Trichostatin A-Mediated Epigenetic Transformation of Adult Bone Marrow-Derived Mesenchymal Stem Cells Biases the In Vitro Developmental Capability, Quality, and Pluripotency Extent of Porcine Cloned Embryos

Figure 2

Evaluation of cytological quality of porcine cloned blastocysts on the basis of simultaneous determination of total nuclear number and detection of apoptotic cell nuclei by terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP (2′-deoxyuridine-5′-triphosphate) nick-end labelling (TUNEL) analysis. Photographs (a) to (d) depict embryos originating from nuclear-transferred (NT) oocytes reconstituted with adult bone marrow-derived mesenchymal stem cells (ABM-MSCs) subjected to epigenetic transformation via trichostatin A (TSA) treatment. Photographs (e) and (f) depict embryos originating from NT oocytes reconstituted with ABM-MSCs not subjected to epigenetic transformation via TSA treatment. In each blastocyst, the cell nuclei of all the blastomeres (both inner cell mass (ICM) and trophectoderm (TE) cells) had been tagged with 4′,6-diamidino-2-phenylindole (DAPI) counterstain and subsequently fluoresced in blue. The cell nuclei of late-apoptotic blastomeres (ICM and/or TE cells) exhibiting internucleosomal DNA fragmentation had been dyed with fluorescein isothiocyanate (FITC) and then fluoresced in bright green. In each photograph, the DAPI-derived blue and FITC-derived green fluorescent signals merge into one another. Photographs (a) to (f) represent the blastocysts displaying different incidence of blastomere apoptosis and thereby varied advancement of internucleosomal DNA fragmentation ((a), (c), (d), and (e) the lack of apoptotic intranuclear DNA fragmentation; (b) and (e) few apoptotic cell nuclei; (f) increased extent of apoptotic intranuclear DNA fragmentation). Images were taken at magnification ×200.

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