Figure 7. Gene and protein expression of the seeded EB constructs after 6 days of culture.

(A) qRT-PCR result at day 6 to depict gene expressions related to early gastrulation. The expression level is normalized to native ESR1 EB at day 6. Higher early differentiation markers –Brachyury, FGF5 and FGF8 were found on ESR1 seeded on dSPT EB scaffold. VE gene – AFP and pluripotency gene – Nanog are downregulated on both seeded constructs compared to native EB. Neuroectoderm marker – Nestin was upregulated on seeded dSPT EB scaffold but downregulated in seeded dRA EB scaffold. (B) Brachyury protein expression was investigated and confirmed by IHC – representative images showing higher cell numbers found positive for Brachyury in ESR1 seeded on dSPT EB scaffold compared to dRA EB scaffold. (C) Cell number area positive for Brachyury were measured and compared among ESR1 seeded on dSPT, dRA EB scaffolds and native ESR1 EB and average Brachyury immunoreactive area was significantly greater on ESR1 seeded on dSPT scaffold. All values are mean ± SD; p<0.05 (*), P<0.01 (**), n = 3, represents individual seeded EBs in 3 experimental repeats.