Abstract
Purpose: Differentiation of BM-MSCs into adult chondrocytes represents a complex physiological mechanism and full characterization of each individual stage through which the BM-MSC differentiate into adult chondrocytes is not yet understood. The physiological micro-environment of the chondrocytes is intensely hypoxic which triggers over-expression SLC2A proteins (GLUTs) in their membranes as a compensatory mechanism for energy production within the glycolytic cycle.
Method: We cultured and differentiated BM-MSC, and adult chondrocytes in hypoxic (5% O2 tension) and normoxic (20% O2) conditions. Within this cell populations we screened for the presence of the 12 GLUT genes as well as quantification of the variation of the 12 GLUTs gene translation by simple pcr and rt-pcr. The expression profile of the GLUT proteins was investigated using western blot analysis and immunohistochemistry. Functional characterization of the GLUTs expressed in the different cell populations was carried out by the means of radio-isotope labeled hexose fluxes done accordingly to the substrate specificity and kinetic properties particular to each SLC2A isoforms.
Results: Our data showed that the functional genotype and phenotype of the adult chondrocyte and hypoxic BM-MSC comprised an extensive expression of fructose-transporting GLUTs as opposed to the glucose-only transporting isoforms expression in normoxic BM-MSC. The flux data showed clear similarities in functional GLUT profiles between BM-MSC cultured in hypoxic conditions, adult chondrocytes. Investigation of the uptake of a panel of five individual sugars (glucose, fructose, 2-deoxy-gluose, 3-orthomethyl-glucose and galactose) in these cellular populations under both hypoxic and normoxic conditions and in the presence and absence of Cytochalasin B (a GLUT1-specific inhibitor) showed that SLC2A class II transporters (GLUTs 5, 7, 9 and 11) play a more important role in the uptake of sugars by the normal hypoxic chondrocytes when compared to the ubiquitously-expressed GLUT1.
Conclusion: Use of this approach allows the correct culturing conditions to be identified that would select for those chondrocyte precursors from the total BM-MSC population that would have the best potential for producing viable articular cartilage. In addition, specific substrates for GLUTs isoforms could be used for physiologic, non-invasive and real time imaging of cartilage, BM-MSC and cartilage autograft by means of Positron Emission Tomography.
Correspondence should be addressed to CEO Doug C. Thomson. Email: doug@canorth.org
