Long-term glycemic control using polymer-encapsulated human stem cell–derived beta cells in immune-competent mice

Journal name:
Nature Medicine
Volume:
22,
Pages:
306–311
Year published:
DOI:
doi:10.1038/nm.4030
Received
Accepted
Published online
Corrected online

The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes1. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically2, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue3. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-β cells), which may represent an unlimited source of human cells for pancreas replacement therapy4. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier5, 6. However, clinical implementation has been challenging because of host immune responses to the implant materials7. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-β cells. SC-β cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells.

At a glance

Figures

  1. SC-[beta] cells encapsulated with TMTD alginate sustain normoglycemia in STZ-treated immune-competent C57BL/6J mice.
    Figure 1: SC-β cells encapsulated with TMTD alginate sustain normoglycemia in STZ-treated immune-competent C57BL/6J mice.

    (a) Top, schematic representation of the last three stages of differentiation of human embryonic stem cells to SC-β cells. Stage 4 cells (pancreatic progenitors 2) co-express pancreatic and duodenal homeobox 1 (PDX-1) and NK6 homeobox 1 (NKX6.1); Stage 5 cells (endocrine progenitors) co-express chromogranin A (ChromA) and NKX6.1; and Stage 6 cells (stem cell–derived beta cells) co-express C-peptide and NKX6.1. Bottom, representative FACS plots (of ten separate differentiations from the HUES8 stem cell line) showing the surface markers on cells at the indicated differentiation stages. Numbers in each quadrant represent the percentages of cells that are positive for the specified markers. (b) Representative bright-field images (n = 15 mice per treatment group) of encapsulated SC-β cells in 500-μm alginate microcapsules (left), 1.5-mm alginate spheres (middle) and 1.5-mm TMTD alginate spheres (right) at different doses. Scale bars, 400 μm. (ce) Blood glucose concentrations measured at the indicated times after transplantation of SC-β cells encapsulated in 500-μm SLG20 alginate microcapsules (c), 1.5-mm SLG20 alginate spheres (d) or 1.5-mm TMTD alginate spheres (e) in the intraperitoneal space of STZ-treated C57BL/6 mice at three different doses of cell clusters (100, 250 and 1,000 clusters per mouse) (n = 5 mice per treatment per experiment; experiments were repeated three times for a total of n = 15 mice per treatment). The red dashed line indicates the blood glucose cutoff for normoglycemia in mice. For reference, 250 clusters equates to ~106 cells. Error bars denote mean ± s.e.m.

  2. SC-[beta] cells encapsulated with TMTD alginate elicit weaker immunological and fibrotic responses in immune-competent C57BL/6J mice.
    Figure 2: SC-β cells encapsulated with TMTD alginate elicit weaker immunological and fibrotic responses in immune-competent C57BL/6J mice.

    (a) Quantification of the indicated cell types associated with the encapsulated SC-β cell implants retrieved 14 d after intraperitoneal transplantation in C57BL/6 mice, as determined by FACS analysis (n = 10 mice per treatment group). TMTD-1.5, 1.5-mm TMTD alginate spheres; SLG-1.5, 1.5-mm SLG20 alginate spheres; SLG-0.5, 500-μm SLG20 microcapsules. Error bars denote mean ± s.e.m. *P < 0.05, **P < 0.001, ***P < 0.0001; one-way analysis of variance (ANOVA) with Bonferroni multiple-comparison correction. Red asterisks specify statistical significance between the indicated groups, and black asterisks specify statistical significance against the SLG-0.5 group. (bd) Representative dark-field (n = 15) (b), bright-field (n = 15) (c) and z-stacked confocal immunofluorescence (DAPI in blue; F-actin in green; α-SMA in red) (n = 15) (d) images of SC-β cell implants in TMTD-1.5 (left), SLG-1.5 (middle) or SLG-0.5 (right) formulations retrieved 90 d after implantation from the STZ-treated C57BL/6J mice in Figure 1c–e (n = 15 mice per group). Scale bars, 2 mm (b), 300 μm (c,d). (e) Proteomic analysis of lysates from implants containing 250 SC-β clusters that were retrieved from the STZ-treated C57BL/6J mice shown in Figure 1c–e 90 d after implantation (n = 4 mice per treatment; analysis was performed on two separate cohorts for a total of n = 8 mice per treatment). Each column in the heat map represents an individual mouse from the respective treatment group; the colored bar denotes the colors used to represent fold expression relative that of the TMTD group. (f) α-SMA protein isolated from implants retrieved from the STZ-treated C57BL/6J mice shown in Figure 1c–e, as measured by immunoblot analysis (n = 5 mice per treatment group). Error bars denote mean ± s.e.m. *P < 0.05, **P < 0.001, ***P < 0.0001; one-way ANOVA with Bonferroni multiple-comparison correction; black asterisks specify statistical significance against the SLG-0.5 group. (g,h) Representative images of encapsulated SC-β cells before implantation (g) or after retrieval 90 d after implantation (h) from the STZ-treated C57BL/6J mice shown in Figure 1c–e (n = 15 mice per group). Left, images after 20× magnification; right, images after 70× magnification. DAPI in blue; insulin in green; glucagon in red. Scale bars, 20 μm. All experiments were done at least three times, with the exception of FACS and proteomics quantification, which were done twice. *P < 0.05, **P < 0.001, ***P < 0.0001; one-way ANOVA with Bonferroni multiple-comparison correction.

  3. SC-[beta] cells encapsulated with TMTD alginate sustain long-term normoglycemia in STZ-treated C57BL/6J mice.
    Figure 3: SC-β cells encapsulated with TMTD alginate sustain long-term normoglycemia in STZ-treated C57BL/6J mice.

    (a) Blood glucose levels in STZ-treated C57BL/6 mice implanted with SC-β cells encapsulated with TMTD alginate at a dose of 250 SC-β clusters/mouse or healthy and non-transplanted C57BL/6 mice. Shown are cohorts of n = 5 mice per treatment group; experiment was repeated three times for a total of n = 15 mice per condition. (b) Blood glucose levels of the mice shown in a together with a cohort of STZ-treated non-implanted mice that were subjected to an intravenous glucose tolerance test (IVGTT) 174 d after implantation. (c) Human C-peptide levels in the blood of the STZ-treated C57BL/6 mice implanted with SC-β cells (used in a). (d) Quantification of mouse insulin extracted from the pancreas of mice (n = 5) in each treatment group. Pancreas from TMTD alginate–treated mice were removed 174 d after implantation, whereas the pancreas of healthy or STZ-treated non-implanted C57BL/6 mice were taken at 8–10 weeks after implantation. (e,f) Representative bright-field (n = 15 mice per treatment) (e) and z-stacked confocal immunofluorescence (DAPI in blue; F-actin in green; α-SMA in red) (n = 15 mice per treatment) (f) images of implants retrieved from the STZ-treated C57BL/6J mice presented in ac that were implanted with SC-β cells encapsulated with TMTD alginate at a dose of 250 SC-β clusters/mouse. Scale bars, 400 μm. (g,h) Masson's trichrome (g) and H&E (h) histological analysis of implants retrieved 174 d after implantation from the STZ-treated C57BL/6J mice presented in ac that were implanted with SC-β cells encapsulated with TMTD alginate at a dose of 250 SC-β clusters/mouse. Scale bars, 2 mm. (i,j) Representative images of immunofluorescence analysis of implants retrieved 174 d after implantation from the STZ-treated C57BL/6J mice mice presented in ac that were implanted with SC-β cells encapsulated with TMTD alginate at a dose of 250 SC-β clusters/mouse. Lower- (20×, left) and higher-magnification (40×, right) images are shown. DAPI in blue; insulin (i) or NKX6.1 (j) in green; glucagon (i) or C-peptide (j) in red. Scale bars, 50 μm. Throughout, error bars denote mean ± s.e.m. n = 5 mice were used per treatment, and all experiments were performed at least three times for a total of n = 15 mice per treatment. In d, ***P < 0.0001; one-way ANOVA with Bonferroni multiple-comparison correction; n.s., not significant.

Change history

Corrected online 18 February 2016
In the version of this article initially published online, the authors omitted acknowledgment recognizing the histology core of the Harvard Stem Cell Institute and several individuals for their assistance. The error has been corrected for the print, PDF and HTML versions of this article.

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Author information

  1. Present addresses: Department of Chemistry, Boston University, Boston, Massachusetts, USA (A.J.V.); Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA (A.R.B.); Division of Endocrinology, Metabolism and Lipid Research, Washington University School of Medicine, St. Louis, Missouri, USA (J.R.M.); Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, Missouri, USA (J.R.M.).

    • Arturo J Vegas,
    • Jeffrey R Millman &
    • Andrew R Bader
  2. These authors contributed equally to this work.

    • Arturo J Vegas &
    • Omid Veiseh

Affiliations

  1. David H Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.

    • Arturo J Vegas,
    • Omid Veiseh,
    • Andrew R Bader,
    • Joshua C Doloff,
    • Jie Li,
    • Michael Chen,
    • Karsten Olejnik,
    • Hok Hei Tam,
    • Siddharth Jhunjhunwala,
    • Erin Langan,
    • Stephanie Aresta-Dasilva,
    • Srujan Gandham,
    • Robert Langer &
    • Daniel G Anderson
  2. Department of Anesthesiology, Boston Children's Hospital, Boston, Massachusetts, USA.

    • Arturo J Vegas,
    • Omid Veiseh,
    • Andrew R Bader,
    • Joshua C Doloff,
    • Jie Li,
    • Michael Chen,
    • Karsten Olejnik,
    • Hok Hei Tam,
    • Siddharth Jhunjhunwala,
    • Erin Langan,
    • Stephanie Aresta-Dasilva,
    • Srujan Gandham,
    • Robert Langer &
    • Daniel G Anderson
  3. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Omid Veiseh,
    • Hok Hei Tam,
    • Robert Langer &
    • Daniel G Anderson
  4. Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA.

    • Mads Gürtler,
    • Jeffrey R Millman,
    • Felicia W Pagliuca &
    • Douglas A Melton
  5. Department of Surgery, Division of Transplantation, University of Illinois at Chicago, Chicago, Illinois, USA.

    • James J McGarrigle,
    • Matthew A Bochenek &
    • Jose Oberholzer
  6. Section on Islet Cell and Regenerative Biology, Research Division, Joslin Diabetes Center, Boston, Massachusetts, USA.

    • Jennifer Hollister-Lock &
    • Gordon C Weir
  7. Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Dale L Greiner
  8. Howard Hughes Medical Institute (HHMI), Harvard University, Cambridge, Massachusetts, USA.

    • Douglas A Melton
  9. Division of Health Science Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Robert Langer &
    • Daniel G Anderson
  10. Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Robert Langer &
    • Daniel G Anderson

Contributions

A.J.V., O.V. and D.G.A. designed experiments, analyzed data and wrote the manuscript. M.G., J.R.M., F.W.P. and D.A.M. provided SC-β cells. A.J.V., O.V., M.G., J.R.M., F.W.P., A.R.B., J.C.D., J.L., M.C., K.O., S.J., E.L., S.A.-D., S.G., J.J.M., M.A.B. and J.H.-L. performed experiments. H.H.T. performed statistical analyses of data sets and aided in the preparation of displays communicating data sets. J.O., D.L.G., G.C.W., D.A.M. and R.L. provided conceptual advice and technical support. R.L. and D.G.A. supervised the study. All of the authors discussed the results and assisted in the preparation of the manuscript.

Competing financial interests

F.W.P., J.R.M., M.G. and D.A.M. declare a financial interest via a patent filed by Harvard University and HHMI on the production of stem cell–derived β-cells. A.J.V., O.V., J.C.D., R.L. and D.G.A. declare financial interests via patents filed by MIT on the material and hydrogel capsule technology.

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