Isolamento e identificação da população de células-tronco mesenquimais derivadas do córdão umbilical e medula óssea de bubalinos post-mortem

Abstract

Mesenchymal stem cells (MSCs) are undifferentiated cells with a high capacity of proliferation and transformation into several specialized cells. They are found in tissues such as bone marrow, umbilical cord, dental pulp, periodontal ligament and adipose tissue. Although MSCs isolation has been already well established, in buffaloes it is still a challenge. In buffaloes, stem cells represent an ideal tool for embryogenesis study in vitro, favoring the development of effective methods for somatic cells cloning, transgenesis and gene regulation. The aim of this study was to evaluate the gene expression of pluripotency and stem cells markers in cells isolated from the bone marrow of buffaloes (Bubalus bubalis) post mortem. After isolation and cultivation, samples were frozen until the assays. Morphological analyses were made through optical and electronic microscopy. The growth curve and immunophenotyping were made by flow cytometry. After 24 hours was observed cell adhesion into culture plates. The cells showed high expansion capacity in vitro, presenting an average of 85% confluence on the fifth day of culture. Flow cytometry showed a small cell population with little granularity. The populations of bone marrow cells were positive for the marker of mesenchymal cells Vimentina (72.7%). In the analysis of the genetic expression by qRT-PCR in cells of the marrow there was no expression of any of the genes analyzed, whereas in umbilical cord cells mRNA transcripts of the genes CD29, CD44, SOX-2 and NANOG were expressed. We can conclude that as mesenchymal stem cells can be isolated post-mortem of adipose tissue and bone marrow of buffaloes (Bubalus bubalis), and that as characteristics presented by umbilical cord cells, confirm that these are mesenchymal stem cells.