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Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells

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Abstract

Transplantation of pancreatic progenitors or insulin-secreting cells derived from human embryonic stem cells (hESCs) has been proposed as a therapy for diabetes. We describe a seven-stage protocol that efficiently converts hESCs into insulin-producing cells. Stage (S) 7 cells expressed key markers of mature pancreatic beta cells, including MAFA, and displayed glucose-stimulated insulin secretion similar to that of human islets during static incubations in vitro. Additional characterization using single-cell imaging and dynamic glucose stimulation assays revealed similarities but also notable differences between S7 insulin-secreting cells and primary human beta cells. Nevertheless, S7 cells rapidly reversed diabetes in mice within 40 days, roughly four times faster than pancreatic progenitors. Therefore, although S7 cells are not fully equivalent to mature beta cells, their capacity for glucose-responsive insulin secretion and rapid reversal of diabetes in vivo makes them a promising alternative to pancreatic progenitor cells or cadaveric islets for the treatment of diabetes.

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Figure 1: Overview of differentiation protocol and characterization of S4–S6.
Figure 2: S6 insulin+ cells co-express key transcription factors, such as NKX6.1, unlike S4 pancreatic progenitor cells.
Figure 3: S6 insulin+ cells develop faster in vivo than S4 pancreatic progenitor cells.
Figure 4: Optimization of S7 differentiation protocol for induction of MAFA expression.
Figure 5: Stage 7 cells have a similar gene expression profile to human islets, and insulin+ cells co-express key beta-cell maturation markers, including MAFA.
Figure 6: Stage 7 cells have certain functional similarities to human beta cells, but cannot be considered mature human beta cells.
Figure 7: S7 cells rapidly reverse diabetes and develop glucose-responsive insulin secretion in vivo.

Change history

  • 16 September 2014

    Erratum: In the version of this article initially published online, there were several errors in the PDF and/or the HTML versions. PDF and HTML versions: In the abstract, the last sentence should have read “in vivo” rather than “in vitro.” In the Methods, under the heading “Differentiation of S4 cells in suspension aggregate cultures,” the units of the cell clump size should have been micrometers rather than millimeters, as follows: “On the last day of culture, S4 cells were treated with 5 mg/ml dispase (Corning, Cat# 354235) for 5 min at 37 °C, followed by gentle pipetting to break into cell clumps (100–200 μm).” In the Methods, under the heading “S4: pancreatic endoderm, PDX1+/NKX6.1+ cells (3 d),” milliliter should have been microliter, as follows: “The resulting cell pellet was resuspended as single cells at a density of ~0.5 × 105 cells/μl on filter inserts (BD, Cat#35-3493 or Corning Cat#3420); 5–10 μl per spot for a total of 0.25–0.5 × 106 cells/spot) at an air-liquid interface.” HTML version: In the legends to Figures 2, 3, 4 and 7, the units of the scale bars should have been micrometers rather than millimeters. The errors have been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

This work was supported in part by funding from the JDRF, the Canadian Institutes of Health Research (CIHR) Regenerative Medicine and Nanomedicine Initiative, and the Stem Cell Network (SCN). J.E.B. was funded by a JDRF postdoctoral fellowship and L'Oreal Canada for Women in Science Research Excellence Fellowship. Y.H.C.Y. was the recipient of an NSERC Post Graduate Scholarship and M.M. was the recipient of a Mitacs Elevate Postdoctoral Fellowship. The CIHR Transplantation Training Program provided funding for J.E.B., N.Q. and M.M. We also thank Stem Cell Technologies for their financial support, T. Webber, S. Karuna and B. Hu for their technical assistance, and G. Warnock and Z. Ao from the Irving K. Barber Human Islet Isolation Laboratory (Vancouver, BC) for providing human islets.

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A. Rezania, J.E.B., J.D.J. and T.J.K. designed experiments, analyzed and interpreted results. J.E.B., P.A., A. Rubin, I.B., A.A., S.O., N.Q., M.M., T.A. and Y.H.C.Y. performed experiments. A. Rezania and T.J.K. conceived the project and supervised its participants. A. Rezania, J.E.B., J.D.J. and T.J.K. wrote the manuscript. A. Rezania, J.E.B., A.A., J.D.J., T.J.K., N.Q., M.M., T.A. and Y.H.C.Y. contributed to manuscript revisions.

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Correspondence to Alireza Rezania or Timothy J Kieffer.

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A. Rezania, P.A., A. Rubin and I.B. are employees of Janssen R&D, LLC; T.J.K. received financial support from Janssen R&D, LLC, for the research described in this article.

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Rezania, A., Bruin, J., Arora, P. et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol 32, 1121–1133 (2014). https://doi.org/10.1038/nbt.3033

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