The use of parthenotegenetic and IVF bovine blastocysts as a model for the creation of human embryonic stem cells under defined conditions

Abstract

Purposes Clinical application of human embryonic stem cells will be possible, when cell lines are created under xeno-free and defined conditions. We aimed to establish methodologies for parthenogenetic activation, culture to blastocyst and mechanical isolation of the inner cell mass (ICM) using bovine oocytes, as a model for derivation and proliferation of human embryonic stem cells under defined xeno-free culture conditions. Methods Cumulus-oocyte-complexes were in vitro matured and activated using Ca 2+ Ionophore and 6-DMAP or in vitro fertilized (IVF). Parthenotes and biparental embryos were cultured to blastocysts, when their ICM was mechanically isolated and placed onto a substrate of fibronectin in StemPro® medium. After attachment, primary colonies were left to proliferate and stained for pluripotency markers, alkaline phosphatase and Oct-4. Results Parthenogenesis and fertilization presented significantly different success rates (91 and 79 %, respectively) and blastocyst formation (40 and 43 %, respectively). ICMs from parthenogenetic and IVF embryos formed primary and expanded colonies at similar rates (39 % and 33 %, respectively). Six out of eight parthenogenetic colonies tested positive for alkaline phosphatase. Three colonies were analyzed for Oct-4 and they all tested positive for this pluripotency marker. Conclusion Our data show that Ca 2+ Ionophore, and 6-DMAP are efficient in creating large numbers of blastocysts to be employed as a model for human oocyte activation and embryo development. After mechanical isolation, parthenogetic derived ICMs showed a good rate of derivation in fibronectin and Stem-Pro forming primary and expanded colonies of putative embryonic stem cells. This methodology may be a good strategy for parthenogenetic activation of discarded human oocytes and derivation in defined conditions for future therapeutic interventions.

BAPTA‐AM dramatically improves maturation and development of bovine oocytes from grade‐3 cumulus‐oocyte complexes

“…Oocytes from each of the groups were submitted to parthenogenetic activation 24 hr after their maturation, using previously published methods (Felmer & Arias, ; Ruggeri et al, ), with slight modifications. Oocytes with extruded first polar bodies were activated for 5 min by 5 mM ionomycin (Calbiochem, San Diego, CA), followed by culture for 4 hr in the dark at 39°C in 5% CO 2 and humidified air in CR1aa medium containing 2 mM 6‐dimethylaminopurine (DMAP) and 1% BSA.…”

Reactive oxygen species production and redox state in parthenogenetic and sperm-mediated bovine oocyte activation

“…Chemical activation presented significantly lower success rates and blastocyst formation compared with IVF (Ruggeri et al 2012), demonstrating that the process of oocyte activation is a major factor for successful production of reconstructed embryos by somatic cell nuclear transfer (Wells et al 1999). ROS generation has been implicated as a major cause of poor development of bovine embryos in vitro.…”

Substrates and supplements for hESCs: a critical review

“…For inner cell mass (ICM) isolation from the trophectoderm, immunosurgery using non-human antibodies and also nonhuman complement is widely used [55]. One feasible alternative to avoid the use of xeno-components is to manipulate the embryos with a pair of flexible metal [67] or insuline needles [58] to open the zona pellucida and isolate the ICM as much as possible from the trophectodermal cells. However, manual dissection demands good expertise and manipulation skills.…”

“…Recently, vitronectin-coated surfaces were successfully employed to derive hESC and iPSC cells [16]. As another possibility, fibronectin alone was used as substrate for derivation of bovine partenogeneticaly generated ESC using a commercially available defined medium (Stem Pro® ) [58]. Alternatively, a culture system was created that supports derivation of the hESC lines as floating clusters in suspension [63].…”