Mesenchymal stem cells have been recently investigated for their potential use in regenerative medicine. It has been suggested that there may be a stem cell population within both umbilical cord matrix and amniotic fluid. However, little knowledge exists about the characteristics of these progenitor cells within these sources in the equine species. This study wanted to investigate an alternative and non-invasive stem cell source for the equine tissue engineering and to learn more about the properties of these cells for future cell banking. Moreover, population of adult stem cells were recently identified in human and lab animal tendons, but no detailed investigations have been made in the equine species. The aim of the study was to compare in vitro the stemness features of horse progenitor cells derived from bone marrow (BM-MSCs), amniotic fluid (AF-MSCs), umbilical cord matrix (EUC-MSCs) and tendon derived progenitor cells (TSPCs). This work defines a protocol for extraction, isolation, expansion and characterization of mesenchymal stem cells from equine bone marrow, amniotic fluid, umbilical cord matrix (Wharton’s jelly) and tendon. Their localization into the tissues from which they were extracted, was reported. During the cell culture, cell expansion, CFU-F assay, doubling time, plasticity and immunophenotype were analyzed. Furthermore, a specific cell labeling was realized that could be used for non-invasive magnetic resonance cell tracking through endosomal incorporation of superparamagnetic iron oxide particles through the in vitro evaluation of the efficiency of this labeling method. In the next future, this technique should facilitate translation of the approach into clinical trials, in particular to track cells in vivo after transplantation and to follow their homing, viability and repair potential during time. The mesenchymal stem cells were grown on control medium, such as DMEM with the addition of basic FGF. Our results pointed out that these cells performed similarly in terms of CFU-F formation and growth kinetic. The immunocytochemical and RT-PCR analysis of MSCs isolated from all tissues showed the presence of antigens such as CD44, CD105, CD29, Oct-4, c-Myc, SSEA4 and HLA-ABC, whereas they were negative for CD34 and HLA-DR. These cells, differentiated into osteogenic, adipogenic, chondrogenic and tenogenic lineages confirming the nature of mesenchymal stem cells. These findings suggest that AF-MSCs appeared to be a readily obtainable and high proliferative cell line that may represent a good model system for stem cell biology. EUC-MSCs need to be further investigated regarding their particular behavior in vitro represented by spheroid formation. Equine TSPCs have high clonogenic properties and proliferating potential, they express stem cell markers and have the capability to be multipotent as well as BM-MSCs. These findings suggest that TSPCs may represent a good model for stem cell biology and could be useful for future tendon regenerative medicine investigations. These data could be useful for optimization of horse’s mesenchymal stem cell isolation and expansion protocol that could be used in the experimental clinic application for tissue regeneration because to their biological properties. The MSCs isolated from equine extra-embryonic tissues and tendons, showed characteristics of stem cells and therefore can be regarded as good candidates to be used in regenerative medicine, with special reference to orthopaedic diseases of horses. Furthermore, future investigations in vivo are useful to evaluate the efficacy of cell labeling with contrast agents for magnetic resonance with particular attention to the muscle-skeletal tissues.

COMPARISON OF EQUINE BONE MARROW-, UMBILICAL CORD MATRIX-, AMNIOTIC FLUID- AND TENDON-DERIVED PROGENITOR CELLS / A.b. Lovati ; Tutor: Fausto Cremonesi ; Coordinator: Angelo Belloli. - : . Universita' degli Studi di Milano, 2010 Dec 15. ((23. ciclo, Anno Accademico 2010.

COMPARISON OF EQUINE BONE MARROW-, UMBILICAL CORD MATRIX-, AMNIOTIC FLUID- AND TENDON-DERIVED PROGENITOR CELLS

A.B. Lovati
2010-12-15

Abstract

Mesenchymal stem cells have been recently investigated for their potential use in regenerative medicine. It has been suggested that there may be a stem cell population within both umbilical cord matrix and amniotic fluid. However, little knowledge exists about the characteristics of these progenitor cells within these sources in the equine species. This study wanted to investigate an alternative and non-invasive stem cell source for the equine tissue engineering and to learn more about the properties of these cells for future cell banking. Moreover, population of adult stem cells were recently identified in human and lab animal tendons, but no detailed investigations have been made in the equine species. The aim of the study was to compare in vitro the stemness features of horse progenitor cells derived from bone marrow (BM-MSCs), amniotic fluid (AF-MSCs), umbilical cord matrix (EUC-MSCs) and tendon derived progenitor cells (TSPCs). This work defines a protocol for extraction, isolation, expansion and characterization of mesenchymal stem cells from equine bone marrow, amniotic fluid, umbilical cord matrix (Wharton’s jelly) and tendon. Their localization into the tissues from which they were extracted, was reported. During the cell culture, cell expansion, CFU-F assay, doubling time, plasticity and immunophenotype were analyzed. Furthermore, a specific cell labeling was realized that could be used for non-invasive magnetic resonance cell tracking through endosomal incorporation of superparamagnetic iron oxide particles through the in vitro evaluation of the efficiency of this labeling method. In the next future, this technique should facilitate translation of the approach into clinical trials, in particular to track cells in vivo after transplantation and to follow their homing, viability and repair potential during time. The mesenchymal stem cells were grown on control medium, such as DMEM with the addition of basic FGF. Our results pointed out that these cells performed similarly in terms of CFU-F formation and growth kinetic. The immunocytochemical and RT-PCR analysis of MSCs isolated from all tissues showed the presence of antigens such as CD44, CD105, CD29, Oct-4, c-Myc, SSEA4 and HLA-ABC, whereas they were negative for CD34 and HLA-DR. These cells, differentiated into osteogenic, adipogenic, chondrogenic and tenogenic lineages confirming the nature of mesenchymal stem cells. These findings suggest that AF-MSCs appeared to be a readily obtainable and high proliferative cell line that may represent a good model system for stem cell biology. EUC-MSCs need to be further investigated regarding their particular behavior in vitro represented by spheroid formation. Equine TSPCs have high clonogenic properties and proliferating potential, they express stem cell markers and have the capability to be multipotent as well as BM-MSCs. These findings suggest that TSPCs may represent a good model for stem cell biology and could be useful for future tendon regenerative medicine investigations. These data could be useful for optimization of horse’s mesenchymal stem cell isolation and expansion protocol that could be used in the experimental clinic application for tissue regeneration because to their biological properties. The MSCs isolated from equine extra-embryonic tissues and tendons, showed characteristics of stem cells and therefore can be regarded as good candidates to be used in regenerative medicine, with special reference to orthopaedic diseases of horses. Furthermore, future investigations in vivo are useful to evaluate the efficacy of cell labeling with contrast agents for magnetic resonance with particular attention to the muscle-skeletal tissues.
CREMONESI, FAUSTO
BELLOLI, ANGELO
equine ; mesenchymal stem cells ; characterization ; cell labeling
Settore VET/10 - Clinica Ostetrica e Ginecologia Veterinaria
COMPARISON OF EQUINE BONE MARROW-, UMBILICAL CORD MATRIX-, AMNIOTIC FLUID- AND TENDON-DERIVED PROGENITOR CELLS / A.b. Lovati ; Tutor: Fausto Cremonesi ; Coordinator: Angelo Belloli. - : . Universita' degli Studi di Milano, 2010 Dec 15. ((23. ciclo, Anno Accademico 2010.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/150197
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