Isolation, culture and characterization of multipotent mesenchymal stem cells from goat umbilical cord blood

Abstract

RESUMO.-[Isolamento, cultura e caracterização de cé-lulas tronco mesenquimais multipotentes provenientes do sangue do cordão umbilical caprino.] As células tronco mesenquimais (MSC) residem em pequenas quantidades em muitos tecidos e órgãos adultos, desempenhando um papel ativo na homeostase destes locais. O isolamento de MSC já foi demonstrado em amostras de medula óssea, tecido adiposo e fluido amniótico de cabras. O sangue de cordão umbilical é considerado uma fonte importante desse tipo de células. No entanto, até o presente momento, não foi demonstrado o isolamento de MSC provenientes do sangue de cordão umbilical de cabras. Dessa forma, o objetivo do presente estudo foi isolar, cultivar e caracterizar células tronco mesenquimais provenientes do sangue do cordão umbilical caprino. As MSC foram isoladas utilizando o gradiente de densidade Ficoll-Paque e cultivadas em DMEM suplementado com 10% ou 20% de FBS. A caracterização desse tipo celular foi realizada através de aná-lise por citometria de fluxo e diferenciação em linhagens celulares mesodermais. A analise no citômetro de fluxo demonstrou a presença de duas populações distintas, um grupo com células maiores e outro com células menores; observando expressão positiva de CD90, CD44 e CD105, e negativa para CD34 nas células maiores; enquanto que as menores foram positivas para CD90 e CD105, mas negativas para CD44 e CD34. Mesenchymal stem cells (MSC) reside in small numbers in many adult tissues and organs, and play an active role in the homeostasis of these sites. Goat derived multipotent MSC have been established from bone marrow, adipose tissues and amniotic fluid. Umbilical cord blood (UCB) is considered an important source of these cells. However, the MSC isolation from the goat UCB has not been demonstrated. Therefore, the aim of the present study was to isolate, culture and characterize goat umbilical cord blood derived mesenchymal stem cells. MSC were isolated from UCB by Ficoll-Paque density centrifugation and cultured in DMEM supplemented with 10% or 20% FBS. FACS analysis was performed and induction lineage differentiation was made to characterize these cells. They exhibited two different populations in flow cytometry, and revealed the positive expression of CD90, CD44 and CD105, but negative staining for CD34 in larger cells, and positive stained for CD90 and CD105, but negative for CD44 and CD34 in the smaller cells. MSC from goat UCB showed capability to differentiate into chondrocytes and osteoblasts when incubated with specific differentiation medium. Present study established that goat mesenchymal stem cells can be derived successfully from umbilical cord blood.

Small Ruminants and Its Use in Regenerative Medicine: Recent Works and Future Perspectives

“…Epigenetic changes can also affect their ability for self-renewal [ 178 ]. Cellular proliferation of gMSCs is improved through culture with higher concentrations of FBS [ 179 ]. As in the case of oMSCs, gMSCs are also characterized according to ISCT criteria.…”

“…Typical markers of MSCs are expressed by gMSCs in general, although with varying intensity. These variations may originate from the collected tissue, collection method, culture conditions, detaching agents and antibodies used for identification, creating the usual problems described in other veterinary species [ 174 , 179 ]. Regarding the differentiation capacity, gMSCs are able to follow the classic tridifferentiation, but under specific conditions they also differentiate in other lines such as neurogenic [ 180 ], myogenic [ 181 ], epithelial [ 182 ] and germ cell-like [ 183 ].…”

Current Status of Canine Umbilical Cord Blood-Derived Mesenchymal Stem Cells in Veterinary Medicine

“…The same authors isolated MSCs with a success rate of 57% from a volume of 65–250 mL of equine UCB [ 77 , 78 ]. Different authors have investigated the isolation of MSCs from UCB of goat and sheep ( Table 3 ) [ 76 , 85 ].…”

“…Successful isolation of MSCs (up to 100%) from equine UCB has been reported with the use of special culture media with a large volume of 42–250 mL [ 77 , 78 ]. Although the isolation of MSCs from sheep and goat was successful, the researchers did not reveal any specific parameters that resulted in an effective number of MSCs [ 75 , 76 , 85 ]. Based on our literature survey, we have demonstrated that a large volume of UCB with some other special determinants is a principal factor to achieve high MSC yield.…”

“…UCB-MSCs can be isolated from different species such as human [ 37 , 58 , 59 , 64 – 74 ], sheep [ 75 , 76 ], equine [ 77 – 79 ], canine [ 80 – 84 ], and goat [ 85 ]. Human UCB-MSCs (hUCB-MSCs) are collected noninvasively at the time of delivery, whereas sheep UCB is collected intrusively by the surgical intrauterine approach [ 75 , 76 ].…”