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Islet transplantation in the subcutaneous space achieves long-term euglycaemia in preclinical models of type 1 diabetes

Nature Metabolism volume 2, pages 1013–1020 (2020)Cite this article

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Abstract

The intrahepatic milieu is inhospitable to intraportal islet allografts1,2,3, limiting their applicability for the treatment of type 1 diabetes. Although the subcutaneous space represents an alternate, safe and easily accessible site for pancreatic islet transplantation, lack of neovascularization and the resulting hypoxic cell death have largely limited the longevity of graft survival and function and pose a barrier to the widespread adoption of islet transplantation in the clinic. Here we report the successful subcutaneous transplantation of pancreatic islets admixed with a device-free islet viability matrix, resulting in long-term euglycaemia in diverse immune-competent and immuno-incompetent animal models. We validate sustained normoglycaemia afforded by our transplantation methodology using murine, porcine and human pancreatic islets, and also demonstrate its efficacy in a non-human primate model of syngeneic islet transplantation. Transplantation of the islet–islet viability matrix mixture in the subcutaneous space represents a simple, safe and reproducible method, paving the way for a new therapeutic paradigm for type 1 diabetes.

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Fig. 1: Pancreatic islets transplanted in the subcutaneous space with IVM promote optimal glucose homeostasis in immuno-incompetent diabetic hosts.
Fig. 2: Pancreatic islets transplanted in the subcutaneous space with IVM promote optimal glucose homeostasis in immune-competent recipients.
Fig. 3: The islet–IVM mixture of pancreatic islets transplanted in the retroperitoneal space renders recipients normoglycaemic.
Fig. 4: IVM imparts enhanced viability to subcutaneously transplanted human islets, as reflected in anti-apoptotic and pro-angiogenic signals.

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Data availability

Raw and processed exosome sequencing data have been submitted to GEO (accession no. GSE145593). Any additional information supporting the data within this paper and the findings of this study are available from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

We thank S. Rostami, B. Koeberlein and G. Quinn for their valuable assistance in data collection and development of animal models. We thank Y. J. Wang for assistance with the islet β-cell BrdU staining experiments. We also acknowledge the support of J. Schug, Technical Director of the Next-Generation Sequencing Core at the University of Pennsylvania, for exosome sequencing analysis. We thank members of the Pancreatic Islet Isolation Team at the University of Pennsylvania (Y. Li, Z. Min and X. Zuo). D.A. thanks the Blavatnik Family Foundation for the Graduate Student Fellowship he received during his MD/PhD training. We also thank the National Institutes of Health for award nos. NIH/NIDDK DK070430, NIH/NIAID AI-102430 and NIH/NIDDK UC4–112217 (HPAP), and the NIDDK IIDP for the grant (Beckman Research Center, no. 10028044) awarded to A.N.

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Author notes

  1. These authors contributed equally: Ming Yu, Divyansh Agarwal.

Authors and Affiliations

  1. Division of Transplantation, Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, PA, USA

    Ming Yu, Divyansh Agarwal, Laxminarayana Korutla, Wei Wang, Chengyang Liu & Ali Naji

  2. Medical Scientist Training Program, Genomics and Computational Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Divyansh Agarwal

  3. Institute for Diabetes, Obesity & Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Catherine L. May, Klaus H. Kaestner & Ali Naji

  4. Department of Genetics, University of Pennsylvania, Philadelphia, PA, USA

    Catherine L. May

  5. Griffith Center for Restorative Surgery, Red Bank, NJ, USA

    Negin Noorchashm Griffith

  6. Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, MN, USA

    Bernhard J. Hering

  7. Division of Vascular Surgery, DeWitt Daughtry Family Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, USA

    Omaida C. Velazquez

  8. Division of Transplant Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    James F. Markmann

  9. Division of Cardiothoracic Surgery, Yale University School of Medicine, New Haven, CT, USA

    Prashanth Vallabhajosyula

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Contributions

A.N. conceptualized the study. O.C.V. provided the initial reagents that led to the development of an IVM. B.J.H. provided porcine islets. M.Y., N.N.G., C.L.M., C.L., D.A. and A.N. designed the transplantation and endocrine function experiments, conducted by M.Y., C.L.M., N.N.G., K.H.K., W.W. and C.L. Data analysis was led by D.A. Islet exosome research work was designed and conducted by L.K. and P.V. D.A. and A.N. wrote the paper with feedback from all authors. J.F.M., C.L. and A.N. supervised the study.

Corresponding authors

Correspondence to Divyansh Agarwal, Chengyang Liu or Ali Naji.

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Peer review information Primary Handling Editor: Christoph Schmitt.

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Extended data

Extended Data Fig. 1 The constituents and their respective concentrations needed to create 1.0 mL of Islet Viability Matrix (IVM).

A visual protocol to make the IVM is described in Supplementary Video 1.

Extended Data Fig. 2 Syngeneic and xenogeneic islet transplantation in the subcutaneous space.

a and c Murine or porcine islet grafts were transplanted with IVM in immunoincompetent diabetic mice, following which non-fasting blood glucose level returned to physiological ranges (<200mg/dl) and remained stable long term. Hyperglycemia promptly resumed upon removal of the grafts (indicated by downward arrows in a and c). Additionally, we established the presence of viable and functional transplanted islets from donor mice b and pigs d in the subcutaneous space by histologic examination and staining for insulin (red) and glucagon (green).

Extended Data Fig. 3 Intraperitoneal glucose tolerance test (GTT) in non-diabetic, normal/healthy mice, compared with immunoincompetent mice with long term survival (>6 months) with subcutaneous islet-IVM grafts.

In each set of experiments, GTT kinetics were evaluated in controls (n = 5) and B6 recipients of a, mouse islets (n = 5) and b, porcine islets (n = 5). GTT was also performed in B6 nude and B6 SCID recipients of c, human islets (n = 5 in each group). Islet grafts in the subcutaneous tissue promptly restore normoglycemia upon glucose challenge. Mean glycemic values for each experimental group are plotted, and the error bars represent the standard deviation. There were no statistically significant differences in glucose regulation between healthy/normal mice and the IVM+ islet transplant groups.

Extended Data Fig. 4 Table comparing the human islet graft survival with IVM in the subcutaneous space, with traditional islet transplant sites used in mice, viz., the kidney capsule and portal vein.

Of note, the same protocol for islet isolation and preparation was followed and N animals were used to for each particular set of transplant experiments. Hand-picked, healthy human islets transplanted in the subcutaneous space with IVM uniformly resulted in normoglycemia, whereas the same number of islets transplanted in the kidney capsule and the portal vein in the genetically identical animals resulted in a delayed restoration of normoglycemia.

Extended Data Fig. 5 SLC2A2 (Glut2) and INSULIN levels were upregulated at day 7 in islet culture with IVM (n=4 animals in each group).

a, All gene expressions were normalized to TBP and expressed as mean ± SEM. * denotes p < 0.05 based on a one-sided Student’s t-test. Each dot represents an individual data point. No p-value correction for multiple hypothesis adjustment was done. The p-values corresponding to SLC2A2 at days 3 and 7 were 0.006 and 0.01 respectively; p=0.008 for INSULIN at day 7. b, Human islets transplanted subcutaneously in immune-incompetent diabetic mice with and without IVM (n=4 mice in both groups) were excised on POD7, and the grafts were immunoassayed for insulin (green), BrdU (red) and counterstained for nuclear DNA with DAPI (blue). Yellow arrows point to β-cells with DNA replication as indicated by BrdU incorporation. One hundred insulin+ cells were counted per section in the IVM+ group, whereas <20 insulin+ cells could be counted in the IVM– cohort. The red structures represent nuclei, albeit some nuclei appear en face or out of plane. c, Quantification of DNA replication rate in IVM+ and IVM– cohorts (n=100 cells in the IVM+ group and 18 cells in the IVM– group) shows increased replication in the IVM+ group (** indicates one-sided Student’s t-test p=4x10-5).

Extended Data Fig. 6 Based on human-into-mouse islet transplant model, immunohistochemical profiling of islets ± IVM for markers of angiogenesis (VEGF), anti-apoptosis (Bcl-2, GLP-1) and endothelial cells (VWF) highlights the increased intensity and stained area of these epitopes in the IVM+ group.

The proportion and intensity of staining, quantified by QuPath using five automated regions of interest (ROIs) per sectioned image, is plotted below each section (* denotes p < 0.05 and ** denotes p < 5x10-5 based on the one-way Kruskal–Wallis H Test; arrow in the GLP-1 IVM– section denotes staining artifact). The particular significant p-values were — VEGF at POD 1 (p=0.009), Bcl-2 at POD1 (p=7x10-8) and POD10 (p=10-9), GLP-1 at POD1 (p=10-8) and POD 10 (p=4x10-9) and VWF at POD 10 (p=2x10-4).

Extended Data Fig. 7 Circulating human TISEs isolated at 6hr, 12hr, and on PODs 1, 3 and 10 from the IVM+ and IVM– cohorts were sequenced.

Differential expression across the five time points analyzed using one-sided Mann-Whitney U, as summarized in the Volcano Plot a. The x-axis is log2 ratio of gene expression levels between the two cohorts; the y-axis is the Benjamin Hochberg-adjusted p-value based on −log10. The colored dots represent the differentially expressed genes (blue = lower expression; red = higher expression in the IVM+ cohort) based on p<0.05 and two-fold expression difference. Gene Set Enrichment Analysis identified the major pathways (FDR ≤ 0.05) that were either upregulated or downregulated in the IVM+ group b. TISEs from IVM+ group showed higher expression of β-cell-specific proteins and anti-apoptotic markers as part of its intraexosomal cargo by Western blot analysis c. TSG101 protein is shown as a canonical exosome marker.

Source data

Extended Data Fig. 8 Morphological and immunohistochemical analysis of autologous cynomolgus monkey islets, implanted in the subcutaneous space, was performed at the time of euthanasia (animal ID# 212077, POD 918).

We found abundant healthy islet cell clusters, exhibiting vivid expression of key markers such as Insulin, Glucagon, Bcl-2, GLP-1, Ki67, VEGF, VWF and Collagen. Due to IACUC regulations and ethical care guidelines for nonhuman primate research, subcutaneous autologous islet transplants without IVM as a control could not be performed in a cynomolgus monkey.

Extended Data Fig. 9 For cynomolgus monkey ID# 210069, the animal’s blood glucose just prior to pancreatectomy was 72 mg/dl; blood glucose monitoring post-transplantation demonstrated persistent hyperglycemia in the animal, which required management by exogenous insulin therapy.

a, Failure to achieve normoglycemia in this monkey can be attributed partly to the suboptimal islet yield and transplantation of a relatively low mass of islets (11,827 IEQs/kg body weight), as well as the infusion of streptozotocin which likely led to the destruction of both native remnant islets as well as subcutaneously transplanted islets. b, In view of the persistent state of insulin dependency and per the recommendation of the IACUC veterinarian, the monkey was subjected to euthanasia on POD 250. During this course, an excisional biopsy of the islet bearing skin was performed on POD 46 and at the time of euthanasia. Both histologic assessments revealed abundant well granulated islet β-cells as well as glucagon-positive α-cells.

Extended Data Fig. 10

Characteristics of human islet donors used in the described experiments.

Supplementary information

Supplementary Information

Supplementary Table 1

Reporting Summary

Supplementary Video 1

A visual protocol for preparation of the IVM, illustrating its individual constituents.

Source data

Source Data Extended Data Fig. 7

Unprocessed immunoblot in support of Extended Data Fig. 7c.

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Yu, M., Agarwal, D., Korutla, L. et al. Islet transplantation in the subcutaneous space achieves long-term euglycaemia in preclinical models of type 1 diabetes. Nat Metab 2, 1013–1020 (2020). https://doi.org/10.1038/s42255-020-0269-7

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