RESUMO
In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos.
Assuntos
Antígenos de Diferenciação/biossíntese , Búfalos/embriologia , Embrião de Mamíferos/embriologia , Células-Tronco Embrionárias/metabolismo , Partenogênese , Células-Tronco Pluripotentes/metabolismo , Animais , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/fisiologia , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Camadas Germinativas/embriologia , Células-Tronco Pluripotentes/citologia , Tretinoína/farmacologiaRESUMO
This study investigated the effects of serum-starvation, total confluence, and roscovitine treatment on cell-cycle synchronization of buffalo ear skin fibroblasts to the G0/G1 stage and on the developmental competence of cloned embryos. Serum starvation of total confluence cultures for 24 h had a higher (p<0.05) proportion of cells at G0/G1 stage (94.4%) compared with serum starved cyclic and nonstarved confluent cultures (76.8 and 86.0%, respectively), whereas differences between cyclic cells with or without serum starvation were not significant. The proportion of cells at G0/G1 was higher (p<0.05) with 20 and 30 µM roscovitine treatment than that with 10 µM (94.4, 96.4, and 86.6%, respectively), which was similar to that for total confluence (86.0%). MTT assay showed that cell viability decreased as dose of roscovitine increased. The blastocyst rate was significantly higher (p<0.05) when nuclear transfer embryos were reconstructed using donors cells from total confluence, confluence serum starved, and roscovitine-treated (20 and 30 µM) groups (48.8, 48.9, 57.9, and 62.9%, respectively) compared to nontreated cyclic cells (20.2%). However, the cleavage rate and total cell number of cloned embryos were similar for all the groups. The number of ICM cells was improved by 30 µM roscovitine treatment (45.25 ± 2.34). The cryosurvival rate of blastocysts derived from cells synchronized with 20 or 30 µM roscovitine was higher compared to that for total confluence group (33.6, 37.8 vs. 23.8%). In conclusion, treatment with 30 µM roscovitine is optimal for harvesting G0/G1 stage cells for producing high quality cloned buffalo embryos, and that it is better than serum-starvation or total confluence for cell synchronization.