Morulae produced by human somatic cell nuclear transfer

Abstract

OBJECTIVE: To create human embryonic stem cell (hESC) lines by somatic cell nuclear transfer (SCNT). Researchers have succeeded in performing SCNT in a number of animals, but not yet in humans. Our goals are to make patient-specific lines with potential therapeutic value as well as disease-specific lines of value to researchers in need of a stable cell line with a particular genetic abnormality. DESIGN: Women undergoing superovulation for fertility treatment at Huntington Reproductive Center (HRC) in Laguna Hills, CA consent to donation of supernumerary oocytes to this project as part of an oocyte sharing program. Somatic cell donors undergo skin biopsy at HRC. All donor cells are transported to and cultured at the University of California Irvine (UCI). MATERIALS AND METHODS: 29 MII oocytes were obtained from 3 patients. Somatic cells were obtained from two cell donors. Using a laser ablator to traverse the zona pellucida and birefringent imaging system to visualize mitotic spindle, the spindle was aspirated and replaced with a somatic cell. Following reconstruction, cells were activated by ionomycin or Ca2+ ionophore treatment, followed by incubation in 6-DMAP and cultured in standard IVF conditions. RESULTS: 21 oocytes were successfully enucleated, reconstructed and activated. 8 of the reconstructed oocytes cleaved. From these, 3 continued to develop to the compact morula stage, then arrested. CONCLUSIONS: Micromanipulation and activation methods are moderately successful. Optimization of micromanipulation techniques and culture conditions is required to progress to blastocyst formation and line derivation.