Assessment of parthenogenetic activation of human metaphase II oocytes for stem cell derivation

Abstract

Objective: Human stem cells derived from embryos provide potential cell based therapies for repair of degenerating or damaged tissue. However, there are many ethical concerns surrounding the use of human embryos for stem cell research. Parthenogenetic activation has been studied extensively in invertebrates, amphibians, and mice. Activation occurred in these models following an increased intracellular calcium transient. Inhibitors of protein kinases have also been used sequentially with calcium ionophores to suppress the second meiotic reductional division allowing production of diploid parthenones. The aim of this study was to assess parthenogenetic activation and blastocyst development of human donor oocytes using calcium ionophore and protein kinase inhibitors. Design: Prospective, experimental study utilizing donor oocytes. Materials/Methods: Consented oocyte donors underwent follicular stimulation with rFSH following treatment with either a GnRH agonist or antagonist. Oocyte retrieval occurred 36 hours after HCG (10,000 IU) injection and the cumulus cells were removed with hyaluronidase(80 IU/ml) one hour later. Metaphase II oocytes were treated with 5 uM calcium ionophore A23187 for 5 minutes at 33 degrees Celcius followed by a 3 hour incubation in 1 mM 6 dimethyl -aminopurine (6-DMAP) at 37 degrees. Activated oocytes with one pronucleus were cultured in IVC-1 media for 72 hours and were then moved to Gen-X Blastocyst media for 48 hours. Blastocysts which displayed a large blastocoel cavity with the clear presence of an ICM were hatched with acidified tyrodes and treated with anti-human trophoectoderm antibody followed by guinea pig complement. Treated cells were moved to 4-well dishes which were previously coated with mitotically inactivated murine embryonic feeder cells (ATCC) in Dulbeco’s modified Eagle’s medium supplented with 20% FBS. Attachment of the inner cell mass cells was evaluated after 24 hours. Results: Seventy-six oocytes were retrieved from five donors. 89% (68/88) were mature, metaphase II oocytes. 59% (40/68) activated following treatment with calcium ionophore and DMAP. 37% (15/40) of the parthenotes progressed to the blastocyst stage and underwent assisted hatching (see photo). Following immunosurgery, 60% (9/15) of the treated blastocysts attached to the feeder cells. Cell masses remained attatched to the feeder cells for 3–7 days before their growth arrested. Conclusions: Activation of human donor oocytes with calcium ionophore and protein kinase inhibitors is an effective method for production of parthenotes displaying the morphological characteristics of in vitro produced blastocysts. Using current methodologies for culture of embryo derived stem cells, blastocysts which underwent immunosurgery attached to feeder cells and remained attached for up to seven days. Genetic analysis of the the parthenotes to determine ploidy is currently underway. This method, as demonstrated in the monkey and mouse, is a very promising approach for the production of stem cell lines and may help to minimimize the ethical concerns surrounding embryonic stem cell research. Supported by: Stemron Corporation; Gaithersburg, MD.