This study was initiated in order to test a mini-invasive method of mesenchymal stem/progenitor cells (MS/PCs) isolation from a rat bone marrow (BM), and subsequently their expansion, differentiation, and evaluation of their immunophenotypic characteristics; and later their preservation as donor cells in an optimal condition for potential autotransplantation. The study group comprised of 6 adult male Sprague-Dawley (S-D) rats, weighing 480—690 g. The rats were anaesthetised by isoflurane with room air in a Plexiglas box and maintained by inhalation of a mixture of isoflurane and O2. Their femurs were surgically exposed and their diaphyses double-trephined. Then BM cells were flushed out by saline with heparin and aspirated into a syringe with a solution of DMEM (Dulbecco’s modified eagle’s medium) and heparin. The mononuclear cells from the BM were isolated by centrifugation and expanded in a standard culture medium supplemented with ES-FBS (es-cell-qualified foetal bovine serum), L-glutamine and rh LIF (recombinant human leukemia inhibitory factor). Following 14 days of passaging cultures, the cells were split into 2 equal parts. The first culture continued with the original medium. The second culture received additional supplementation with a human FGFβ (fibroblast growth factor beta) and EGF (epidermal growth factor). The populations of these cells were analysed by light-microscopy, then the mean fluorescence intensities (MFIs) of CD90 and Nestin were evaluated by a tricolour flow cytometry using monoclonal antibodies. The type of general anaesthesia used proved to be appropriate for the surgical phase of the experiments. All rats survived the harvesting of the BM without complications. The total number of mononuclear cells was 1.5—4.0 × 106 per sample and the proportion of CD90/Nestin expressing cells was < 1 %. Following 14 days of expansion, the cells became larger, adherent, with fibrillary morphology; the proportion of cells expressing CD90/Nestin increased to almost 25 %, i. e. they earned basic phenotypic characteristics of MSCs. Throughout the further cultivation a gradual decrease of the CD90/Nestin expression occurred. This suggested that the suitability of rat bone marrow derived MS/PCs for replacement therapy would probably be the highest between days 12—15 of cultivation and then would diminish.
If the inline PDF is not rendering correctly, you can download the PDF file here.
1. Animal Protection Act of Slovakia No. 15/1995 part 39 (In Slovak) 1250—1255.
2. Caplan A. I. Dennis J. E. 2006: Mesenchymal stem cells as trophic mediators. J. Cell. Biochem. 98 1076—1084.
3. Chesier S. H. Kalani M. Y. S. Lim M. Ailles L. Huhn S. L. Weissman I. L. 2009: A neurosurgeon’s guide to stem cells cancer stem cells and brain tumor stem cells. Neurosurgery 65 237—250.
4. Čížková D. Rosocha J. Vanický I. Jergová S. Čížek M. 2006: Transplants of human mesenchymal stem cells improve functional recovery after spinal cord injury in the rat. Cell. Mol. Neurobiol. 26 1167—1180.
5. Danišovič Ľ. Boháč M. Zamborský R. Oravcová L. Provazníková Z. Csölönyiová M. Varga I. 2016: Comparative analysis of mesenchymal stromal cells from different tissue sources in respect to articular cartilage tissue engineering. Gen. Physiol. Biophysics 35 207—214.
6. Dezawa M. Ishikawa H. Itokazu Y. Yoshihara T. Hoshino M. Takeda S. et al. 2005: Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science 309 314—317.
7. Dominici M. Blane K. L. Mueller L. Slaper-Cortenbach I. Marini F. C. Krause D. S. et al. 2006: Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8 315—317.
8. Greyson W. L. Zhano F. Brunnell B. Ma T. 2007: Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells. Biochem. Biophys. Res. Commun. 358 948—953.
9. Kalanin P. Flešárová S. 2006: Neuron damage elicited by cardiac arrest in a dog brain. Folia Veterinaria 50 73—75.
10. Kim B. G. Hwang D. H. Lee S. I. Kim E. J. Kim S. N. 2007: Stem cell-based cell therapy for spinal cord injury. Cell Transplant. 16 355—364.
11. Maženský D. Flešárová S. 2016: Importance of the arterial blood supply to the rabbit and guinea pig spinal cord in experimental ischemia. In Schaller B. (Ed.):Ischemic Stroke — Updates. Tech. Croatia 59—86.
12. Michalczyk K. Ziman M. 2005: Nestin structure and predicted function in cellular cytoskeletal organisation. Histol. Histopathol. 20 665—671.
13. Phinney D. G. Prockop D. J. 2007: Mesenchymal stem/multipotent stromal cells: The state of transdifferentiation and modes of tissue repair — current views. Stem Cells 11 2896—2902.
14. Pittinger M. F. Mackay A. M. Beck S. C. Jaiswal R. H. Douglas R. Mosca J. D. et al. 1999: Multilineage potential of adult human mesenchymal stem cells. Science 284 143—147.
15. Rider D. A. Dombrowski C. Sawyer A. A. Ng G. H. B. Leong D. Hutmacher D. W. et al. 2008: Autocrine fibroblast growth factor 2 increases the multipotentiality of human adipose-derived mesenchymal stem cells. Stem Cells 26 1598—1608.
16. Shroff G. Agarwal P. Mishra A. Sonowal N. 2015: Human embryonic stem cells in treatment of spinal cord injury: A prospective study. J. Neurol. Res. 5 213—220.
17. Slovinská L. Székiová E. Blaško J. Devaux S. Salzet M. Čížková D. 2015: Comparison of dynamic behaviour and maturation of neural multipotent cells derived from different spinal cord developmental stages: an in vitro study. Acta Neurobiol. Exp. (Wars.) 75 107—114.
18. Soleimani M. Nadri S. A. 2009: A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nature Protocols 4 102—106.
19. Šulla I. Bačiak L. Juránek I. Cicholesová T. Boldižár M. Balik V. Lukáčová N. 2014: Assessment of motor recovery and MRI correlates in a porcine spinal cord injury model. Acta Vet. Brno 83 393—397.
20. Šulla I. Balik V. Petrovičová J. Almášiová V. Holovská K. Oroszová Z. 2016: A rat spinal cord injury model. Folia Veterinaria 60 41—46.
21. Tropel P. Platet N. Platel J. C. Noël D. Albrieux M. Benabid A. L. Berger F. 2006: Functional neuronal differentiation of bone marrow-derived mesenchymal stem cells. Stem Cells 24 2868—2876.
22. Žilka N. Žilková M. Kaznerová Z. Šarišský M. Cigánková V. Novák M. 2011: Mesenchymal stem cells rescue the Alzheimer’s disease cell model from cell death induced by misfolded tau. Neuroscience 193 330—337.