Preservation of epithelial progenitor cells from collagenase-digested oral mucosa during ex vivo cultivation.

To avoid xenogeneic infection, we report a novel protocol for producing animal-derived component-free oral mucosal epithelial cells (OMECs) sheet for transplantation, in which collagenase was used to replace dispase II/trypsin-EDTA for digesting oral mucosal tissue, and human platelet-derived PLTMax to replace fetal bovine serum. The resulting epithelial aggregates were expanded on de-epithelialized amniotic membranes without 3T3 feeder cells, and serum-free EpiLife was used to reduce contamination by submucosal mesenchymal cells. The OMEC sheets thus generated showed similar positive keratin 3/76-positive and keratin 8-negative staining patterns compared with those generated by the original protocol. Colony formation efficiency assay, BrdU label retention assay, and p63 and p75NTR immunostaining results indicated that higher proliferative potentials and more progenitor cells were preserved by the modified protocol. TaqMan array analysis revealed that the transcription of integrin-linked kinase (ILK) was up-regulated along with an increase in ß-catenin signaling and its downstream cell cycle modulators, cyclin D1 and p27KIP1. Furthermore, ILK silencing led to the inhibition of nuclear ß-catenin accumulation, suppressed p63 expression, and reduced the expression of cyclin D1 and p27KIP1; these observations suggest that ILK/ß-catenin pathway may be involved in cell proliferation regulation during the ex vivo expansion of OMECs for transplantation purposes.

Influence of age on adenomatous polyposis coli and p53 mutation frequency in sporadic colorectal cancer-rarity of co-occurrence of mutations in APC, K-ras, and p53 genes.

INTRODUCTION: Previous studies have investigated the possibility that specific mutations may be related to specific clinicopathological features. However, most previous investigations included only an average age (40-80 years) group of sporadic colorectal cancers and, moreover, studied only a single gene in isolation. Therefore, the influence of age on these mutation frequencies remains unclear, despite age being considered a risk factor for genetic mutation. MATERIALS AND METHODS: This study included 122 sporadic colorectal cancers from three different age groups and analyzed mutation frequencies of adenomatous polyposis coli (APC), K-ras, and p53 genes and microsatellite instability to determine their mutation frequencies and relationships with clinicopathological features. RESULTS: Significantly lower p53 mutation frequencies were observed among young (32 years old or younger) and old (86 years old or older) patient groups compared with an average age (39-85 years old) patient group (14.3% and 19.2% versus 51.5%, P<0.001). APC mutation frequency (11.8%) was significantly lower in highly aggressive (Dukes' stage D) tumors ( P=0.003) than in the other stage tumors (Dukes' stage A, B, and C). Additionally, simultaneous occurrence of all three genetic alterations in an individual tumor was rare (below 5%). Statistical analysis further confirmed that mutation number in Dukes' D tumors occurred less frequently than expected in other stage tumors ( P=0.008). CONCLUSIONS: Genetic alterations of sporadic colorectal cancers have different relationships with age or tumor stage. Additionally, most sporadic colorectal tumors do not necessarily require following the widely accepted genetic model, because the three key genetic mutations, APC, K-ras, and p53, rarely occur simultaneously.