Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

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.

References

  1. 1.

    Kawahara, T. et al. Portal vein thrombosis is a potentially preventable complication in clinical islet transplantation. Am. J. Transpl. 11, 2700–2707 (2011).

    CAS  Article  Google Scholar 

  2. 2.

    Moberg, L. et al. Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation. Lancet 360, 2039–2045 (2002).

    CAS  Article  Google Scholar 

  3. 3.

    Walsh, T. J., Eggleston, J. C. & Cameron, J. L. Portal hypertension, hepatic infarction, and liver failure complicating pancreatic islet autotransplantation. Surgery 91, 485–487 (1982).

    CAS  PubMed  Google Scholar 

  4. 4.

    Hering, B. J. et al. Phase 3 trial of transplantation of human islets in type 1 diabetes complicated by severe hypoglycemia. Diabetes Care 39, 1230–1240 (2016).

    CAS  Article  Google Scholar 

  5. 5.

    Ricordi, C. & Strom, T. B. Clinical islet transplantation: advances and immunological challenges. Nat. Rev. Immunol. 4, 259–268 (2004).

    CAS  Article  Google Scholar 

  6. 6.

    Smink, A. M., Faas, M. M. & de Vos, P. Toward engineering a novel transplantation site for human pancreatic islets. Diabetes 62, 1357–1364 (2013).

    CAS  Article  Google Scholar 

  7. 7.

    Ao, Z., Matayoshi, K., Lakey, J. R., Rajotte, R. V. & Warnock, G. L. Survival and function of purified islets in the omental pouch site of outbred dogs. Transplantation 56, 524–529 (1993).

    CAS  Article  Google Scholar 

  8. 8.

    Vlahos, A. E., Cober, N. & Sefton, M. V. Modular tissue engineering for the vascularization of subcutaneously transplanted pancreatic islets. Proc. Natl Acad. Sci. USA 114, 9337–9342 (2017).

    CAS  Article  Google Scholar 

  9. 9.

    Pepper, A. R. et al. A prevascularized subcutaneous device-less site for islet and cellular transplantation. Nat. Biotechnol. 33, 518–523 (2015).

    CAS  Article  Google Scholar 

  10. 10.

    Cantarelli, E. et al. Bone marrow as an alternative site for islet transplantation. Blood 114, 4566–4574 (2009).

    CAS  Article  Google Scholar 

  11. 11.

    Pepper, A. R. et al. Long-term function and optimization of mouse and human islet transplantation in the subcutaneous device-less site. Islets 8, 186–194 (2016).

    CAS  Article  Google Scholar 

  12. 12.

    Pepper, A. R. et al. Transplantation of human pancreatic endoderm cells reverses diabetes post transplantation in a prevascularized subcutaneous site. Stem Cell Rep. 8, 1689–1700 (2017).

    CAS  Article  Google Scholar 

  13. 13.

    Odorico, J. et al. Report of the key opinion leaders meeting on stem cell-derived beta cells. Transplantation 102, 1223–1229 (2018).

    Article  Google Scholar 

  14. 14.

    Juang, J.-H., Hsu, B. R.-S. & Kuo, C.-H. Islet transplantation at subcutaneous and intramuscular sites. Transplant. Proc. 37, 3479–3481 (2005).

    Article  Google Scholar 

  15. 15.

    Lacy, P. E., Hegre, O. D., Gerasimidi-Vazeou, A., Gentile, F. T. & Dionne, K. E. Maintenance of normoglycemia in diabetic mice by subcutaneous xenografts of encapsulated islets. Science 254, 1782–1784 (1991).

    CAS  Article  Google Scholar 

  16. 16.

    Smink, A. M. & de Vos, P. Therapeutic strategies for modulating the extracellular matrix to improve pancreatic islet function and survival after transplantation. Curr. Diab. Rep. 18, 39 (2018).

    Article  Google Scholar 

  17. 17.

    Van Deijnen, J. H., Van Suylichem, P. T., Wolters, G. H. & Van Schilfgaarde, R. Distribution of collagens type I, type III and type V in the pancreas of rat, dog, pig and man. Cell Tissue Res. 277, 115–121 (1994).

    Article  Google Scholar 

  18. 18.

    Vlahos, A. E. et al. Endothelialized collagen based pseudo-islets enables tuneable subcutaneous diabetes therapy. Biomaterials 232, 119710 (2020).

    CAS  Article  Google Scholar 

  19. 19.

    Stephens, C. H. et al. In situ type I oligomeric collagen macroencapsulation promotes islet longevity and function in vitro and in vivo. Am. J. Physiol. Endocrinol. Metab. 315, E650–E661 (2018).

    CAS  Article  Google Scholar 

  20. 20.

    Parsons, R. F. et al. Murine islet allograft tolerance upon blockade of the B-lymphocyte stimulator, BLyS/BAFF. Transplantation 93, 676–685 (2012).

    CAS  Article  Google Scholar 

  21. 21.

    Lucas-Clerc, C., Massart, C., Campion, J. P., Launois, B. & Nicol, M. Long-term culture of human pancreatic islets in an extracellular matrix: morphological and metabolic effects. Mol. Cell. Endocrinol. 94, 9–20 (1993).

    CAS  Article  Google Scholar 

  22. 22.

    Weber, L. M. & Anseth, K. S. Hydrogel encapsulation environments functionalized with extracellular matrix interactions increase islet insulin secretion. Matrix Biol. 27, 667–673 (2008).

    CAS  Article  Google Scholar 

  23. 23.

    Jiang, K. et al. 3-D physiomimetic extracellular matrix hydrogels provide a supportive microenvironment for rodent and human islet culture. Biomaterials 198, 37–48 (2019).

    CAS  Article  Google Scholar 

  24. 24.

    Kale, J., Osterlund, E. J. & Andrews, D. W. BCL-2 family proteins: changing partners in the dance towards death. Cell Death Differ. 25, 65–80 (2018).

    CAS  Article  Google Scholar 

  25. 25.

    Portha, B., Tourrel-Cuzin, C. & Movassat, J. Activation of the GLP-1 receptor signalling pathway: a relevant strategy to repair a deficient beta-cell mass. Exp. Diabetes Res. 2011, 376509 (2011).

    PubMed  PubMed Central  Google Scholar 

  26. 26.

    Xiong, X., Shao, W. & Jin, T. New insight into the mechanisms underlying the function of the incretin hormone glucagon-like peptide-1 in pancreatic β-cells: the involvement of the Wnt signaling pathway effector β-catenin. Islets 4, 359–365 (2012).

    Article  Google Scholar 

  27. 27.

    Tomas, A., Jones, B. & Leech, C. New insights into beta-cell GLP-1 receptor and cAMP signaling. J. Mol. Biol. 432, 1347–1366 (2020).

    CAS  Article  Google Scholar 

  28. 28.

    Vallabhajosyula, P. et al. Tissue-specific exosome biomarkers for noninvasively monitoring immunologic rejection of transplanted tissue. J. Clin. Invest. 127, 1375–1391 (2017).

    Article  Google Scholar 

  29. 29.

    Wu, J. & Yan, L.-J. Streptozotocin-induced type 1 diabetes in rodents as a model for studying mitochondrial mechanisms of diabetic β cell glucotoxicity. Diabetes Metab. Syndr. Obes. 8, 181–188 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Scholz, J. L. et al. BLyS inhibition eliminates primary B cells but leaves natural and acquired humoral immunity intact. Proc. Natl Acad. Sci. USA 105, 15517–15522 (2008).

    CAS  Article  Google Scholar 

  31. 31.

    Ricordi, C. et al. National Institutes of Health-sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: manufacture of a complex cellular product at eight processing facilities. Diabetes 65, 3418–3428 (2016).

    CAS  Article  Google Scholar 

  32. 32.

    Liu, C. et al. B lymphocyte-directed immunotherapy promotes long-term islet allograft survival in nonhuman primates. Nat. Med. 13, 1295–1298 (2007).

    CAS  Article  Google Scholar 

  33. 33.

    Avrahami, D. et al. Targeting the cell cycle inhibitor p57Kip2 promotes adult human β cell replication. J. Clin. Invest. 124, 670–674 (2014).

    CAS  Article  Google Scholar 

  34. 34.

    Bankhead, P. et al. QuPath: open source software for digital pathology image analysis. Sci. Rep. 7, 16878 (2017).

    Article  Google Scholar 

  35. 35.

    Agarwal, D., Mukherjee, S., Bhattacharya, B. B. & Zhang, N. R. Distribution-free multisample test based on optimal matching with applications to single cell genomics. J. Am. Stat. Assoc. https://doi.org/10.1080/01621459.2020.1791131 (2020).

  36. 36.

    Wang, J., Vasaikar, S., Shi, Z., Greer, M. & Zhang, B. WebGestalt 2017: a more comprehensive, powerful, flexible and interactive gene set enrichment analysis toolkit. Nucleic Acids Res. 45, W130–W137 (2017).

    CAS  Article  Google Scholar 

Download references

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.

Author information

Affiliations

Authors

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 or Chengyang Liu or Ali Naji.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Primary Handling Editor: Christoph Schmitt.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing