Horse bone marrow mesenchymal stem cells express embryo stem cell markers and show the ability for tenogenic differentiation by in vitro exposure to BMP-12 - PubMed (original) (raw)

Horse bone marrow mesenchymal stem cells express embryo stem cell markers and show the ability for tenogenic differentiation by in vitro exposure to BMP-12

Stefania Violini et al. BMC Cell Biol. 2009.

Abstract

Background: Mesenchymal stem cells (MSCs) have been recently investigated for their potential use in regenerative medicine. MSCs, in particular, have great potential, as in various reports they have shown pluripotency for differentiating into many different cell types. However, the ability of MSCs to differentiate into tendon cells in vitro has not been fully investigated.

Results: In this study, we show that equine bone marrow mesenchymal stem cells (BM-MSCs), defined by their expression of markers such as Oct4, Sox-2 and Nanog, have the capability to differentiate in tenocytes. These differentiated cells express tendon-related markers including tenomodulin and decorin. Moreover we show that the same BM-MSCs can differentiate in osteocytes, as confirmed by alkaline phosphatase and von Kossa staining.

Conclusion: As MSCs represent an attractive tool for tendon tissue repair strategies, our data suggest that bone marrow should be considered the preferred MSC source for therapeutic approaches.

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Figures

Figure 1

Figure 1

BM-derived MSCs express stem cell marker protein Oct4. Cells (5th passage) were fixed and incubated with antibodies directed against Oct4. Immunoreactivity was detected with avidin, biotinylated horseradish peroxydase and 3,3'-Diaminobenzidine (DAB) (1B). 1A: negative control, incubated only with secondary antibody. Magnification 20×.

Figure 2

Figure 2

Gene expression analysis of BM-MSCs. The products from RT-PCR analysis of GAPDH (lane 1), Oct4 (lane 2), CD34 (lane 3), SOX-2 (lane 4) and Nanog (lane 5) mRNA expression at day 14 (passage 5) in BM-MSCs, showing the expression of mesenchymal stem cell-related markers and lack of expression of CD34.

Figure 3

Figure 3

QRT-PCR of BM-MSCs for the Nanog, Oct4, Sox2 and GAPDH genes. Plot of QRT-PCR of Nanog (violet), Oct4 (green), Sox2 (blue) and GAPDH (red) on cDNA from BM-MSC cells. Cycle number is shown on the X-axis and emission intensity of a fluorescent reporter (Rn) is shown on the Y-axis. Fixed threshold is indicated as horizontal line.

Figure 4

Figure 4

Gene expression analysis of BM-MSC derived tenocytes. Products from RT-PCR analysis of tenomodulin (lane 1), P19 lipocalin (lane 3), GAPDH (lane 3) and decorin (lane 4) mRNA expression following 14 days stimulation of BM-MSC cells with BMP-12, resulting in differentiation into tenocytes.

Figure 5

Figure 5

Morphology of equine BM-MSC-derived tenocytes cultured in monolayer at day 20th. The cells at 5th passage in growth medium added with BMP-12 under phase contrast microscopy exhibited heterogeneous morphology with most cells fibroblast-like (A) and other elongated cells (B). Magnification 10×.

Figure 6

Figure 6

BM-MSCs osteogenic induction: alkaline phosphatase (AP) and von Kossa staining. After osteogenic induction cells showed a completely different morphology compared to untreated MSCs. (A, C): untreated control cells showing no staining for AP and von Kossa respectively (Magnification 10×). (B, D): AP and von Kossa positive staining in bone marrow-derived osteogenic cells. Magnification 20×.

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