[Identification and isolation of mesenchymal stem cells from human fetal pancreas].

Objective: To isolate and identify the phenotype and biological characteristics of pancreas derived mesenchymal stem cell.

Methods: Fresh pancreas of 4-5 months old aborted fetus was dissected free from connective tissue, and was cut into small pieces. The adherent cells were harvested and subcultured, after the third subculture, the cells were used for examination. Cell cycle was analyzed by measuring DNA content by FACScan flow cytometer. Phenotype of MSCs was analyzed by immunohistochemical SA technique and differentiated cells were identified by relevant specific staining.

Results: Fetal pancreas derived cells gave rise to a population of adherent cells characterized by the presence of a predominant cell type with a typical fibroblast like morphology. By transmission electron microcopy, MSC had few endoplasmic reticulums and mitochondrias. During the log phase of growth, MSC proliferated with a two fold population at 30 h. MSC can be ex vivo expanded by successive cycles of trypsinization, seeding, and culture. Under these conditions, MSC had capability of passaging up to 30 times without displaying significant changes in morphology, with 2-fold increase in cell number after each passage. This indicates the high ex vivo expansion potential of MSC. The results showed that the yield of CFU-Fs was above 200 clones even after the 6th passage. Cell cycle analysis by flow cytometry revealed that more than 83% of cells were in the G0/G1 phases, while a small population of cells were actively engaged in proliferation (S + G2 + M = 17%). We also showed that more than 86% of cells were positive stained by FITC labeled CD44, CD29, CD13, and only about 1% of cells were positive for CD34, HLA-DR. Expression of collagen I, III was positive while vWF was negative. In the differentiation study, we found culture-expanded pancreas MSCs could be directed into the osteogenic lineage as detected by osteoblastic morphology, expression of alkaline phosphatase, modulation of osteocalcin mRNA production and the formation of a mineralized extracellular matrix. We also found that MSCs could give rise to the adipogenic and chondrogenic lineage as evidenced by accumulation of lipid-rich vacuoles within cells and the expression of lipoprotein lipase mRNA or the expression of collagen II and the deposition of proteoglycans.

Conclusion: Mesenchymal stem cells existing in human pancreas can be isolated by their adherent ability and should be essential to sustain a steady supply of primitive cells in tissue remodeling.