass required to reverse diabetes in mice. These cells significantly improved islet metabolic function and revascularisation [
10].
Islet engraftment is a slow process, and while vascular sprouting, angiogenesis and revascularisation occur within 1–2 weeks after transplantation, the maturation of the blood vessels is likely to take several weeks to even months. Although VEGF can boost the process of islet revascularisation, this protein is also critical for maintenance of the intra-islet endothelial cell pool [
40]. We found a significant improvement in the development of a new islet capillary network in mice where islets were co-transplanted with islet–human MAPCs. Indeed, higher numbers of capillary-like structures with a lining of endothelial cells (detected with mouse-specific endomucin antibody) were found on the periphery and in the intra-islet space of the islet–human MAPC composites at week 5 post-transplantation. This suggests that host-derived vessels are directly feeding transplanted islets and that close proximity and even direct contact between the transplanted pancreatic islets and human MAPCs is of critical importance for the improved glucose control, diabetes reversal rate and increased revascularisation. Absence of human
Cd31 (
Pecam1) mRNA expression in the islet grafts further supports the idea that human MAPCs are not incorporated into new capillaries but possibly secrete growth factors to initiate angiogenesis and to support functional tissue survival (data not shown). These observations are in full agreement with previous observations that the major role of human MAPCs is to provide angiogenic growth factors in the first days after implantation, after which they are cleared rapidly from the body, without leading to immune activation [
18,
37].
The present data encouraged the use of human MAPCs in islet transplantation protocols as our results demonstrate the improvement of islet graft morphology and function by transplantation of islet–human MAPC composites, possibly via the promotion of graft revascularisation mediated by human MAPCs.
Acknowledgements
Special thanks go to J. Laureys and F. Coun (Laboratory of Clinical and Experimental Endocrinology, Katholiecke Universiteit Leuven (KULEUVEN)) for technical advice and assistance. The Ventana staining service was provided by InfraMouse (KULEUVEN – VIB) through a Hercules type 3 project (ZW09-03). We also thank E. Radaelli and A. Francis (InfraMouse) for their help with fluorescence stainings. Some of the data were presented as abstracts at the 1st National Diabetes Excellence Summit, Brussels, Belgium (April 25 2015), the Joint Meeting of The Islet Study Group & Beta Cell Workshop, Jerusalem, Israel (May 3–7, 2015), the 17th congress of the European Society for Organ Transplantation congress, Brussels, Belgium (September 13–16, 2015) and the 25th meeting of the Belgian Endocrine Society, Antwerp, Belgium (October 23–24, 2015).
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