Journal: PLoS ONE
Article Title: Manipulation of Signaling Thresholds in “Engineered Stem Cell Niches” Identifies Design Criteria for Pluripotent Stem Cell Screens
doi: 10.1371/journal.pone.0006438
Figure Lengend Snippet: In the first experiment (A–C), mESCs were cultured in colonies with diameters (D) ranging from 50–200 µm. Pitch (P), the centre-to-centre distance between colonies, was kept constant for the two smaller colonies and was increased to accommodate the larger D = 200 µm colony. A) Average local cell density calculations for each pattern type demonstrating the co-relation between colony diameter and the local cell density. B) Predicted and actual measurements of single-cell pStat3 levels as function of increasing colony diameter. White bars indicate cultures with no LIF supplementation, black bars with 500 pM LIF supplementation, and grey bars with 600 nM JAK inhibitor. C) Experimental measurements of Oct-4 intensity demonstrated the co-relation between colony diameter, pStat3, and Oct-4 expression with LIF deprivation. In the second experiment (D–F), mESCs were cultured in colonies with fixed diameter, D = 100 µm, while pitch (P) ranged from 200-500 µm. D) Average local cell density calculations for each pattern type demonstrating the inverse co-relation between pitch and the local cell density. E) Predicted and actual measurements of single-cell pStat3 levels as function of decreasing pitch. F) Experimental measurements of Oct-4 intensity demonstrated the inverse co-relation between pitch and Oct-4 expression. In the last experiment (G–I), both colony diameter and pitch were altered to increase the degree of clustering while keeping the total available area for seeding constant. G) Average local cell density calculations for each pattern type demonstrating that the local cell density was within experimental error between pattern types. H) Predicted and actual measurements of single-cell pStat3 levels as function of degree of clustering. I) Experimental measurements of Oct-4 intensity demonstrating a positive co-relation between pStat3 and Oct-4. Representative bright field images of micro-patterned mESC cultures can be found in Supplementary .
Article Snippet: Cells were seeded in KOSR-based media with LIF for 4 hrs, after which media was replaced with KOSR-based media with or without 500 pM LIF or 600 nM of Jak inhibitor 1 (Calbiochem).
Techniques: Cell Culture, Expressing
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