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

Dattena, Maria and Chessa, Bernardo and Lacerenza, D. and Accardo, Carla and Pilichi, Susanna and Mara, Laura and Chessa, F. and Vincenti, L. and Cappai, Pietro (2005) Isolation, culture, and characterization of embryonic cell lines from vitrified sheep blastocysts. Molecular Reproduction and Development, Vol. 73 (1), p. 31-39. eISSN 1098-2795. Article.

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DOI: 10.1002/mrd.20378

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.

Item Type:Article
ID Code:2492
Status:Published
Refereed:Yes
Uncontrolled Keywords:Blastocysts, ICM, embryonic cell culture, sheep
Subjects:Area 07 - Scienze agrarie e veterinarie > VET/05 Malattie infettive degli animali domestici
Divisions:001 Università di Sassari > 01 Dipartimenti > Patologia e clinica veterinaria
002 Altri enti e centri di ricerca del Nord Sardegna > Istituto zootecnico e caseario per la Sardegna, Olmedo
Publisher:Wiley-Liss
eISSN:1098-2795
Deposited On:18 Aug 2009 10:07

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