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“Three is the magic number”: Human oocytes reprogram somatic cells to a pluripotent state

From Nature
By Stuart P. Atkinson

When induced pluripotent stem cell (iPSC) technology broke onto the scene in 2007, the attainment of a practical and ethical source of patient specific pluripotent stem cells seemed to be within our grasp. The use of somatic cell nuclear transfer (SCNT) for the derivation of human embryonic stem cells (hESCs) was all but relegated to the past, along with its ethical and technical difficulties. Further, as noted in an editorial in Nature, previous fraudulent claims in this field had added a degree of unwarranted scepticism to research undertaken in this field. However, iPSC derivation and its detailed analysis has shown that iPSC may have only a limited use in a therapeutic context due to differences between blastocyst-derived stem cells at the level of gene expression, epigenetic patterning, differentiation potential and genomic integrity. It is suggested that these differences may lead to dysfunctionality and/or tumorigenicity and have led some groups to return to SCNT-mediated techniques. The group of Dieter Egli from the The New York Stem Cell Foundation Laboratory, New York initiated a rigorous study designed to understand the obstacles to blastocyst development in SCNT in order that we can use this process to generate bona fide patient-specific hESCs. In their paper, published in Nature (Noggle et al), the group found that transfer of the somatic genome into an enucleated oocyte failed to develop and this was associated with an apparent arrest of developmentally-associated gene expression. They found however, that if the oocyte genome is left in place upon somatic genome transfer, the embryo can develop to the blastocyst stage and hESCs can be derived.

Oocytes enucleated via removal of the spindle-chromosome complex were used for the transfer of somatic cell genomes from the skin cells of a male diabetic (T1D) or a male adult labelled with a GFP or a H2B:GFP transgene under the control of the ubiquitously expressed CAGGS promoter. In all oocytes, the somatic genome became condensed and 71% developed past the cleavage stage following activation. However, as previously observed (Egli et al), the embryos arrested at the 6-10 cell stage. As a control, somatic nuclei were transferred without removal of the oocyte genome, so that 6-8 hours after artificial activation and upon formation of two interphase nuclei within a single cell, either genome could be specifically extracted. Activated oocytes containing only the somatic genome formed 4–12 cells, but all arrested without reactivating the GFP transgene, while 57% of the activated oocytes containing only the oocyte genome developed to the blastocyst stage allowing the generation of pluripotent parthenogenetic stem cells.

Extensive transcriptional activity from the zygotic genome normally starts at the 4–8 cell stage (Braude et al) or on day 3 of development, coinciding with the stage of developmental arrest after somatic genome transfer. Transcriptomic analysis found that cells from the 6–12 cell stage after genome exchange most closely resembled fertilized eggs from the 1-cell to the 6–8-cell stage in media containing the RNA polymerase II inhibitor alpha amanitin, a state of transcriptional activity inhibition. Of the genes known to be upregulated upon zygotic genome activation, only 16% were upregulated following genome transfer and 8% following amanitin treatment, while 70% were more than fivefold upregulated in parthenogenetic stem cells. Further, it was shown that developmental arrest was not associated with the continued expression of somatic cell genes. As the developmental defects observed could be caused by the removal of vital factors during oocyte genome removal, the authors transferred a somatic cell genome but did not remove the oocyte genome. Excitingly, this led to the continuation of development of the embryo to the compacted morula stage and efficient development to the blastocyst stage (21%) indicating that the somatic cell genome did not interfere with development to this stage.

This then allowed for the isolation of the inner cell mass and the derivation of two lines; soPS1 (somatic cell genome, oocyte genome pluripotent stem cell) from a type 1 diabetic male and soPS2 from a healthy male subject. Karyotypic analysis found the cells to be triploid, consistent with the presence of a diploid somatic cell genome and the haploid genome of the oocyte. soPS1 was observed to harbour an additional chromosome 17 and a chromosome 15 and 17 translocation, and upon passaging soPS1 also gained chromosome 12 and 17 (30% at p23), common aberrations observed in cultured ESCs (Draper et al), while soPS2 was karyotypically stable over more than 20 passages. Mitochondrial genomes were of oocyte donor origin without any sign of heteroplasmy in either cell line. Appropriate in vivo and in vitro differentiation was observed for both lines, while transcriptomic analysis showed their similarity to other pluripotent cell types including NYSCF1, a stem cell line derived from an IVF blastocyst, the parthenogenetic stem cell line pPS1, and iPSC lines derived from both skin cell donors. Of 1,327 genes found to be differentially expressed between the soPS2 and the donor fibroblast, 463 were present at fivefold higher levels in the stem cells than in the fibroblasts (including LIN28A, POU5F1, SOX2, NANOG and LEFTY1) and 670 transcripts were decreased by a factor of five or more in soPS2 (including many fibroblast specific genes; FAP, PAPPA, MMP3, CTHRC1 and SNAI2). soPS2 and soPS1 were very similar to NYSCF1 with only 28 genes and 24 genes expressed at higher levels, respectively. These included GAL, NNAT, SRY, ZFP42, NODAL, CER1 and LEFTY2 perhaps reflecting spontaneous differentiation rather than incomplete reprogramming of the somatic cell genome.

Consistent with reprogramming of the somatic cell genome to a pluripotent state, methylation of DNA at the Nanog promoter was low in soPS cells and high in the somatic donor cells with no demethylation occurring at the imprinted PEG3 locus, suggesting specific demethylation. Further detailed analysis through genome-wide digital allelotyping, to distinguish gene expression from the somatic cell-derived genome and the oocyte-derived genome in soPS cells, demonstrated that reprogramming had occurred at other loci and that there was no bias of oocyte mediated gene expression vs. somatic nucleus mediated expression.

This is a spectacular finding which may allow us to produce bona fide patient-specific hESCs in the future, allowing us to bypass many of the problems associated with iPSC generation. Complications may arise from the additional 23 chromosomes present and the further descriptions of the behaviour of these cells and detailed comparisons with traditionally derived hESCs and hiPSCs will need to be undertaken. However, even if it is proved that these cells cannot ultimately be used in a therapeutic context, they will allow further research into the genetic elements from the maternal genome which are required for reprogramming of the somatic genome. Also, as widely noted in the press, ethical and legal concerns will also be raised as this type of cell derivation requires the use of donated human oocytes and ultimately the destruction of embryos. Such questions have already begun to be addressed, such as in the recent edition of Cell Stem Cell regarding oocyte donation compensation payments and how we can continue to move forward in fields like this whilst in keeping with ethical concerns.

 

References

Braude P, Bolton V, Moore S.
Human gene expression first occurs between the four- and eight-cell stages of preimplantation development.
Nature. 1988 Mar 31;332(6163):459-61.

Draper JS, Smith K, Gokhale P, et al.
Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells.
Nat Biotechnol. 2004 Jan;22(1):53-4.

Egli D, Chen AE, Saphier G, et al.
Reprogramming within hours following nuclear transfer into mouse but not human zygotes.
Nat Commun. 2011 Oct 4;2:488.

Noggle S, Fung H, Gore A, et al.
Human oocytes reprogram somatic cells to a pluripotent state
Nature 478, 70–75 (06 October 2011).

 

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Human somatic cell nuclear transfer: Three genomes is the charm

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