Establishment of Trophectoderm Cell Lines from Buffalo (<i>Bubalus bubalis</i>) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them

<div><p>Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF.</p></div

Effect of fetal fibroblast CM on growth of TE cells.

<p>Data from 3 trials. Data are Mean ± SEM. Values with different superscripts (a and b) within the same column differ significantly (P<0.05).</p><p>Effect of fetal fibroblast CM on growth of TE cells.</p

Global CDX2 levels in different types of embryos.

<p>Global level of CDX2 in cloned embryos produced using trophoblast cells, adult fibroblasts and fetal fibroblasts as donor cells and those produced by IVF. Bars marked with an asterisk differ significantly from corresponding values (P<i><</i>0.05).</p

Immunostaining of CDX2.

<p>(A) IVF-derived hatched blastocysts (100X) and (B) TE cells produced under feeder-free conditions showing positive expression of CDX2 (200X). Scale bar = 100 μm.</p

Growth analysis of presumptive TE cells from different sources.

<p>Growth rate of (A) primary colonies on fetal fibroblast feeder layer and (B) TE cells obtained from IVF-derived hatched blastocysts and HMC-derived cloned blastocysts on MaxGel ECM under feeder-free conditions. Bars/graph points marked with an asterisk differ significantly from the corresponding value. *(P<0.01); **(P<0.001).</p

Characterization of presumptive TE cells by immunofluorescence for cytoskeletal proteins.

<p>TE cells produced under feeder-free conditions showing the expression of cytokeratin-18, keratin and tubulin but not that of vimentin, by immunofluorescence staining.</p

Effect of FGF2 on expression level of trophoblast-specific genes.

<p>Bars with different superscripts differ significantly (P<0.05).</p

Global H3K18ac and H3K27me3 levels in different types of donor cells and embryos derived from them.

<p>(A) Global level of H3K18ac and H3K27me3 in trophoblast cells, adult fibroblasts and fetal fibroblasts. (B) Global level of H3K18ac and H3K27me3 in cloned embryos produced using trophoblast cells, adult fibroblasts and fetal fibroblasts as donor cells and those produced by IVF. Bars marked with an asterisk or different superscripts differ significantly from corresponding values (P<i><</i>0.05).</p

Derivation of presumptive TE cells.

<p>(A) An IVF-derived blastocyst seeded on buffalo fetal fibroblast feeder layer; (B) A primary colony of TE cells (40X, Scale bar = 500 μm); (C) A primary colony showing inner cell mass and endoderm cells (100X); (D) Endoderm colonies indicated by the arrow mark showing tight colony morphology (100X), Inset: Endoderm colony at 400X (Scale bar = 50 μm); (E) TE cells at passage 20 (200X) and (F) TE cells digested with accutase showing loosening of cells and thread-like structures which are the parts of tight junctions (400X, Scale bar = 50 μm). Scale bar = 100 μm, unless otherwise mentioned in Figure.</p

Morphology of presumptive TE cells under feeder and feeder-free conditions.

<p>Growth of IVF-derived trophoblast cells under (A) feeder and (B) feeder-free conditions. Subculture of trophoblast cells under feeder-free conditions led to development of vesicles of 100–500 μm diameter, which get dissociated (C). Following subculture, the trophoblast cells formed confluent monolayer on feeder layer (D) and dome shaped colonies under feeder-free conditions (E). Scale bar = 500 μm. Single dome observed under 100X (F). Scale bar = 100 μm.</p