in-vitro-colony-assays-for-characterizing-tri-potent-progenitor-cells

<em>In Vitro</em> Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Beckman Research Institute of City of Hope

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.

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

Progenitor cells in the adult pancreas are potential sources of endocrine beta cells for treating type 1 diabetes. Previously, we identified tri-potent progenitor cells in the adult (2-4month-old) murine pancreas that were capable of self-renewal and differentiation into duct, acinar, and endocrine cells in vitro. These progenitor cells were named pancreatic colony-forming units (PCFUs). However, because PCFUs are a minor population in the pancreas (~1%) they are difficult to study. To enrich PCFUs, strategies using cell-surface marker analyses and fluorescence-activated cell sorting were developed. We found that CD133(high)CD71(low) cells, but not other cell populations, enriched PCFUs by up to 30 fold compared to the unsorted cells. CD133(high)CD71(low) cells generated primary, secondary, and subsequent colonies when serially re-plated in Matrigel-containing cultures, suggesting self-renewal abilities. In the presence of a laminin hydrogel, CD133(high)CD71(low) cells gave rise to colonies that contained duct, acinar, and Insulin(+)Glucagon(+) double-hormonal endocrine cells. Colonies from the laminin hydrogel culture were implanted into diabetic mice, and five weeks later duct, acinar, and Insulin(+)Glucagon(-) cells were detected in the grafts, demonstrating tri-lineage differentiation potential of CD133(high)CD71(low) cells. These CD133(high)CD71(low) cells will enable future studies of putative adult pancreas stem cells in vivo.

Article	Year
Postnatal Pancreas of Mice Contains Tripotent Progenitors Capable of Giving Rise to Duct, Acinar, and Endocrine Cells In Vitro. Stem cells and development\| PubMed ID: 25941840	2015
Cells with surface expression of CD133(high)CD71(low) are enriched for tripotent colony-forming progenitor cells in the adult murine pancreas. Stem cell research\| PubMed ID: 26691820	2016

H. Teresa Ku

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