Bone Morphogenetic Protein-12 Induces Tenogenic Differentiation of Mesenchymal Stem Cells Derived from Equine Amniotic Fluid

Abstract

Tendon injuries are common in race horses, and mesenchymal stem cells (MSCs) isolated from adult and foetal tissue have been used for tendon regeneration. In the present study, we evaluated equine amniotic fluid (AF) as a source of MSCs and standardised methodology and markers for their in vitro tenogenic differentiation. Plastic-adherent colonies were isolated from 12 of 20 AF samples by day 6 after seeding and 70-80% cell confluency was reached by day 17. These cells expressed mesenchymal surface markers [cluster of differentiation (CD)73, CD90 and CD105] by reverse transcription (RT)-polymerase chain reaction (PCR) and immunocytochemistry, but did not express haematopoietic markers (CD34, CD45 and CD14). In flow cytometry, the expression of CD29, CD44, CD73 and CD90 was observed in 68.83 ± 1.27, 93.66 ± 1.80, 96.96 ± 0.44 and 93.7 ± 1.89% of AF-MSCs, respectively. Osteogenic, chondrogenic and adipogenic differentiation of MSCs was confirmed by von Kossa and Alizarin red S, Alcian blue and oil red O staining, respectively. Upon supplementation of MSC growth media with 50 ng/ml bone morphogenetic protein (BMP)-12, AF-MSCs differentiated to tenocytes within 14 days. The differentiated cells were more slender, elongated and spindle shaped with thinner and longer cytoplasmic processes and showed expression of tenomodulin and decorin by RT-PCR and immunocytochemistry. In flow cytometry, 96.7 ± 1.90 and 80.9 ± 6.4% of differentiated cells expressed tenomodulin and decorin in comparison to 1.6 and 3.1% in undifferentiated control cells, respectively. Our results suggest that AF is an easily accessible and effective source of MSCs. On BMP-12 supplementation, AF-MSCs can be differentiated to tenocytes, which could be exploited for regeneration of ruptured or damaged tendon in race horses.

Multidifferentiation Potential of Human Mesenchymal Stem Cells from Adipose Tissue and Hamstring Tendons for Musculoskeletal Cell-Based Therapy

“…As little is known about the maturation of tendons and the related tendon cell niche factors, little information is available for developing an efficient tenogenic differentiation medium. In previous in vitro studies on embryonic stem cells, ASCs and BMSCs, the tenogenic induction has been attempted using a variety of growth factors, including bone morphogenic proteins (BMP-2 and -5, as well as BMP-12, -13 and -14, also known as GDF-7, -6 and -5, respectively), IGF-1, TGF-β1 and TGF-β3 [8][9][10][34][35][36][37]. The influence of BMP-12 on tenogenic differentiation was evaluated in several types of MSCs [37,38], but only a few studies were conducted on ASCs [8,10] and, to our knowledge, none were conducted on human cells.…”

Stem Cells from Foetal Adnexa and Fluid in Domestic Animals: An Update on Their Features and Clinical Application

“…Positivity was found only for AD2 clone, while clone 5F/B9 did not cross‐react (Gulati et al. ). Anyway, for AD2 clone, cross‐reactivity was not found in equine (Iacono et al.…”

“…However, Mohanty's research group stated that equine AFMSCs reacted with AD2 clone, but not with 5F/B9 clone (Gulati et al. ), showing opposing results for the same species. Anyway, differences in the culture medium, such as the content of FBS, the timeframe of plastic adherent culture and the cell harvesting technique could influence the cell characteristics (Paebst et al.…”

Can a Biodegradable Implanted Bilayered Drug Delivery System Loaded with BMP-2/BMP-12 Take an Effective Role in the Biological Repair Process of Bone–Tendon Injuries? A Preliminary Report

“…One of these proteins is bone morphogenic protein- (BMP-) 2, a protein growth factor that triggers signal transmission and induces osteoblast formation. Another protein growth factor, BMP-12, activates tenoblasts [7, 8]. …”