395 ISOLATION AND CHARACTERIZATION OF PORCINE ADIPOSE-DERIVED ADULT STEM CELLS

Abstract

A dipose-derived adult stem (ADAS) cells have been proposed as an alternative to embryonic stem cells for tissue engineering applications. Stromal cells isolated from human adipose tissue are self-renewing, can be induced to differentiate along several mesenchymal tissue lineages, and are characterized by a high expression of CD34 and CD44 stem cell markers. The objective of this study was to develop a protocol for the isolation and culture of porcine ADAS cells and to determine stem cell-like characteristics such as the ability to differentiate into multiple cell lineages and expression of CD34 and CD44. Ten grams of subcutaneous fat were collected from mature pigs, finely minced, and washed with PBS. The tissue was homogenized with 1% collagenase in PBS with 0.1% bovine serum albumin in a shaker incubator at 37°C for 2 h. The cell suspension was filtered and stained with Hoechst 33342 to determine the number of nucleated cells in the suspension. Adherent cells were cultured in DMEM supplemented with 10% fetal bovine serum and incubated at 39°C in 5% CO₂. At 90% confluence, cells were passaged by trypsinization and cultured for 21 days in lineage-specific cocktails previously demonstrated to induce differentiation. Stains specific for intracellular lipids, glycosaminoglycans, and calcium deposition were used to detect adipocyte, osteocyte, and chondrocyte differentiation, respectively. mRNA from undifferentiated and differentiated cells was isolated using a Dynabeads mRNA Kit (Dynal, Lake Success, NY, USA), and cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). RT-PCR was performed with primers for Poly A Polymerase (PAP; reference gene), CD34, and CD44. PCR reaction products were subjected to electrophoresis and analyzed by Quantity One software (Bio-Rad). An average of 2 300 000 nucleated cells/mL was isolated from the fat sample; 26% of the cells were adherent, and the cells completed a cell cycle approximately every 2.8 days. After culture in differentiation medium, 57% of cultures stained positive with Nile red (adipocytes), 87% stained with Safranin O (chondrocytes), and 50% stained with Alizarin Red (osteocytes). The number of pixels in each electrophoresis band was used to determine the relative levels of gene expression and is represented as a ratio of CD34 or CD44 to PAP. In undifferentiated cells, the band intensity ratio for CD34 : PAP was 1.54 and that for CD44 : PAP was 0.96. Ratios for differentiated cells were as follows (CD34 : PAP, CD44 : PAP): adipocytes (0.38, 1.1), chondrocytes (0.24, 1.0), osteocytes (0.63, 0.99), and fibroblasts (0.38, 1.14). The CD34 expression level was lower than that of CD44 among the differentiated cells. CD34 expression was higher for ADAS compared with differentiated cells; however, CD44 did not follow this pattern. CD34 but not CD44 expression appears to be a good marker for differentiation in these cells.