ABSTRACT: Phenotypic heterogeneity has been observed among mesenchymal stem/stromal cell (MSC) populations, but specific genes associated with this variability have not been defined. To study this question, we analyzed two distinct MSC populations isolated from the same umbilical cord blood (UCB) sample. These populations (UCB1 and UCB2) are from a single donor, minimizing differences contributed by genetic background. We characterized these UCB-MSCs for cell morphology, growth kinetics, immunophenotype and differentiation potential. UCB1 displayed rapid growth kinetics, higher population doublings, and increased adipogenic lineage differentiation compared to UCB2. To identify the MSC-specific and developmental genes associated with these phenotypic differences, we performed expression analysis using Affymetrix HG-U133 microarrays and compared them to bone marrow (BM) MSCs. First, hepatocyte growth factor (HGF) and stromal derived factor 1 (SDF1/CXCL12) were up -regulated in UCB1 cells, potentially contributing to the higher growth kinetics observed in this circulating cell population. Second, we observed that peroxisome proliferation activated receptor gamma (PPARG), a marker for adipogenic differentiation, was significantly increased in undifferentiated UCB1 cells. Moreover, significant expression of gene markers of blastocyst and gatrulation embryonic stages were detected in UCB1 and UCB2 cells, as were selected markers of early hematopoiesis, chondrogenesis, and cardiac differentiation. Comparison of UCB1, UCB2, and BM by microarray analysis clearly demonstrated clusters of developmental genes that displayed significant differences among these cells. Quantitative PCR analysis of selected genes validated the microarray results. Comparison of different UCB-derived adherent cells from a single donor has identified gene profiles potentially useful for therapeutic evaluation of MSC populations. Experiment Overall Design: In order, to identify developmental genes whose expression differences distinguished UCB1 and UCB2 cell MSCs, we carried out microarray analysis using Affymetrix HG-U133 microarrays. For comparison, adult BM-derived MSCs (Cambrex, Lot #4F0312) were analyzed using the same cell culture conditions and confluence as UCB-MSCs. RNA was prepared from three biological replicates for UCB1, UCB2, and BM MSCs, for a total of nine samples analyzed on the HG-U133A and HG-U133B arrays.
