Isolation, culture, and characterization of embryonic cell lines from vitrified sheep blastocysts

Abstract

This study was conducted to isolate, to culture, and to characterize embryonic cell lines from in vitro produced vitrified sheep blastocysts. Embryos were produced and vitrified at the expanded blastocyst stage. Ten inner cell masses arising from day 6-7 blastocysts were isolated by immunosurgery, disaggregated, and cultured onto mitomocin-C-inactivated mouse STO fibroblasts (MIF). After 5 or 6 days of culture the primary cell colonies were disaggregated, seeded in a new MIF, and cultured for 3 or 4 days to form new colonies called Passage 1. These cells were then disaggregated and cultured for other two passages. The primary cell colonies and Passage 2 colonies expressed stage specific embryonic markers SSEA-1, SSEA-3, and SSEA-4, and were alkaline phosphatase positive. In the absence of feeder layer and human leukemia inhibitory factor (LIF), these cells differentiated into variety of cell types and formed embryoid bodies. When cultured for an extended period of time, embryoid bodies differentiated into derivatives of three embryonic germ (EG) layers. These were characterized by detection of specific markers for differentiation such early mesoderm (FE-C6), embryonic myosin (F1-652), neural precursor (FORSE-1), and endoderm (anti-cytokeratin 18). To our knowledge, this is the first time that embryonic cell lines from in vitro produced and vitrified ovine blastocysts have been isolated and examined for detection of SSEA markers, and embryoid bodies have been cultured and examined for specific cell surface markers for differentiation.

Developmental Competence of Buffalo (Bubalus bubalis) Pluripotent Embryonic Stem Cells Over Different Homologous Feeder Layers and the Comparative Evaluation with Various Extracellular Matrices

“…Morphologically, ICM-derived undifferentiated primary colonies were densely packed with clear border on all the feeders and synthetic matrices while differentiated colonies showed cystic EBs at their periphery. Our findings were similar to Verma et al (11) (24), however in pig (25), dog (26) and sheep (27) ESCs express SSEA-1. Contradictory reports are available in bovines on the presence of SSEA-1 in ESCs, some studies showed its presence (14, 28) while others did not (29).…”

Putative Embryonic Stem Cell Lines from Pig Embryos

“…Thus, it would be desirable, for economic reasons, to use embryos produced from in vitro matured and fertilized oocytes. However, up to now, only a few ES-like lines have been established in farm animals from in vitro produced material [33][34][35]. The first attempt to use in vitro blastocysts in the pig was described by Miyoshi et al [36].…”

“…The use of frozen-thawed (FT) and ICSI produced blastocysts for production of porcine stem cell lines has yet to be reported in spite of the fact that the above techniques have been used (with and without success) for production ES cell lines in other species (horse FT [10]; sheep FT [35]; primate ICSI [43]; human ICSI [44]; human FT [45]). Another possibility is to establish ES cell lines from blastocysts produced by nuclear transfer.…”

Development, Characterization, and Pluripotency Analysis of Buffalo (Bubalus bubalis) Embryonic Stem Cell Lines Derived fromIn Vitro–Fertilized, Hand-Guided Cloned, and Parthenogenetic Embryos

“…SSEA1, SSEA4, and tumor rejection antigens (TRA-1-60 and TRA-1-81) are regarded as markers of human (Carpenter et al, 2003;Henderson et al, 2002), primate (Liu et al, 2004;Thomson et al, 1995), and bovine ESCs (Dattena et al, 2006;Mitalipova et al, 2001;Saito et al, 2002). The FOXD3 forkhead transcription factor is required for maintaining pluripotent cells in early mouse embryo and for establishment of murine ESC lines (Liu and Labosky, 2008), whereas CD90 has been used as human ESC marker (Fang et al, 2005) as well as a marker for spermatogonial and mesenchymal stem cells (Kobayashi et al, 2009).…”