Gene Expression Profiles of Human Adipose Tissue-Derived Mesenchymal Stem Cells Are Modified by Cell Culture Density

<div><p>Previous studies conducted cell expansion <i>ex vivo</i> using low initial plating densities for optimal expansion and subsequent differentiation of mesenchymal stem cells (MSCs). However, MSC populations are heterogeneous and culture conditions can affect the characteristics of MSCs. In this study, differences in gene expression profiles of adipose tissue (AT)-derived MSCs were examined after harvesting cells cultured at different densities. AT-MSCs from three different donors were plated at a density of 200 or 5,000 cells/cm². After 7 days in culture, detailed gene expression profiles were investigated using a DNA chip microarray, and subsequently validated using a reverse transcription polymerase chain reaction (RT-PCR) analysis. Gene expression profiles were influenced primarily by the level of cell confluence at harvest. In MSCs harvested at ∼90% confluence, 177 genes were up-regulated and 102 genes down-regulated relative to cells harvested at ∼50% confluence (<i>P</i><0.05, FC>2). Proliferation-related genes were highly expressed in MSCs harvested at low density, while genes that were highly expressed in MSCs harvested at high density (∼90% confluent) were linked to immunity and defense, cell communication, signal transduction and cell motility. Several cytokine, chemokine and growth factor genes involved in immunosuppression, migration, and reconstitution of damaged tissues were up-regulated in MSCs harvested at high density compared with MSCs harvested at low density. These results imply that cell density at harvest is a critical factor for modulating the specific gene-expression patterns of heterogeneous MSCs.</p></div

Phase-contrast micrograph and cell density of AT-MSCs from three different donors in CC1 or CC2.

<p>(<b>A</b>) Morphological appearance of AT-MSC donors 7 days after plating at 200 cells/cm² (CC1) or 5,000 cells/cm² (CC2). All cells exhibited a spindle shaped or fibroblastic morphology. (<b>B</b>) The number of cell divisions and (<b>C</b>) total cell numbers at the time of harvest of MSCs cultured under different conditions. Data are the mean ± SD from three separate experiments.</p

Hierarchical cluster analysis of differentially expressed genes in AT-MSCs from three different donors in CC1 or CC2.

<p>The microarray data for 47,323 genes were filtered by applying two criteria for significance, <i>P</i><0.05 and fold change (FC)>2, between the two cell densities (CC1 and CC2) at harvest for each MSC donor. (<b>A</b>) The selected data represented by hierarchical clustering of the normalized Ct of 279 genes on MSCs using individual samples (177 with increased expression, 102 with decreased expression). Each row represents a single gene, while each column represents the gene expression levels for a cell culture. The color coded gene expression levels range from red for the highest level of expression to green for the lowest. (<b>B</b>) Hierarchical cluster analysis of 17 differentially expressed cytokine, chemokine and growth factor genes. (<b>C</b>) Hierarchical cluster analysis of 33 differentially expressed proliferation-associated genes. CC1, cultures plated with an initial cell density of 200 cells/cm² and a culture duration of 7 days; CC2, cultures plated with an initial cell density of 5,000 cells/cm² and a culture duration of 7 days.</p

RT-PCR analysis of differentially expressed cytokine, chemokine and proliferation-associated genes in AT-MSC from different donors and different cell densities.

<p>The expression profile of selected genes from the microarray data was validated by semi-quantitative RT-PCR using independent samples harvested 7days after plating at different cell densities as distinct from that for microarray analysis. Quantitative gene expression data of each candidate gene indicates mRNA expression relative to GAPDH mRNA. Band intensity was normalized against that of GAPDH mRNA. Semi-quantitative RT-PCR analysis was independently performed using different MSC samples but the samples for microarray analysis. CC1, cultures plated with an initial cell density of 200 cells/cm² and a culture duration of 7 days; CC2, cultures plated with an initial cell density of 5,000 cells/cm² and a culture duration of 7 days.</p

Differentially expressed cytokine genes in AT-MSCs from three different donors, cultured to low or high density, as determined by microarray analysis.

<p>Viable second-passage AT-MSCs plated at 200 cells/cm² (CC1 MSCs) or 5,000 cells/cm² (CC2 MSCs) were incubated for 7 days by which time they reached ∼50% or ∼90% confluence, respectively. After harvesting, total mRNA was isolated from pooled samples of MSCs from three donors and used in the microarray analysis. Microarray data were filtered by applying two criteria for significance, P<0.05 and FC>2 between culture conditions.</p

Differentially expressed cell proliferation-associated genes in AT-MSCs from three different donors, cultured to low or high density, as determined by microarray analysis.

<p>Viable second-passage AT-MSCs plated at 200 cells/cm² (CC1 MSCs) or 5,000 cells/cm² (CC2 MSCs) were incubated for 7 days, by which time they reached ∼50% or ∼90% confluence, respectively. After harvesting, mRNA from three donor pooled samples of AT-MSCs was used in the microarray analysis. Microarray data were filtered by applying two criteria for significance, P<0.05 and FC>2 between culture conditions.</p

Characterization of AT-MSCs from three different donors.

<p>(<b>A</b>) The immunophenotype of AT-MSCs from three donors was analyzed by flow cytometry. The expression of surface antigens was plotted against appropriate IgG isotype controls (black histogram). MSCs used for the analyses were positive for CD73, CD90 and CD105, and negative for CD14, CD34 and CD45 (clear histogram). The histograms presented are representative of 3 independent experiments. (<b>B</b>) Differentiation of AT-MSCs from three donors. Cells were incubated for 14–21 days in the presence of specific differentiation agents for osteoblasts, chondrocytes, and adipocytes. Alkaline phosphatase staining shows mineralization of the extracellular matrix. Toluidine Blue staining shows the deposition of proteoglycans and lacunae. Differentiation into the adipocyte lineage was demonstrated by staining with Oil Red O. (Magnification: ×100).</p

AT-MSC donor demographics: gender, age, weight, height, and tissue-harvesting site (THS).

<p>AT-MSC donor demographics: gender, age, weight, height, and tissue-harvesting site (THS).</p