Abstract
Previous studies conducted cell expansion ex vivo 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 (P<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.
Highlights
Human mesenchymal stem cells (MSCs) can be isolated from a wide variety of tissues [1] and are promising candidates for cellbased transplantation and regenerative medicine therapies [2,3,4]
Human MSCs were isolated from adult human adipose tissue (AT) that were taken from the thigh during cosmetic surgery
FACS analysis showed that adipose tissue-derived MSCs (AT-MSCs) derived from three different donors were positive for the typical MSC antigens (CD73, CD90, and CD105) but negative for typical hematopoietic antigens (CD14, CD34, and CD45) (Fig. 1A)
Summary
Human mesenchymal stem cells (MSCs) can be isolated from a wide variety of tissues [1] and are promising candidates for cellbased transplantation and regenerative medicine therapies [2,3,4]. MSCs express a wide variety of cytokines, chemokines and growth factors that are important for cell migration, homing and immunomodulation, following reconstitution of damaged tissues [11,14,16,17,18] Based on their functional effects, the difference in the secretion of these molecules by MSCs could possibly have a critical effect on the results of specific application for cell therapy. In this regards, it is important to identify the best subpopulation of cells and determine how the cells are expanded and characterized ex vivo and when they should be used clinically. Ex vivo expansion of MSCs is one of the alternatives for overcoming the heterogeneity and recent reports suggest that low initial plating densities could be beneficial for optimal ex vivo expansion and subsequent differentiation of MSCs [19,20,21]
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