Phenotypical and functional characteristics of mesenchymal stem cells derived from equine umbilical cord blood

Abstract

Mesenchymal stem cells (MSCs) offer promise as therapeutic aid in the repair of tendon and ligament injuries in race horses. Fetal adnexa is considered as an ideal source of MSCs due to many advantages, including non-invasive nature of isolation procedures and availability of large tissue mass for harvesting the cells. However, MSCs isolated from equine fetal adnexa have not been fully characterized due to lack of species-specific markers. Therefore, this study was carried out to isolate MSCs from equine umbilical cord blood (UCB) and characterize them using cross-reactive markers. The plastic-adherent cells could be isolated from 13 out of 20 (65 %) UCB samples. The UCB derived cells proliferated till passage 20 with average cell doubling time of 46.40 ± 2.86 h. These cells expressed mesenchymal surface markers but did not express haematopoietic/ leucocytic markers by RT-PCR and immunocytochemistry. The phenotypic expression of CD29, CD44, CD73 and CD90 was shown by 96.36 ± 1.28, 93.40 ± 0.70, 73.23 ± 1.29 and 46.75 ± 3.95 % cells, respectively in flow cytometry, whereas, reactivity against the haematopoietic antigens CD34 and CD45 was observed only in 2.4 ± 0.20 and 0.1 ± 0.0 % of cells, respectively. Osteogenic and chondrogenic differentiation could be achieved using established methods, whereas the optimum adipogenic differentiation was achieved after supplementing media with 15 % rabbit serum and 20 ng/ml of recombinant human insulin. In this study, we optimized methodology for isolation, cultural characterization, differentiation and immunophenotyping of MSCs from equine UCB. Protocols and markers used in this study can be employed for unequivocal characterization of equine MSCs.

Differences in the intrinsic chondrogenic potential of equine umbilical cord matrix and cord blood mesenchymal stromal/stem cells for cartilage regeneration

“…Various neonatal tissues have been described to isolate MSCs in horse, including umbilical cord blood (UCB-MSCs) 8 – 11 , umbilical cord matrix (UCM-MSCs), commonly referred as “Wharton’s jelly” 12 , amniotic tissue 13 , or placenta 14 . Such cells meet the standards recommended by the International Society of Cellular Therapy (ISCT) for MSCs 15 , including plastic adherence, multilineage differentiation potential, and a particular immunophenotype.…”

Resorbable electrospun polydioxanone fibres modify the behaviour of cells from both healthy and diseased human tendons

“…Further work may demonstrate whether changes in electrospun scaffold design can influence diseased tendon cells to express healthy tendon matrix proteins such as type I and type III collagen. We found no significant difference between the groups in the expression of type VI collagen, scleraxis or tenomodulin (data not shown) after 7 d of in vitro culture (Shukunami et al, 2006). This study was limited to early in vitro outcomes and more comprehensive study is required to investigate the effect of biophysical stimulation on extracellular matrix production.…”

“…Flow cytometry offers one way in which fibroblasts can be sub-classified. However, previous studies have so far revealed only a handful of cell surface proteins that are preferentially expressed on fibroblasts as opposed to other derivatives of mesenchymal stem cells (Cappellesso-Fleury et al, 2010;Halfon et al, 2011;Mohanty et al, 2014). These include CD106, CD26, CD10 and CD9.…”

<b>Estudo <i>in vitro</i> do potencial de diferenciação condrogênico e osteogênico de células mesenquimais obtidas de líquido e membrana sinovial de equinos</b>

“…A medula óssea foi a primeira fonte estudada na Medicina para isolamento de CTMs, posteriormente, outras tecidos demonstraram a presença de tais células, como no tecido Em equinos as CTMs foram obtidas a partir da medula óssea, tecido adiposo (DE MATTOS CARVALHO et al, 2009), cordão umbilical (TOUPADAKIS et al, 2010RANERA et al, 2011;CARRADE HOLT et al, 2012;BARBERINI et al, 2014), sangue do cordão umbilical (KOCH et al, 2007;MOHANTY et al, 2014) Em cultura a característica morfológica de CTMs é apresentada com crescimento em monocamada (PITTENGER et al, 1999), aderência ao plástico, aparência fibroblastóide ou fusiforme e células alongadas (KOCH et al, 2007;LOVATI et al, 2011), com núcleo eucromático, oval grande e central, nucléolo proeminente, associado a um abundante citoplasma, observado à microscopia. Maiores detalhes são detectados com microscopia eletrônica, exibindo organelas bem desenvolvidas incluindo, mitocôndrias, complexo de Golgi, lisossomos, vacúolos lipídicos cheios ou vazios, grande quantidade de ribossomos livres no citoplasma e retículo endoplasmático rugoso dilatado, além de filamentos de actina organizados em suas extremidades (TAGAMI et al, 2003).…”