ABSTRACT
PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.
Subject(s)
Animals , Rabbits , Cell Separation/methods , Mesenchymal Stem Cells/cytology , Olfactory Mucosa/cytology , /physiology , /physiology , Thy-1 Antigens/physiology , Cells, Cultured , Colony-Forming Units Assay , Cryopreservation , Cell Differentiation/physiology , Cell Plasticity/physiology , Cell Proliferation/physiology , Ethmoid Bone/cytology , Flow Cytometry , Green Fluorescent Proteins/metabolism , Olfactory Mucosa/growth & developmentABSTRACT
PURPOSE: To evaluate different protocols to isolate stem cells from ovine umbilical cord blood and adipose tissue. METHODS: There were used 5 samples of umbilical blood and 5 samples of perirenal adipose tissue from 10 female sheep. All the samples were obtained through surgery, to harvest aseptic samples. There were used 3 protocols for obtainment and culture of umbilical cord blood stem cells and 4 protocols for ovine adipose tissue stem cells. RESULTS: It was possible to observe only one successful protocol for the obtainment of umbilical cord blood stem cells. When analyzing the techniques used to obtain adipose tissue stem cells, only one of the methods was effective as well. Through colony forming unit assay, there were obtained 58 colonies of cells after seven days in culture. Flow citometry tests revealed the cells were positive to CD44 and exhibited negative reaction to CD38, CD45, CD41/61. These cells showed a growth curve with very well defined phases LOG, LAG and PLATEAU. This phases are typically seem in mesenchymal stem cells growth curves. CONCLUSIONS: The isolation and culture of mesenchymal stem cells from ovine umbilical cord blood are complex and request more detailed assays. Stem cells from fat tissue sheep showed mesenchymal characteristics, according to their cell growth curve, ability to origin colonies of fibroblastoid cells and positive reactivity with the antibody CD44 by flow citometry.
OBJETIVO: Testar diferentes protocolos para o isolamento de células tronco a partir de sangue de cordão umbilical e tecido adiposo de ovinos. MÉTODOS: Foram utilizadas cinco amostras de sangue de cordão umbilical e cinco amostras de tecido adiposo perirrenal de 10 fêmeas de ovelha. A coleta das amostras foi realizada através de procedimento cirúrgico para coleta do material de forma mais asséptica possível. Foram realizados três protocolos de isolamento e cultivo das células-tronco do cordão umbilical e quatro protocolos para o isolamento e cultivo das células-tronco de gordura de ovinos RESULTADOS: Somente um dos protocolos utilizados para o isolamento das células-tronco de cordão umbilical foi efetivo. Dos quatro protocolos utilizados para isolamento das células-tronco de gordura, da mesma forma, apenas um obteve sucesso. Foi realizado o ensaio de unidades formadoras de colônias destas células, sendo contadas 58 colônias ao final de sete dias. Na citometria de fluxo essas células mostraram-se positivas para CD44 e negativas para CD38, CD45, CD41/61. Estas células apresentaram curva de crescimento com fases de LOG, LAG e PLATEAU bem definidas, características das curvas de crescimento das células-tronco de origem mesenquimal. CONCLUSÕES: O isolamento e cultivo das células-tronco mesenquimais do cordão umbilical de ovinos é de difícil realização, exigindo maiores ensaios e estudos profundos. Células tronco do tecido adiposo de ovelhas demonstraram características mesenquimais, de acordo com a curva de crescimento, habilidade de formação de colônias, células com morfologia fibroblastóide e reação positiva ao anticorpo CD44.