The less differentiated the donor cells are used in nuclear transfer (NT), the more easily are they reprogrammed by the recipient cytoplasm. In this context, mesenchymal stem cells (MSCs) appear as an alternative to donor nuclei for NT. The amniotic fluid and adipose tissue are sources of MSCs that have not been tested for the production of cloned embryos in cattle. The objective of this study was to isolate, characterize, and use MSCs derived from amniotic fluid (MSC-AF) and adipose tissue (MSC-AT) to produce cloned calves. Isolation of MSC-AF was performed using in vivo ultrasound-guided transvaginal amniocentesis, and MSC-AT were isolated by explant culture. Cellular phenotypic and genotypic characterization by flow cytometry, immunohistochemistry, and RT-PCR were performed, as well as induction in different cell lineages. The NT was performed using MSC-AF and MSC-AT as nuclear donors. The mesenchymal markers of MSC were expressed in bovine MSC-AF and MSC-AT cultures, as evidenced by flow cytometry, immunohistochemistry, and RT-PCR. When induced, these cells differentiated into osteocytes, chondrocytes, and adipocytes. Embryo production was similar between the cell types, and two calves were born. The calf from MSC-AT was born healthy, and this fact opens a new possibility of using this type of cell to produce cloned cattle by NT.

Wharton's jelly is a source of mesenchymal stem cells (MSCs) that had not yet been tested for bovine embryo production by nuclear transfer (NT). Thus, the objective of this study was to isolate, characterize and test MSCs derived from Wharton's jelly for embryo and pregnancy RESUMO A geleia de Wharton é uma fonte de células tronco mesenquimais (CTMs) que ainda não havia sido testada para a produção de embriões bovinos por transferência nuclear (TN). O objetivo deste estudo foi isolar, caracterizar e testar as CTMs derivadas da geleia de Wharton para produção de embriões e gestações por transferência nuclear em bovinos. O cordão umbilical foi coletado durante o nascimento e as células derivadas da geleia de Wharton (CGWs) foram isoladas por explante e cultivadas em

ContentsThe aim of this work was to investigate the methylation and hydroxymethylation status of mesenchymal stem cells (MSC) from amniotic fluid (MSC-AF), adipose tissue (MSC-AT) and fibroblasts (FIB-control) and to verify the effect of trichostatin A (TSA) on gene expression and development of cloned bovine embryos produced using these cells. Characterization of MSC from two animals (BOV1 and BOV2) was performed by flow cytometry, immunophenotyping and analysis of cellular differentiation genes expression. The cells were used in the nuclear transfer in the absence or presence of 50 nM TSA for 20 hr in embryo culture. Expression of HDAC1, HDAC3and KAT2A genes was measured in embryos by qRT-PCR. Methylation results showed difference between animals, with MSC from BOV2 demonstrating lower methylation rate than BOV1. Meanwhile, MSC-AF were less hydroxymethylated for both animals.MSC-AF from BOV2 produced 44.92 ± 8.88% of blastocysts when embryos were exposed to TSA and similar to embryo rate of MSC-AT also treated with TSA (37.96 ± 15.80%). However, when methylation was lower in FIB compared to MSC, as found in BOV1, the use of TSA was not sufficient to increase embryo production.MSC-AF embryos expressed less HDAC3 when treated with TSA, and expression of KAT2A was higher in embryos produced with all MSC and treated with TSA than embryos produced with FIB. The use of MSC less methylated and more hydroxymethylated in combination with embryo incubation with TSA can induce lower expression of HDAC3 and higher expression of KAT2A in the embryos and consequently improve bovine embryo production. K E Y W O R D Sadipose cells, amniotic cells, bovine cloning, DNA methylation, histone acetylation