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Stem Cells 2004;22:790-797 www.StemCells.com
© 2004 AlphaMed Press

Derivation of Human Embryonic Stem Cells from Day-8 Blastocysts Recovered after Three-Step In Vitro Culture

Miodrag Stojkovica, Majlinda Lakoa, Petra Stojkovica, Rebecca Stewartb, Stefan Przyborskib, Lyle Armstrongb, Jerry Evansa, Mary Herbertc, Louise Hyslopa, Sajjad Ahmada, Alison Murdochc, Tom Strachana

a Institute of Human Genetics, University of Newcastle, Newcastle upon Tyne, United Kingdom;
b School of Biological and Biomedical Sciences, University of Durham, Durham, United Kingdom;
c Newcastle Fertility Centre at Life, International Centre for Life, Newcastle upon Tyne, United Kingdom

Key Words. Human embryonic stem cells • In vitro culture • Pluripotency • Differentiation

Correspondence: Miodrag Stojkovic, M.D., Institute of Human Genetics, University of Newcastle, Central Parkway, Newcastle upon Tyne, NE1 3BZ, U.K. Telephone: 44-191-219-4746; Fax: 44-191-219-4747; e-mail: miodrag.stojkovic{at}ncl.ac.uk

Human embryonic stem cells (hESCs) have been derived from the inner cell mass (ICM) of day 5–7 blastocysts and hold great promise for research into human developmental biology and the development of cell therapies for the treatment of human diseases. We report here that our novel three-step culture conditions successfully support the development of day-8 human blastocysts, which possess significantly (p <.01) more ICM cells than day-6 blastocysts. Plating of ICMs isolated from day-8 blastocysts resulted in the formation of a colony with hESC morphology from which a new hESC line (hES-NCL1) was derived. Our stem cell line is characterized by the expression of specific cell surface and gene markers: GTCM-2, TG343, TRA1-60, SSEA-4, alkaline phosphatase, OCT-4, NANOG, and REX-1. Cytogenetic analysis of the hESCs revealed that hES-NCL1 line has a normal female (46, XX) karyotype. The pluripotency of the cell line was confirmed by the formation of teratomas after injection into severely combined immunodeficient mice and spontaneous differentiation under in vitro conditions.




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