Vertebral Bone Marrow-Derived Mesenchymal Stromal Cells from Osteoporotic and Healthy Patients Possess Similar Differentiation Properties In Vitro
Abstract
:1. Introduction
2. Results
2.1. Morphology and Proliferation Rate of BMSCs from Osteoporotic and Non-Osteoporotic Control Donors
2.2. Phenotypic Analysis and Immunomodulatory Capacity
2.3. Osteogenic, Adipogenic, and Chondrogenic Differentiation
2.4. Alkaline Phosphatase Intensity and Activity during Osteogenic Differentiation Process
2.5. Assessment of Osteogenic Differentiation
2.6. Osteoblast Marker Gene Expression
3. Discussion
4. Materials and Methods
4.1. Tissue Donors and Isolation of Bone Marrow-Derived MSCs
4.2. Morphologic Analysis
4.3. MTT Assay
4.4. Flow Cytometric Analysis
4.5. Immunomodulatory Capacity
4.6. Adipogenic Differentiation
4.7. Chondrogenic Differentiation
4.8. Osteogenic Differentiation
4.9. Alkaline Phosphatase Measurement, Optical Density Measurement, and Free Phosphate Assay
4.10. Real-Time Polymerase Chain Reaction
4.11. Statistics
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
ALP | Alkaline phosphatase |
ALPL | Alkaline phosphatase gene |
BMSC | Bone marrow-derived mesenchymal stromal cells |
CD | Cluster of differentiation |
cDNA | Complementary deoxyribonucleic acid |
CFSE | Carboxyfluorescein succinimidyl ester |
COL1A1 | Collagen, type I, alpha 1 |
DAPI | 4′,6-Diamidino-2-phenylindole |
ddCT | Delta-delta-Ct |
DMEM | Dulbecco’s modified Eagle’s medium |
DPBS | Dulbecco’s phosphate-buffered saline |
ECM | Extracellular matrix |
EDTA | Ethylenediaminetetraacetic acid |
FBS | Fetal bovine serum |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase |
hMSC | Healthy mesenchymal stromal cells |
ISCT | International Society for Cellular Therapy |
mRNA | Messenger ribonucleic acid |
MSC | Mesenchymal stromal cells |
OD | Optical density |
oMSC | Osteoporotic mesenchymal stromal cells |
PBMC | Peripheral blood mononuclear cell |
PBS | Phosphate-buffered saline |
PFA | Paraformaldehyde |
RT-PCR | Real-time polymerase chain reaction |
RUNX2 | Runt-related transcription factor 2 |
SOX9 | SRY-Box Transcription Factor 9 |
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Gene | Primer Sequence | Product Length | Accession Number |
---|---|---|---|
GAPDH | fwd: 5′CTCTGCTCCTCCTGTTCGAC3′ rev: 5′ACCAAATCCGTTGACTCCGA3‘ | 109 bp | NM_002046.5 |
ALPL | fwd: 5′TTTATAAGGCGGCGGGGGTG3′ rev: 5′AGCCCAGAGATGCAATCGAC3′ | 198 bp | NM_000478.5 |
COL1A1 | fwd: 5′TGCTCGTGGAAATGATGGTG3′ rev: 5′CCTCGCTTTCCTTCCTCTCC3′ | 449 bp | NM_000088.3 |
RUNX2 | fwd: 5′GCGCATTCCTCATCCCAGTA3′ rev: 5′GGCTCAGGTAGGAGGGGTAA3′ | 176 bp | NM_001024630.3 |
SOX9 | fwd:5′AGGAAGTCGGTGAAGAACGG3′ rev: 5′AAGTCGATAGGGGGCTGTCT3′ | 275 bp | NM_000346.3 |
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Haddouti, E.-M.; Randau, T.M.; Hilgers, C.; Masson, W.; Pflugmacher, R.; Burger, C.; Gravius, S.; Schildberg, F.A. Vertebral Bone Marrow-Derived Mesenchymal Stromal Cells from Osteoporotic and Healthy Patients Possess Similar Differentiation Properties In Vitro. Int. J. Mol. Sci. 2020, 21, 8309. https://doi.org/10.3390/ijms21218309
Haddouti E-M, Randau TM, Hilgers C, Masson W, Pflugmacher R, Burger C, Gravius S, Schildberg FA. Vertebral Bone Marrow-Derived Mesenchymal Stromal Cells from Osteoporotic and Healthy Patients Possess Similar Differentiation Properties In Vitro. International Journal of Molecular Sciences. 2020; 21(21):8309. https://doi.org/10.3390/ijms21218309
Chicago/Turabian StyleHaddouti, El-Mustapha, Thomas M. Randau, Cäcilia Hilgers, Werner Masson, Robert Pflugmacher, Christof Burger, Sascha Gravius, and Frank A. Schildberg. 2020. "Vertebral Bone Marrow-Derived Mesenchymal Stromal Cells from Osteoporotic and Healthy Patients Possess Similar Differentiation Properties In Vitro" International Journal of Molecular Sciences 21, no. 21: 8309. https://doi.org/10.3390/ijms21218309