In Vitro Expansion and Differentiation of Islet Precursor Cells from Cultured Neonatal Porcine Pancreatic Tissue

Ahn, Yu Bae; Yoon, Kun Ho; Seo, Sun Hee; Ko, Seung Hyun; Song, Ki Ho; Han, Je Ho; Yoo, Soon Jip; Son, Hyun Sik; Kang, Moo Il; Cha, Bong Yun; Lee, Kwang Woo; Son, Ho Young; Kang, Sung Koo

Abstract

BACKGROUND: Neonatal porcine pancreas is an attractive alternative source for islet transplantation because of its growth potential and availability. Porcine neonatal pancreatic cell clusters (NPCCs) consist mainly of protodifferentiated cells expressing both the duct cell marker pancytokeratin and islet hormones. In this study, we investigated to expand and mature the pancreas duct cells contained in porcine NPCCs with extracellular matrix.
METHODS
For NPCCs, pancreas obtained from neonatal pigs were minced, digested with collagenase and cultured overnight. Then NPCCs were further dispersed to small cell groups and cultured on HTB-9 extracellular matrix: the tissue attached and formed monolayer patches. At the 3rd and 8th days, tissue was fixed, immunostained for pancytokeratin (panCK), vimentin (VT) and islet hormones.
RESULTS
During 5 days culture, the total cell numbers increased 3.2 fold on the matrix, and 1.6 fold on the sticky dish, respectively. Insulin positive cells (Ins+) were 6.0% of total cells at day 3 and increased 1.6 fold in numbers at day 8. There was significant increase in DNA content of NPCCs in monolayers on both sticky dishes and HTB-9 matrix. In contrast, insulin content of both groups decreased during culture periods. Until 8 days of culture after dispersion of porcine NPCC, most duct cells costained with panCK and VT.
CONCLUSION
We observed NPCCs were composed of many of duct cells which were known to be endocrine precursor cells and monolayer culture of NPCC withextracellular matrix resulted in the proliferation and differentiation of pancreatic duct cells.