Generation of Pancreatic Progenitor Cells

Pancreatic progenitor cells are a crucial intermediate stage in the development of pancreatic beta cells, which play a vital role in glucose homeostasis. The protocols on their differentiation can serve as a valuable tool for researchers aiming to produce pancreatic progenitor cells for further study.

In this protocol, Creative Biolabs outlines a step-by-step guide for differentiating pluripotent stem cells into pancreatic progenitor cells. Specific methods may vary depending on the source of stem cells and research goals. Always consult with our experts for cell line-specific details.

Materials Required

• Stem cells
• Cell culture medium for maintaining pluripotent stem cells
• Dulbecco's Modified Eagle's Medium (DMEM)/F12 for differentiation purposes
• Growth factors and cytokines: Activin A, Noggin, Wnt3a, EGF, FGF2, and others
• Insulin-Transferrin-Selenium (ITS) supplement for pancreatic progenitor cell differentiation
• B27 supplement
• Poly-L-Ornithine and Laminin
• A basal medium for differentiation

Procedure

Maintenance of Pluripotent Stem Cells

Maintain stem cells on Matrigel-coated culture plates. Ensure the culture medium is changed daily. Passage cells when they reach 70-80% confluence. Culture pluripotent stem cells in pancreatic differentiation medium supplemented with Activin A and HGF for 2 days. Change the medium every day for the next 4 days.

Formation of Definitive Endoderm

After 6 days, switch to a differentiation medium containing RPMI 1640, ITS, and Activin A. Continue daily medium changes for 3 days. Monitor cell morphology for the appearance of definitive endoderm-like cells.

Pancreatic Progenitor Formation

Transition the cells to a differentiation medium containing ITS, KGF, and EGF. Maintain the culture for 6-8 days with daily medium changes. Verify the presence of pancreatic progenitor-like cells.

Maturation of Pancreatic Progenitor Cells

Culture cells in RPMI 1640 medium supplemented with FBS and ITS. Keep cells for 6-10 days with daily medium changes. Assess the expression of pancreatic markers (e.g., PDX1, NKX6.1, INS) by immunostaining. Perform glucose-stimulated insulin secretion assays to evaluate the functionality of the differentiated cells.

Notes

• Regularly monitor the pluripotent stem cells to ensure their quality and health. Use cells within low passage numbers for better differentiation efficiency.
• The differentiation protocol might require optimization for specific cell lines or experimental conditions. Adjust growth factors and cytokine concentrations accordingly.
• Pay attention to the timing of medium changes and culture conditions, as these are crucial for successful differentiation.
• Comprehensive characterization of the derived pancreatic progenitor cells is crucial for confirming their identity and functionality. Perform immunocytochemistry, gene expression analysis, and functional assays as needed.

This protocol provides a foundational guide for the differentiation of pluripotent stem cells into pancreatic progenitor cells. Researchers can adapt and optimize this protocol to suit their specific research goals and application requirements. Careful attention to cell quality, medium composition, and analytical techniques will be instrumental in achieving successful differentiation and characterization of pancreatic progenitor cells.

Reference

1. McGaugh, Emily C., and M. Cristina Nostro. "Efficient differentiation of pluripotent stem cells to NKX6-1+ pancreatic progenitors." JoVE (Journal of Visualized Experiments) 121 (2017): e55265.

For Research Use Only. Not For Clinical Use.