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Published August 13, 2015 | Published + Supplemental Material
Journal Article Open

Postnatal Pancreas of Mice Contains Tripotent Progenitors Capable of Giving Rise to Duct, Acinar, and Endocrine Cells In Vitro


Postnatal pancreas is a potential source for progenitor cells to generate endocrine β-cells for treating type 1 diabetes. However, it remains unclear whether young (1-week-old) pancreas harbors multipotent progenitors capable of differentiating into duct, acinar, and endocrine cells. Laminin is an extracellular matrix (ECM) protein important for β-cells' survival and function. We established an artificial extracellular matrix (aECM) protein that contains the functional IKVAV (Ile-Lys-Val-Ala-Val) sequence derived from laminin (designated aECM-lam). Whether IKVAV is necessary for endocrine differentiation in vitro is unknown. To answer these questions, we cultured single cells from 1-week-old pancreas in semi-solid media supplemented with aECM-lam, aECM-scr (which contains a scrambled sequence instead of IKVAV), or Matrigel. We found that colonies were generated in all materials. Individual colonies were examined by microfluidic reverse transcription-polymerase chain reaction, immunostaining, and electron microscopy analyses. The majority of the colonies expressed markers for endocrine, acinar, and ductal lineages, demonstrating tri-lineage potential of individual colony-forming progenitors. Colonies grown in aECM-lam expressed higher levels of endocrine markers Insulin1, Insulin2, and Glucagon compared with those grown in aECM-scr and Matrigel, indicating that the IKVAV sequence enhances endocrine differentiation. In contrast, Matrigel was inhibitory for endocrine gene expression. Colonies grown in aECM-lam displayed the hallmarks of functional β-cells: mature insulin granules and glucose-stimulated insulin secretion. Colony-forming progenitors were enriched in the CD133^(high) fraction and among 230 micro-manipulated single CD133^(high) cells, four gave rise to colonies that expressed tri-lineage markers. We conclude that young postnatal pancreas contains multipotent progenitor cells and that aECM-lam promotes differentiation of β-like cells in vitro.

Additional Information

© 2015 Mary Ann Liebert, Inc. Received for publication January 6, 2015, Accepted after revision May 4, 2015, Online Ahead of Editing: May 5, 2015, Online Ahead of Print: June 9, 2015, Published in Volume: 24 Issue 17: August 13, 2015. This work is supported in part by National Institutes of Health (NIH) grants R01DK081587 and R01DK099734 to H.T.K.; U01DK089533 to A.D.R; and California Institute for Regenerative Medicine (CIRM) grant RB5-07398 to D.A.T. N.G. is supported by a CIRM predoctoral fellowship as part of an institutional grant to City of Hope. We thank the Light Microscopy Core, Electron Microscopy Core, and the Analytical Cytometry Core at City of Hope for providing technical assistance. The authors declare no conflicts of interest.

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Supplemental Material - Supp_Fig1.pdf

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