Hum. Reprod. Advance Access published online on October 24, 2007
Human Reproduction, doi:10.1093/humrep/dem335
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Mechanical isolation of the inner cell mass is effective in derivation of new human embryonic stem cell lines
1 Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden 2 Department of Biosciences and Nutrition at Novum, Karolinska University Hospital Huddinge, SE 141 86 Stockholm, Sweden 3 Department of Molecular Medicine and Surgery, Cardiothoracic Surgery and Anaesthesiology Unit, Karolinska Institutet, Stockholm, Sweden 4 Department of Molecular Medicine and Surgery, Clinical Genetics Unit, Karolinska Institutet, Stockholm, Sweden
5 Correspondence address. E-mail: outi.hovatta{at}ki.se
BACKGROUND: For clinical grade human embryonic stem cell (hESC) lines, a robust derivation system without any substances having animal origin would be required. We have gradually improved our hESC derivations. Human skin fibroblasts were used as feeder cells in derivation of all our 25 permanent fully characterized hESC lines. In the first four derivations, fetal calf serum was used as a supplement in the medium, thereafter, serum replacement medium was used. Immunosurgery generally used for isolation of the inner cell mass (ICM) still involves animal serum and complement.
METHODS: We developed a practical mechanical isolation method for the ICM. Two flexible metal needles with sharpened tips, 0.125 mm in diameter, were used to open the zona pellucida and extract the ICM under a stereomicroscope. Immunohistochemical and karyotype characterization of the new hESC lines was carried out, and pluripotency was tested in vitro (immunocytochemistry and RT–PCR) and in vivo (teratoma growth).
RESULTS: Five hESC lines were obtained from 19 supernumerary blastocysts collected in 2005–2006 (26%), whereas in similar conditions, we obtained 16 lines from 100 blastocysts (16%) using immunosurgery in 2003–2005. The new lines had a normal karyotype and tissues originating from the three embryonic germ cell layers were present.
CONCLUSIONS: Mechanical isolation of the ICM proved to be an effective way to derive new hESC lines. The technique is fast, does not require any extra investment and the xeno-components of immunosurgery could be avoided.
Key words: human embryonic stem cells/inner cell mass/mechanical isolation/human skin fibroblasts/derivation
These authors contributed equally to this work. Submitted on April 14, 2007; resubmitted on September 4, 2007; accepted on September 18, 2007.
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