Production of Functional Glucagon-Secreting α-Cells From Human Embryonic Stem Cells

  1. Timothy J. Kieffer2,3
  1. 1BetaLogics Venture, Centocor Research and Development, Skillman, New Jersey;
  2. 2Laboratory of Molecular and Cellular Medicine, Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada;
  3. 3Department of Surgery, University of British Columbia, Vancouver, British Columbia, Canada.
  1. Corresponding author: Timothy J. Kieffer, tim.kieffer{at}ubc.ca.
  1. A.R., M.J.R., and R.D.W. contributed equally to this work.

Abstract

OBJECTIVE Differentiation of human embryonic stem (hES) cells to fully developed cell types holds great therapeutic promise. Despite significant progress, the conversion of hES cells to stable, fully differentiated endocrine cells that exhibit physiologically regulated hormone secretion has not yet been achieved. Here we describe an efficient differentiation protocol for the in vitro conversion of hES cells to functional glucagon-producing α- cells.

RESEARCH DESIGN AND METHODS Using a combination of small molecule screening and empirical testing, we developed a six-stage differentiation protocol for creating functional α-cells. An extensive in vitro and in vivo characterization of the differentiated cells was performed.

RESULTS A high rate of synaptophysin expression (>75%) and robust expression of glucagon and the α-cell transcription factor ARX was achieved. After a transient polyhormonal state in which cells coexpress glucagon and insulin, maturation in vitro or in vivo resulted in depletion of insulin and other β-cell markers with concomitant enrichment of α-cell markers. After transplantation, these cells secreted fully processed, biologically active glucagon in response to physiologic stimuli including prolonged fasting and amino acid challenge. Moreover, glucagon release from transplanted cells was sufficient to reduce demand for pancreatic glucagon, resulting in a significant decrease in pancreatic α-cell mass.

CONCLUSIONS These results indicate that fully differentiated pancreatic endocrine cells can be created via stepwise differentiation of hES cells. These cells may serve as a useful screening tool for the identification of compounds that modulate glucagon secretion as well as those that promote the transdifferentiation of α-cells to β-cells.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • Received April 22, 2010.
  • Accepted September 20, 2010.

Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

| Table of Contents

This Article

  1. Diabetes vol. 60 no. 1 239-247
  1. Online Appendix
  2. All Versions of this Article:
    1. db10-0573v1
    2. db10-0573v2
    3. 60/1/239 most recent