Basal and inducible Osterix expression reflect equine mesenchymal progenitor cell osteogenic capacity

Abstract

Introduction: Mesenchymal stem cells are characterized by their capacities for extensive proliferation through multiple passages and, classically, tri-lineage differentiation along osteogenic, chondrogenic and adipogenic lineages. This study was carried out to compare osteogenesis in equine bone marrow-, synovium- and adipose-derived cells, and to determine whether osteogenic capacity is reflected in the basal expression of the critical osteogenic transcription factors Runx2 and Osterix.

Methods: Bone marrow, synovium and adipose tissue was collected from six healthy 2-year-old horses. Cells were isolated from these sources and expanded through two passages. Basal expression of Runx2 and Osterix was assessed in undifferentiated third passage cells, along with their response to osteogenic culture conditions.

Results: Bone marrow-derived cells had significantly higher basal expression of Osterix, but not Runx2. In osteogenic medium, bone-marrow cells rapidly developed dense, multicellular aggregates that stained strongly for mineral and alkaline phosphatase activity. Synovial and adipose cell cultures showed far less matrix mineralization. Bone marrow cells significantly up-regulated alkaline phosphatase mRNA expression and enzymatic activity at 7 and 14 days. Alkaline phosphatase expression and activity were increased in adipose cultures after 14 days, although these values were less than in bone marrow cultures. There was no change in alkaline phosphatase in synovial cultures. In osteogenic medium, bone marrow cultures increased both Runx2 and Osterix mRNA expression significantly at 7 and 14 days. Expression of both transcription factors did not change in synovial or adipose cultures.

Discussion: These results demonstrate that basal Osterix expression differs significantly in progenitor cells derived from different tissue sources and reflects the osteogenic potential of the cell populations.

Keywords: Osterix; Runx2; bone formation; mesenchymal stem cells; osteogenesis.

Figure 1

Basal expression of Runx2 (A) and Osterix (B) mRNAs in bone marrow- (BM), synovial- (SYN) and adipose- (ADI) third passage cells in control medium. Mean expression levels in BM samples were set at “1” in each analysis. Asterisks indicate mean + SE values significantly different from BM levels of expression (ANOVA n = 6; P < 0.05).

Figure 2

Representative microscopic images of bone marrow- [BM: (A, D)], synovial- [SYN: (B, E)] and adipose- [ADI: (C, F)] third passage cells in control (A–C) and osteogenic (D–F) medium after seven days.

Figure 3

Representative microscopic images of bone marrow- [BM: (A, D)], synovial- [SYN: (B, E)] and adipose- [ADI: (C, F)] third passage cells in control (A–C) and osteogenic (D–F) medium after 14 days, stained with Alizarin Red solution to demonstrate the presence of ionized calcium deposition in basal (A–C) and osteogenic (D–F) cultures.

Figure 4

Representative microscopic images of bone marrow- [BM: (A, D)], synovial- [SYN: (B, E)] and adipose- [ADI: (C, F)] third passage cells in control (A–C) and osteogenic (D–F) medium after 14 days, stained with von Kossa stain to demonstrate the presence of ionized phosphate deposition in basal (A–C) and osteogenic (D–F) cultures.

Figure 5

Representative microscopic images of bone marrow- [BM: (A, D)], synovial- [SYN: (B, E)] and adipose- [ADI: (C, F)] third passage cells in control (A–C) and osteogenic (D–F) medium after 14 days, stained with ALP solution to demonstrate the presence of cell-associated ALP activity.

Figure 6

ALP mRNA expression in bone marrow-, synovium- and adipose tissue-derived cells in control (white columns) or osteogenic (black columns) medium after 7 or 14 days. Within each cell type, asterisks indicate mean + SE values significantly different from control levels of expression. Columns designated with different upper-case letters are significantly different in osteogenic cultures (two-way ANOVA n = 6; P < 0.05).

Figure 7

ALP enzymatic activities in bone marrow-, synovium- and adipose tissue-derived cells in control (white columns) or osteogenic (black columns) medium after 7 or 14 days. Within each cell type, asterisks indicate mean + SE values significantly different from control levels of expression Columns designated with different lower-case letters are significantly different in control cultures. Columns designated with different upper-case letters are significantly different in osteogenic cultures (two-way ANOVA n = 6; P < 0.05).

Figure 8

Runx2 mRNA expression in bone marrow-, synovium- and adipose tissue- derived cells in control (white columns) or osteogenic (black columns) medium after 7 or 14 days. Within each cell type, asterisks indicate mean + SE values significantly different from control levels of expression. Columns designated with different upper-case letters are significantly different in osteogenic cultures (two-way ANOVA n = 6; P < 0.05).

Figure 9

OSX mRNA expression in bone marrow-, synovium- and adipose tissue- derived cells in control (white columns) or osteogenic (black columns) medium after 7 or 14 days. Within each cell type, asterisks indicate mean + SE values significantly different from control levels of expression columns designated with different lower-case letters are significantly different in control cultures. Columns designated with different upper-case letters are significantly different in osteogenic cultures (two-way ANOVA n = 6; P < 0.05).