Differentiation of synovial CD‐105⁺ human mesenchymal stem cells into chondrocyte‐like cells through spheroid formation

Abstract

Mesenchymal stem cells (MSCs) have the capacity to differentiate into several cell lineages, some of which can generate bone, cartilage, or adipose tissue. The presence of MSCs in the synovial membrane was recently reported. Data from comparative studies of MSCs derived from various mesenchymal tissues suggest that MSCs from synovial membranes have a superior chondrogenesis capacity. Previous chondrogenic differentiation studies have used the total population of MSCs, including cells with several MSC markers, such as CD44, CD90, CD105, or CD73. However the chondrogenic capacity of an individual population of MSCs has not been examined. Our aim was to study the chondrogenic capacity of the cellular MSC subset, CD105(+), derived from synovial membrane tissues of patients with osteoarthritis (OA) and normal donors. The tissues were digested with a cocktail of collagenase/dispase and the isolated MSCs were seeded into plates. The subpopulation of CD105(+)-MSCs was separated using a magnetic separator. The MSCs were then differentiated towards chondrocyte-like cells using a specific medium to promote spheroid formation. Spheroids were collected after 14, 28, and 46 days in chondrogenic medium and stained with hematoxylin, eosin, Safranin O or Alcian blue to evaluate the extracellular matrix. Immunohistochemistry was performed to study collagen types I (COLI) and II (COLII) and aggrecan expression. Phenotypic characterization of the isolated CD105(+)-MSCs shows that these cells are also positive for CD90 and CD44, but negatives for CD34 and CD45. In addition, this cellular subset expressed Sox-9. Spheroids appeared after 7 days in culture in the presence of chondrogenic medium. Our studies show no differences between MSCs obtained from OA and normal synovial membranes during chondrogenesis. The morphological analysis of spheroids revealed characteristics typical of chondrocyte cells. The intensity of Safranin O, Alcian blue and aggrecan staining was positive and constant throughout the culture period. However, the intensity of COL2 staining was higher at 28 days (84.29 +/- 0.1 U) than at 46 days (61.28 +/- 01 U), while COL1 staining was not detected in any samples analyzed. These results were confirmed by reverse transcriptase-polymerase chain reaction assays. We conclude that the cellular subset of CD105(+)-MSCs has chondrogenic capacity. The study also show the similar chondrogenic capacity of CD105(+)-MSCs cultured from normal and OA synovial membranes.

Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties

“…Classically hMSCs were isolated and expanded as adherent monolayer cultures, but it was soon recognized that centrifugation of the cells to form micropellets or large aggregates greatly enhanced their chondrogenic differentiation that slowly occurred over several weeks (18,27). However, several recent publications demonstrated that culture of MSCs in 3D or as spheroids for shorter periods of time improved their therapeutic potential by increased expression of genes such as CXCR4 to promote adhesion to endothelial cells or of IL-24 that has tumor suppressing properties (20,22,23).…”

“…Recently there has been a series of publications on aggregation of MSCs either as a procedure for enhancing chrondrogenic differentiation of the cells (17)(18)(19) or to increase their therapeutic potential (20)(21)(22)(23). Because aggregated hMSCs were detected in the pulmonary microemboli observed after i.v.…”

Isolation and characterization of primary bone marrow mesenchymal stromal cells

“…Isolation is traditionally based on the typical capacity of MSCs to adhere to plastic surfaces. [18][19][20] One disadvantage of isolating cells by adherence, however, is the nonspecificity of this approach despite the use of selected sera and media for culture initiation. This is due to the fact that MSCs represent only a very rare fraction of the starting cell population that contains other, more abundant cells types, several of which also adhere to standard cell culture surfaces.…”

Effects of endothelial cells on human mesenchymal stem cell activity in a three-dimensional in vitro model

“…Data gained from 3D in vitro methods may help to bridge the gap between traditional 2D culture studies and in vivo pre-clinical animal models (Rangarajan et al, 2004;Griffi th and Swartz, 2006;Yamada and Cukierman, 2007;Pampaloni et al, 2007;Hutmacher, 2010). Signifi cant alterations in cell behaviour have been identifi ed, when they are grown in 3D compared to 2D conditions; including changes in cell morphology (Cukierman et al, 2001), differentiation capacity (Arufe et al, 2009;Wang et al, 2009b), replicative ability (Grayson et al, 2006), cell signalling (Cukierman et al, 2002;Green and Yamada, 2007) as well as signifi cant increases in their therapeutic potential (Tapp et al, 2008;Wang et al, 2009a;Dittmer et al, 2009;Bartosh et al, 2010;Frith et al, 2010). For example, we have previously shown that MSCs grown in dynamic 3D culture conditions can promote osteogenic and adipogenic differentiation potential (Frith et al, 2010).…”