In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis

Abstract

One approach to resolving the complexities of chondrogenesis is to examine simplified systems in vitro. We analyzed cartilage differentiation by human adult stem cells from bone marrow stroma. Marrow stromal cells were cultured as micromass pellets for 21 days in serum-free medium containing transforming growth factor (TGF)-beta3, dexamethasone, and bone morphogenetic protein (BMP)-6. Assays for pulse-labeled [3H]DNA and for total DNA indicated that there was little proliferation and a progressive loss of cells in the pellets. There were continuous increases in mRNAs for cartilage matrix (proteoglycans and COL2, -9, -10, and -11), receptors [fibroblast growth factor 2 (FGFR2) and parathyroid hormone-related peptide receptor (PTHrP-R)], and transcription factors (SOX5, -6, and -9) as demonstrated by histochemical and microarray assays. Reverse transcription-PCR assays for 11 mRNAs confirmed the microarray data. SOX4, vascular endothelial growth factor (VEGF), and matrix metalloproteinase 14 (MMP14) increased at day 1 and decreased thereafter, suggesting roles early in chondrogenesis. Also, forkhead, CD10, and MMP13 increased up to day 7 and decreased thereafter, suggesting roles in an intermediate stage of chondrogenesis. In addition, two collagens (COL3A1 and COL16A1), a signaling molecule (WNT11), a homeobox homolog (BAPX1), a receptor (IL-1R1), an IGFs modulator (IGFBP5), and a mettaloproteinase (MMP16) increased progressively up to about day 14, suggesting roles later in chondrogenesis. Our results indicate that the simplicity of the system makes it possible to define in detail the cellular and molecular events during chondrogenesis.

Figure 1

Time course for synthesis of cartilage by MSCs. (A) Macro picture of differentiating MSC pellet at days 7, 14, and 21. A 1-mm scaled ruler is shown. (B) Wet weight of pellets at days 1, 7, 14, and 21. Data are expressed as mean ± SD (n = 3). (C) Histology of paraffin sections stained with toluidine blue sodium borate. The purple color is indicative of proteoglycans, and the blue color is background. Proteoglycan content increased and cell density decreased throughout the course of differentiation.

Figure 2

Cell proliferation and death during chondrocyte differentiation. (A) DNA per pellet; prepared from 20 pellets at day 0, and from 40 pellets each at 7, 14, and 21 days. The amount of DNA decreased throughout the course of differentiation. (B) Specific activity of [³H]DNA per 100 ng DNA in cells prelabeled with [³H]thymidine. This result suggests MSCs proliferate between day 0 and day 7 but not thereafter. (C) Representative model of MSCs proliferation and death during chondrocyte differentiation in pellet culture system.

Figure 3

Time sequence of gene expression assayed by RT-PCR. RT-PCR was used to further verify the data from the microarray (COL2A1, COL10A1, COMP, SOX4, SOX5, SOX9, PTHrP-R, BMP-2) and to examine the expression of genes that were not on microarray chip (link protein, SOX6, PTHrP, and BMP-6).