Objective: The ability to isolate human trophoblast stem cells (TSC) from third-trimester placentae is key to understanding major pregnancy pathologies, but has proved challenging to date. We have previously used the side-population technique to isolate TSC-like cells from first trimester placentae that have gene expression characteristics and the differentiation potential of a TSC population. The same side-population technique can also isolate these cells from third-trimester placentae, but it is apparent that different trophic requirements are required to sustain cell viability at this gestation. This work aimed to optimise extracellular matrices and cytokine supplementation to improve third-trimester side-population trophoblast viability and differentiation.
Methods: The Hoechst side-population technique was used to isolate trophoblasts from normal third-trimester placentae. The effect of different extracellular matrices (5μg/mL Collagen-IV, or 10μg/mL Laminin-521), or the addition of 25ng/mL Decorin and/or 50ng/mL IL-8 on cell adherence, viability, and growth over 14 days of culture was assessed using ilastik and CellProfiler software.
Results: Twice as many third-trimester side-population trophoblasts attached and spread across the culture surface on Laminin-521 (41±2.000 SEM, n=3) than on Collagen-IV (17.33±6.333 SEM, n=3, p=0.1000), and most cells were entirely lost from the Collagen-IV surface by day 14 in culture. Compared to controls (0.008 mm2±0.0007 SEM, n=3), addition of either decorin (0.188mm2±0.0038 SEM, n=3, p<.0001) or IL-8 (0.241mm2±0.0043 SEM, n=3, p<.0001) significantly increased the size of side-population colonies over 14 days. Combining decorin and IL-8 supplementation enabled third-trimester side-population trophoblasts to be maintained for at least 30 days of culture (n=3). Preliminary data suggests side-population cells can differentiate into syncytin-1 positive syncytiotrophoblast (n=2) or HLA-G positive extravillous trophoblast (n=1).
Conclusion: The optimisation of conditions to allow 2D culture and manipulation of third-trimester side-population trophoblasts in an undifferentiated state underlie further analyses of potential trophoblast dysfunction in pregnancy pathologies.