Human Embryos Derived by Somatic Cell Nuclear Transfer Using an Alternative Enucleation Approach

Abstract

Somatic cell nuclear transfer (SCNT) was used to generate patient-specific embryonic stem cells (ESCs) from blastocysts cloned by nuclear transfer (ntESCs). In this study, a total of 135 oocytes were obtained from 12 healthy donors (30-35 years). Human oocytes, obtained within 2 h following transvaginal aspiration, were enucleated using a Spindle Imaging System to position the spindle and chromosomes that lay on the metaphase plate, and a Zona Infrared Laser Optical System was used to open a single hole in the zona pellucida at the ~ 2 o'clock position. Human fibroblasts and lymphocytes were used to construct SCNT embryos. Nearly half (26 of 58) of the oocytes were fused after electrofusion and embryo development rates were 96.2% (two-cell, 25 of 26), 92.3% (four-cell, 24 of 26), 61.5% (eight-cell, 16 of 26), 34.6% (16-cell, 9 of 26), 26.9% (morula, 7 of 26), and 19.2% (blastocyst, 5 of 26), respectively, following incubation in improved G-series sequential medium. One cloned blastocyst was used for STR-DNA identification and genetic polymorphism analysis of mtDNA, and STR-DNA analysis of all cloned blastocysts indicated they were derived from SCNT. Quantitative analysis showed that mtDNA of cloned embryos reflected the change tendency of those observed in human IVF embryos. Our research provides an alternative enucleation approach for producing human SCNT-derived blastocysts, and may aid in providing a new method for human therapeutic cloning.

Laser Pulse Application in IVF

“…The 1.48 uM diode laser may be used in conjunction with oocyte enucleation procedures in a similar manner as with ICSI and assisted hatching. A small hole is drilled in the zona pellucida, through which the nucleus is removed while leaving most of the cytoplasm (Li et al, 2009). A picosecond pulsed 405 nm diode laser also effectively aids in enucleation with extremely short pulse duration of 1-2 seconds.…”

Embryonic Stem Cells for Therapies – Challenges and Possibilities

“…Upon stimulation, the re-programmed cell divides to form a blastocyst with an inner cell mass that has identical nuclear genetic composition as the nucleus donor. Although this approach has worked to generate ESCs from different animals such as mice, rabbits, cats, sheep, cattle, pigs, goats [reviewed in (Wilmut et al, 2002)] and even primates (Byrne et al, 2007), no hESC has been generated through this approach as it remains a highly inefficient process and the use of human oocytes is ethically controversial (French et al, 2008;J. Li et al, 2009b).…”

Induced Pluripotent Stem Cells : It Looks Simple but Can Looks Deceive?

“…This procedure was technically demanding and inefficient in animals and was known to produce offspring of less than optimal quality when used for reproductive cloning but perhaps more importantly it had never been successfully applied to humans. In 2005, the first human embryo derived by SCNT was reported by our group [2] and this has been supplemented by the data of two other groups [3, 4] in the following few years but to date none of these have been successful in deriving the necessary embryonic stem cells (ESC) from such embryos. So what were we to do?…”