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Comparative analysis of nuclear transfer embryo-derived mouse embryonic stem cells. Part II: gene regulation.

ABSTRACT: In a mouse model nuclear transfer embryo-derived embryonic stem cell lines (ntESCs) of various genetic backgrounds and donor cell types were compared with reference ESCs and analyzed comprehensively at molecular level as a second part of a larger study. Expression profiles of ntESCs established by different NT-methods (piezoelectric microinjection or zona-free) were indistinguishable. However, expression profiling analyses identified differentially regulated genes between reference ESCs and ntESCs from different genetic backgrounds. A number of pluripotency and stemness marker genes significantly differed at the mRNA level between the cell lines. However, cluster and lineage analyses revealed that such differences had no effect on cell differentiation and cell fate. Regardless of the donor cell type, gene expression profiles of ntESCs were more similar to each other than to their counterpart fertilized embryo-derived ESCs of the same genotype. Overall, the results indicated that expression profile differences may be related to the genotype rather than to technical variations.

SUBMITTER: Kobolak J

PROVIDER: S-EPMC3275096 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular "uncoupling". Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters re-differentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way.

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Comparative analysis of nuclear transfer embryo-derived mouse embryonic stem cells. Part II: gene regulation.

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