Canine Mesenchymal Stem Cells Derived from Bone Marrow: Isolation, Characterization, Multidifferentiation, and Neurotrophic Factor Expression in vitro원문보기
The purpose of this study is to characterize canine mesenchymal stem cells (MSCs) derived from bone marrow (BM) for use in research on the applications of stem cells in canine models of development, physiology, and disease. BM was harvested antemortem by aspiration from the greater tubercle of the h...
The purpose of this study is to characterize canine mesenchymal stem cells (MSCs) derived from bone marrow (BM) for use in research on the applications of stem cells in canine models of development, physiology, and disease. BM was harvested antemortem by aspiration from the greater tubercle of the humerus of 30 normal beagle dogs. Canine BM-derived MSCs were isolated according to methods developed for other species and were characterized based on their morphology, growth traits, cell-surface antigen profiles, differentiation repertoire, immunocytochemistry results, and neurotrophic factor expression in vitro. The canine MSCs exhibited a fibroblast-like morphology with a polygonal or spindle-shaped appearance and long processes; further, their cell-surface antigen profiles were similar to those of their counterparts in other species such as rodents and humans. The canine MSCs could differentiate into osteocytes and neurons on incubation with appropriate induction media. RT-PCR analysis revealed that these cells expressed NGF, bFGF, SDF-1, and VEGF. This study demonstrated that isolating canine MSCs from BM, stem-cell technology can be applied to a large variety of organ dysfunctions caused by degenerative diseases and injuries in dogs. Furthermore, our results indicated that canine MSCs constitutively secrete endogenous factors that enhance neurogenesis and angiogenesis. Therefore, these cells are potentially useful for treating dogs affected with various neurodegenerative diseases and spinal-cord injuries.
The purpose of this study is to characterize canine mesenchymal stem cells (MSCs) derived from bone marrow (BM) for use in research on the applications of stem cells in canine models of development, physiology, and disease. BM was harvested antemortem by aspiration from the greater tubercle of the humerus of 30 normal beagle dogs. Canine BM-derived MSCs were isolated according to methods developed for other species and were characterized based on their morphology, growth traits, cell-surface antigen profiles, differentiation repertoire, immunocytochemistry results, and neurotrophic factor expression in vitro. The canine MSCs exhibited a fibroblast-like morphology with a polygonal or spindle-shaped appearance and long processes; further, their cell-surface antigen profiles were similar to those of their counterparts in other species such as rodents and humans. The canine MSCs could differentiate into osteocytes and neurons on incubation with appropriate induction media. RT-PCR analysis revealed that these cells expressed NGF, bFGF, SDF-1, and VEGF. This study demonstrated that isolating canine MSCs from BM, stem-cell technology can be applied to a large variety of organ dysfunctions caused by degenerative diseases and injuries in dogs. Furthermore, our results indicated that canine MSCs constitutively secrete endogenous factors that enhance neurogenesis and angiogenesis. Therefore, these cells are potentially useful for treating dogs affected with various neurodegenerative diseases and spinal-cord injuries.
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문제 정의
The purpose of this study is to characterize canine BM-derived MSCs for use in research on the applications of stem cells in canine models of development, physiology, and disease.
제안 방법
In this study, the MSCs expressed 4 neurotrophic growth factors (NGF, bFGF, VEGF, and SDF-1) prior to neuronal differentiation. The present findings indicated that canine MSCs constitutively secrete neurotrophic factors that enhance neurogenesis and angiogenesis prior to differentiation.
Staining to detect ALP activity is a rapid and simple test that does not require any special equipment. In this study, the canine MSCs stained positively with ALP stain following incubation for 5 days in osteogenic induction medium containing BMP-2. This result indicated successful osteogenic differentiation.
Subsequently, the specimens were analyzed by flow cytometry (FACScalibur flow cytometer, Beckton Dickinson, USA) using the CellQuest software (BD bioscience, USA).
We evaluated neurotrophic factors, including GDNF, BDNF, VEGF, TGF-β, bFGF, SDF-1, and NGF, in the MSCs before (MSC group) and after neuronal differentiation.
데이터처리
0 KO, USA). Repeated measure Kruskal-Wallis test (nonparametric method) followed by Bonferroni corrected Mann- Whitney U test (nonparametric method) were used to compare the results of three samples.
성능/효과
Following neuronal differentiation, VEGF expression increased significantly in the first subgroup (lane 2), while SDF-1 expression increased significantly in the second subgroup (lane 3) (*p<0.05, lane 2 versus lane 1, **p<0.05, lane 3 versus lane 1, †p<0.05, lane 2 versus lane 3 and ‡p<0.05, lane 3 versus lane2).
In conclusion, we demonstrated that by isolating canine MSCs from BM, stem-cell technology can be applied to a large variety of organ dysfunctions caused by degenerative diseases and injuries in dogs. Furthermore, this study indicated that canine MSCs constitutively secrete endogenous factors that enhance neurogenesis and angiogenesis.
Another defining feature of MSCs is the ability to aquire multiple cellular phenotypes on exposure to appropriate stimuli. In this study, the ability of MSCs to differentiate into cells of mesodermal and ectodermal origin was confirmed by successful osteogenic and neuronal differentiation.
후속연구
However, we observed that the immunocytochemical characteristics and neurotrophic-factor expression differed between the canine MSCs that differentiated into neurons following induction by the 2 different methods. Future studies will focus on a more detailed analysis of neuronal differentiation in canine MSCs.
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