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Production of Mouse Embryonic Stem Cell Lines from Maturing Oocytes by Direct Conversion of Meiosis into Mitosis

From the March 2011 Issue of Stem Cells

Paper Commentary by Stuart P. Atkinson

Induced pluripotent stem cells (iPSC) have the potential to provide a patient specific source of cells that can be used in cellular therapy. However doubts about their similarity to embryonic stem cells (ESCs) have arisen, both at the gene expression and epigenetic level, and also with their initial differentiation capabilities and the capacity of the differentiated cells to function properly. So what other potential sources do we have? Currently derived human ESC lines (hESC) may not have the genetic diversity required, whilst derivation of additional hESC lines has associated ethical (and funding) problems. Meanwhile, hESC-derivation from somatic cell nuclear transfer (SCNT) has yet to yield results and does not look likely to in the short term. For many, iPSCs still represent the future of stem cell research and stem cell derivation. However, another source of pluripotent cells which are often overlooked may be available, in the form of parthenogenetic stem cells. Parthenogenetic ESCs are derived from activated oocytes at the metaphase II stage and could provide a patient specific source of ESCs for the donor female, and perhaps relatives of the said donor (Hikichi et al. and Kim et al.). However donated oocytes at this stage of development are generally used in assisted reproduction and are therefore of scarce availability, a problem also associated with SCNT. However, Josef Fulka Jr’s group at the Institute of Animal Science, Pratelstvi, Prague have now demonstrated a new method of creating parthenogenetic ESCs from metaphase I oocytes, which are often discarded during the course of IVF, by using Butyrolactone I (BL1). This study (Fulka et al.) is presented in the March Edition of Stem Cells.

BL1 is a Cdk1 (Cdc2) and Cdk2 inhibitor and inhibits the meiotic maturation of oocytes. Treatment of metaphase I mouse oocytes with BL1 leads to the expulsion of the first polar body and the lack of formation of the typical second metaphase plate followed by chromosome decondensation and pseudopronuclei formation. Replication of the pseudopronuclei was observed at 16-17 hours after treatment, although 8 hours of treatment with BL1 was deemed to be sufficient. These oocytes then cleaved and formed 2-cell stage embryos after the removal of BL1, with the first cleavage metaphase exhibiting perfect spindles and appropriately arranged mitotic chromosomes. 20% to 30% of these cells reached blastocyst stage at 4 days, with inner cell mass (ICM) cells positive for Oct4 and Nanog and trophoblast cells positive for Cdx2. Conversion of the cells of the ICM to ESC was established with a 70-80% success rate and the resultant ESCs (BL1-ESCs) were stable in culture. Fourteen of the sixteen ESC lines established were karotypically normal over long term culture, suggesting that BL1 does not induce chromosome segregation abnormalities. In the remaining 2 abnormal cell lines, one had a fusion of two chromosome 19´s while the other demonstrated trisomy of chromosome 8, a frequently observed abnormality in ESC lines (Liu et al.). This demonstrates that mouse parthenogenetic ESC can be derived by the inhibition of meiosis and the conversion to mitosis following BL1 treatment. But to what extent are these BL1-ESCs similar to “normally” derived ESCs?

Further studies demonstrated that imprinting, that is, the specific methylation of parent-of-origin gene alleles, was essentially the same as observed for metaphase I oocytes and long term study of one derived BL1-ESC line demonstrated this to be stable with extended passaging. Previous studies of parthenogenetic cells have suggested that epigenetic instability is commonplace and may affect cell function (Hori et al. and Li et al.). Gene expression analysis showed that the BL1-ESC lines where highly similar to control mouse ESC lines that had been derived from fertilised oocytes. Genes which were significantly altered were linked to trophectodermal or haematopoietic lineages, but those genes that were altered were cell specific amongst the different BL1-ESC lines studied and indicates that these changes were not due to a single overarching mechanism and probably arose due to stochastic events.

One noted potential problem with parthenogenetic ESCs is a deficit in differentiation capability. However, when embryoid bodies were formed from the BL1-ESCs, appropriate differentiation towards all three germ layers was observed, while teratoma analysis showed a similar differentiation capability. Several high-percentage chimeric mice were also obtained following the injection of BL1-ESCs into blastocysts but, importantly, no germ line transmission was observed.

The next part of the study focused on the basic cell biology of BL1 treatment and the exit from metaphase 1 directly into mitosis. Deeper analysis showed chromosome decondensation and possible premature sister chromatid separation after BL1 treatment, similar to what has been previously observed to occur for chromosomes in metaphase II. Chromosome segregation during anaphase I however, was normal while levels of phosphorylated serine 10 of histone H3 (pS10 H3, associated with chromosome condensation) was observed to be similar between BL1 treated oocytes and controls. The authors went on to show that BL1 treatment induces the localisation of DNA replication licensing factors Mcm7 and Cdc6 to the chromosomes soon after metaphase I exit, two proteins vital for chromosome replication. In control oocytes, Cdc6 is not observed in chromosomes before meiosis II. Therefore, BL1 treatment induces the ability of the chromosomes to replicate, a state which does not normally occur during meiosis I.

Overall, this study demonstrates that mouse ESC lines can be faithfully generated from oocytes at metaphase I following treatment with Butyrolactone I which acts by converting meiosis into mitosis. Further, this research highlights a potential new source of hESCs from a source (metaphase I oocytes) normally discarded during IVF and even though these cells show a lack of germ line transmission, they may be useful if they can be shown to differentiate towards clinically relevant functional cell types.



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