Figure 2. Differentiation of hESCs cured with Plasmocin^TM.

H5 884 cells treated with Plasmocin^TM 25 µg/ml during 14 days (Curative treatment) and H5 cells were differentiated using the EBs protocol and then: (A) mRNA levels of oct-4 and nanog, ectoderm (pax-6), mesoderm (brachyury and nkx2.5) and endoderm (α-fetoprotein) markers were analyzed by RT-Real Time PCR at days 0, 4, 7 and 14 of the differentiation protocol. Rpl7 expression was used as normalizer. Graph shows mRNA fold induction relative to day 0. The mean ± S.E. from three independent experiments are shown. (B) Cells were grown on gelatin coated plates from day 7 to 14 of differentiation and then ectoderm (neural rosettes), cardiac mesoderm (contractile EBs) and endoderm structures were stained with primary antibodies that recognize Nestin (ectoderm), cTnT (cardiac mesoderm) and AFP (endoderm) markers. Figure shows representative images. Scale bars = 100 µm. (C) Cured H5 884 cells, which express GFP gene under the control of the Brachyury promoter, were differentiated using the EBs protocol. At day 4 of the onset of differentiation GFP green fluorescence was photographed using an inverted fluorescence microscope. Figure shows representative images. Arrows indicate GFP positive regions or EBs. Scale bars = 100 µm. Abbreviations: BF, Bright field; Pax-6, Pair box protein 6; Nkx2.5, NK2 homeobox 5; cTnT, Cardiac troponin T; AFP, Alpha-fetoprotein; GFP, Green fluorescent protein.