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Production of pancreatic hormone–expressing endocrine cells from human embryonic stem cells

Abstract

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor—en route to cells that express endocrine hormones. The hES cell–derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal β-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell–derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.

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Figure 1: Schematic of differentiation procedure and protein expression for some key markers of pancreatic differentiation.
Figure 2: Immunofluorescence analysis of transitions from definitive endoderm to gut tube and from gut tube to posterior foregut.
Figure 3: Immunofluorescence analysis of transitions from pancreas to endocrine precursor and from endocrine precursor to hormone-expressing cells.
Figure 4: Immunofluorescence analysis of hormone-expressing cells.
Figure 5: Flow cytometry analysis of insulin-expressing cells.
Figure 6: High insulin content and robust C-peptide release.

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Acknowledgements

We thank Matthias Hebrok, Mike German and Alberto Hayek for critical review of the manuscript; Alberto Hayek for total RNA from 11- and 14-week fetal human pancreas; and Michael McCaffery at John Hopkins University for performing the EM analyses. SU5402 was a gift from A. Terskikh, mouse anti-NKX6-1 (F55A12) and rabbit anti-NKX6-1 were gifts from Ole Madsen, and rabbit anti-NGN3 was a gift from Mike German. The CyT203 and CyT49 cell lines were derived with partial funding from the Juvenile Diabetes Research Foundation.

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Correspondence to Emmanuel E Baetge.

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Supplementary information

Supplementary Fig. 1

Endocrine differentiation from hES cells is critically dependent on the DE lineage. (PDF 578 kb)

Supplementary Fig. 2a

Endocrine differentiation from hES cells via DE but not neural or extra-embryonic lineages. (PDF 529 kb)

Supplementary Fig. 2b

Endocrine differentiation from hES cells via DE but not neural or extra-embryonic lineages. (PDF 523 kb)

Supplementary Fig. 3

Comparison of mRNA expression levels in hES-derived cells to fetal and adult human pancreas. (PDF 469 kb)

Supplementary Fig. 4

Amylase expressing cells are intermingled with endocrine cells. (PDF 1213 kb)

Supplementary Fig. 5

Endocrine clusters stain with dithizone. (PDF 1054 kb)

Supplementary Fig. 6

Numerous secretory granules in hES cell-derived cells. (PDF 825 kb)

Supplementary Fig. 7

Differentiation of 5 different hES cell lines to insulin-expressing cells. (PDF 541 kb)

Supplementary Fig. 8

MAFB and insulin are co-expressed in hES cell-derived cells and human fetal pancreas. (PDF 1199 kb)

Supplementary Table 1

Real-time PCR primers. (PDF 199 kb)

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D'Amour, K., Bang, A., Eliazer, S. et al. Production of pancreatic hormone–expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 24, 1392–1401 (2006). https://doi.org/10.1038/nbt1259

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