Abstract:Until now, the isolation and characterisation of bovine bone marrow-derived mesenchymal stem cells (bBM-MSCs) have not been established, which prompted us to optimise the differentiation protocol for bBM-MSCs. In this study, bBM-MSCs were freshly isolated from three six-month-old cattle and used for pluripotent or phenotypic characterisation by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometer. Initially, the cells were adherent to plastic surfaces and exhibited spindle-like morpholog… Show more
“…BM-MNCs were collected into another centrifuge tube, and then washed twice with PBS. Isolation of MNCs from BM was also performed using same volume of sodium carbonate buffer solution (0.1%) for lyses of erythrocytes (Lee et al 2015 ) . Particularly, 1 ml volume of BM was used on the same volume of buffer solution for separation of MNCs at low speed centrifugation for 20 min.…”
Section: Methodsmentioning
confidence: 99%
“…This media contained factors essential for adipogenesis as glucose (4.5 g/L), dexamethasone (1 μM), indomethacin (0.5 mM), 3-isobutyl-1-methylxanthine (0.5 mM) and insulin (10 μg/ml). The percent of factors was conducted as previously described (Lee et al 2015 ). All materials used were purchased from Sigma-Aldrich, USA.…”
Section: Methodsmentioning
confidence: 99%
“…These cells are responsible for tissues and organs regeneration and immunomodulation (Abo-Aziza and Zaki 2018 ). MSCs have been obtained from approximately all adult tissue types as whole umbilical cord (Zhang et al 2017 ), umbilical cord blood (Abo-Aziza et al 2018 ) , Wharton’s jelly (Hammam et al 2016 ), bone marrow (Abo-Aziza et al 2019a ; Abo-Aziza et al 2019b ) , placenta, amniotic fluid (Park et al 2012 ) and adipose tissue (Lee et al 2015 ). Each source has been reported to vary in its proliferative and multilineage potential (Brevini and Gandolfi 2013 ) .…”
Clinical applications of cell therapy and tissue regeneration under different conditions need a multiplicity of adult stem cell sources. Up to date, little is available on the comparative isolation, characterization, proliferation, rapid amplification, and osteogenic/adipogenic differentiation of rat mesenchymal stem cells (MSCs) isolated from living bulge cells of the hair follicle (HF) and bone marrow (BM) from the same animal. This work hopes to use HF-MSCs as an additional adult stem cell source for research and application. After reaching 80% confluence, the cell counting, viability %, and yields of HF-MSCs and BM-MSCs were nearly similar. The viability % was 91.41 ± 2.98 and 93.11 ± 3.06 while the cells yield of initial seeding was 33.15 ± 2.76 and 34.22 ± 3.99 and of second passage was 28.76 ± 1.01 and 29.56 ± 3.11 for HF-MSCs and BM-MSCs respectively. Clusters of differentiation (CDs) analysis revealed that HF-MSCs were positively expressed CD34, CD73 and CD200 and negatively expressed CD45. BM-MSCs were positively expressed CD73 and CD200 and negatively expressed of CD34 and CD45. The proliferation of HF-MSCs and BM-MSCs was determined by means of incorporation of Brd-U, population doubling time (PDT) assays and the quantity of formazan release. The percentage of Brd-U positive cells and PDT were relatively similar in both types of cells. The proliferation, as expressed by the quantity of formazan assay in confluent cells, revealed that the quantity of release by BM-MSCs was slightly higher than HF-MSCs. Adipogenic differentiated BM-MSCs showed moderate accumulation of oil red-O stained lipid droplets when compared to that of HF-MSCs which exhibited high stain. The total lipid concentration was significantly higher in adipogenic differentiated HF-MSCs than BM-MSCs (P < 0.05). It was found that activity of bone alkaline phosphatase and calcium concentration were significantly higher (P < 0.01 and P < 0.05 respectively) in osteogenic differentiated BM-MSCs than that of HF-MSCs. The present findings demonstrate that the HF-MSCs are very similar in most tested characteristics to BM-MSCs with the exception of differentiation. Additionally; no issues have been reported during the collection of HF-MSCs. Therefore, the HF may represent a suitable and accessible source for adult stem cells and can be considered an ideal cell source for adipogenesis research.
“…BM-MNCs were collected into another centrifuge tube, and then washed twice with PBS. Isolation of MNCs from BM was also performed using same volume of sodium carbonate buffer solution (0.1%) for lyses of erythrocytes (Lee et al 2015 ) . Particularly, 1 ml volume of BM was used on the same volume of buffer solution for separation of MNCs at low speed centrifugation for 20 min.…”
Section: Methodsmentioning
confidence: 99%
“…This media contained factors essential for adipogenesis as glucose (4.5 g/L), dexamethasone (1 μM), indomethacin (0.5 mM), 3-isobutyl-1-methylxanthine (0.5 mM) and insulin (10 μg/ml). The percent of factors was conducted as previously described (Lee et al 2015 ). All materials used were purchased from Sigma-Aldrich, USA.…”
Section: Methodsmentioning
confidence: 99%
“…These cells are responsible for tissues and organs regeneration and immunomodulation (Abo-Aziza and Zaki 2018 ). MSCs have been obtained from approximately all adult tissue types as whole umbilical cord (Zhang et al 2017 ), umbilical cord blood (Abo-Aziza et al 2018 ) , Wharton’s jelly (Hammam et al 2016 ), bone marrow (Abo-Aziza et al 2019a ; Abo-Aziza et al 2019b ) , placenta, amniotic fluid (Park et al 2012 ) and adipose tissue (Lee et al 2015 ). Each source has been reported to vary in its proliferative and multilineage potential (Brevini and Gandolfi 2013 ) .…”
Clinical applications of cell therapy and tissue regeneration under different conditions need a multiplicity of adult stem cell sources. Up to date, little is available on the comparative isolation, characterization, proliferation, rapid amplification, and osteogenic/adipogenic differentiation of rat mesenchymal stem cells (MSCs) isolated from living bulge cells of the hair follicle (HF) and bone marrow (BM) from the same animal. This work hopes to use HF-MSCs as an additional adult stem cell source for research and application. After reaching 80% confluence, the cell counting, viability %, and yields of HF-MSCs and BM-MSCs were nearly similar. The viability % was 91.41 ± 2.98 and 93.11 ± 3.06 while the cells yield of initial seeding was 33.15 ± 2.76 and 34.22 ± 3.99 and of second passage was 28.76 ± 1.01 and 29.56 ± 3.11 for HF-MSCs and BM-MSCs respectively. Clusters of differentiation (CDs) analysis revealed that HF-MSCs were positively expressed CD34, CD73 and CD200 and negatively expressed CD45. BM-MSCs were positively expressed CD73 and CD200 and negatively expressed of CD34 and CD45. The proliferation of HF-MSCs and BM-MSCs was determined by means of incorporation of Brd-U, population doubling time (PDT) assays and the quantity of formazan release. The percentage of Brd-U positive cells and PDT were relatively similar in both types of cells. The proliferation, as expressed by the quantity of formazan assay in confluent cells, revealed that the quantity of release by BM-MSCs was slightly higher than HF-MSCs. Adipogenic differentiated BM-MSCs showed moderate accumulation of oil red-O stained lipid droplets when compared to that of HF-MSCs which exhibited high stain. The total lipid concentration was significantly higher in adipogenic differentiated HF-MSCs than BM-MSCs (P < 0.05). It was found that activity of bone alkaline phosphatase and calcium concentration were significantly higher (P < 0.01 and P < 0.05 respectively) in osteogenic differentiated BM-MSCs than that of HF-MSCs. The present findings demonstrate that the HF-MSCs are very similar in most tested characteristics to BM-MSCs with the exception of differentiation. Additionally; no issues have been reported during the collection of HF-MSCs. Therefore, the HF may represent a suitable and accessible source for adult stem cells and can be considered an ideal cell source for adipogenesis research.
“…Bovine MSCs derived from different tissues have been shown to be positive for mesenchymal markers related to adhesion such as CD29, CD166, CD105, surface enzymes such as CD73, receptors such as CD44, and glycoproteins such as CD90 [1, 16, 22, 45–48]. Interestingly, bovine MSC also expressed pluripotency markers such as OCT4, SOX2, and NANOG [1, 16, 21, 45, 46], supporting the idea that MSCs have the potential to be pluripotent and differentiate into three germ layers, which was previously shown by the successful differentiation of bovine MSC into osteoblasts, lipoblasts, hepatocytes, islet cells, and neurocytes [22]. Fig.…”
“…In cattle, bone marrow has been the source for MSC in several studies [16, 46, 50–52]. In this procedure, marrow cells are aspirated from calves and isolated for further analysis.…”
Section: Sources Of Bovine Mesenchymal Cellsmentioning
Mesenchymal stem cells (MSCs) have received a great deal of attention over the past 20 years mainly because of the results that showed regeneration potential and plasticity that were much stronger than expected in prior decades. Recent findings in this field have contributed to progress in the establishment of cell differentiation methods, which have made stem cell therapy more clinically attractive. In addition, MSCs are easy to isolate and have anti-inflammatory and angiogenic capabilities. The use of stem cell therapy is currently supported by scientific literature in the treatment of several animal health conditions. MSC may be administered for autologous or allogenic therapy following either a fresh isolation or a thawing of a previously frozen culture. Despite the fact that MSCs have been widely used for the treatment of companion and sport animals, little is known about their clinical and biotechnological potential in the economically relevant livestock industry. This review focuses on describing the key characteristics of potential applications of MSC therapy in livestock production and explores the themes such as the concept, culture, and characterization of mesenchymal stem cells; bovine mesenchymal stem cell isolation; applications and perspectives on commercial interests and farm relevance of MSC in bovine species; and applications in translational research.
Bovine mesenchymal stromal cells (MSCs) display important features that render them valuable for cell therapy and tissue engineering strategies, such as self‐renewal, multi‐lineage differentiation, as well as immunomodulatory properties. These cells are also promising candidates to produce cultured meat. For all these applications, it is imperative to unequivocally identify this cell population. The isolation and in vitro tri‐lineage differentiation of bovine MSCs is already described, but data on their immunophenotypic characterization is not yet complete. The currently limited availability of monoclonal antibodies (mAbs) specific for bovine MSC markers strongly hampers this research. Following the minimal criteria defined for human MSCs, bovine MSCs should express CD73, CD90, and CD105 and lack expression of CD14 or CD11b, CD34, CD45, CD79α, or CD19, and MHC‐II. Additional surface proteins which have been reported to be expressed include CD29, CD44, and CD106. In this study, we aimed to immunophenotype bovine adipose tissue (AT)‐derived MSCs using multi‐color flow cytometry. To this end, 13 commercial Abs were screened for recognizing bovine epitopes using the appropriate positive controls. Using flow cytometry and immunofluorescence microscopy, cross‐reactivity was confirmed for CD34, CD73, CD79α, and CD90. Unfortunately, none of the evaluated CD105 and CD106 Abs cross‐reacted with bovine cells. Subsequently, AT‐derived bovine MSCs were characterized using multi‐color flow cytometry based on their expression of nine markers. Bovine MSCs clearly expressed CD29 and CD44, and lacked expression of CD14, CD45, CD73, CD79α, and MHCII, while a variable expression was observed for CD34 and CD90. In addition, the mRNA transcription level of different markers was analyzed using reverse transcription quantitative polymerase chain reaction. Using these panels, bovine MSCs can be properly immunophenotyped which allows a better characterization of this heterogenous cell population.
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