Reprogramming of Human Somatic Cells Using Human and Animal Oocytes

Abstract

There is renewed interest in using animal oocytes to reprogram human somatic cells. Here we compare the reprogramming of human somatic nuclei using oocytes obtained from animal and human sources. Comparative analysis of gene expression in morula-stage embryos was carried out using single-embryo transcriptome amplification and global gene expression analyses. Genomic DNA fingerprinting and PCR analysis confirmed that the nuclear genome of the cloned embryos originated from the donor somatic cell. Although the human-human, human-bovine, and human-rabbit clones appeared morphologically similar and continued development to the morula stage at approximately the same rate (39, 36, and 36%, respectively), the pattern of reprogramming of the donor genome was dramatically different. In contrast to the interspecies clones, gene expression profiles of the human-human embryos showed that there was extensive reprogramming of the donor nuclei through extensive upregulation, and that the expression pattern was similar in key upregulation in normal control embryos. To account for maternal gene expression, enucleated oocyte transcriptome profiles were subtracted from the corresponding morula-stage embryo profiles. t-Test comparisons (median-normalized data @ fc>4; p<0.005) between human in vitro fertilization (IVF) embryos and human-bovine or human-rabbit interspecies somatic cell transfer (iSCNT) embryos found between 2400 and 2950 genes that were differentially expressed, the majority (60-70%) of which were downregulated, whereas the same comparison between the bovine and rabbit oocyte profiles found no differences at all. In contrast to the iSCNT embryos, expression profiles of human-human clones compared to the age-matched IVF embryos showed that nearly all of the differentially expressed genes were upregulated in the clones. Importantly, the human oocytes significantly upregulated Oct-4, Sox-2, and nanog (22-fold, 6-fold, and 12-fold, respectively), whereas the bovine and rabbit oocytes either showed no difference or a downregulation of these critical pluripotency-associated genes, effectively silencing them. Without appropriate reprogramming, these data call into question the potential use of these discordant animal oocyte sources to generate patient-specific stem cells.

Nuclear–cytoplasmic incompatibility and inefficient development of pig–mouse cytoplasmic hybrid embryos

“…Control SPNT cow and sheep embryos as expected developed to the blastocyst stage; however, none of the cow-sheep or sheep-cow iSPNT embryos developed beyond the 8-to 16-cell stage, again the stage at which EGA occurs in both species. Taken together, our results support the argument that development of inter-species nuclear transfer embryos is highly inefficient with developmental arrest occurring at the stage of EGA specific to recipient cytoplast species (Chung et al 2009, Lagutina et al 2010. These recent reports have clearly demonstrated that the donor nucleus in iSCNT is only partially remodeled by the heterologous cytoplasm, and iSCNT embryos need to overcome nuclear-cytoplasmic compatibility issues before any further embryonic development to the blastocyst or later stages can be expected (Chung et al 2009, Lagutina et al 2010.…”

“…However, all intra-species SCNT embryos produced under the same conditions used for iSCNT developed to blastocyst stage. Our results were not different from several other recent reports (Chung et al 2009, Lagutina et al 2010, who had also reported that iSCNT embryos failed to reactivate the embryonic genome and arrested at the stage of EGA specific to the recipient oocyte. Conversely, inter-genus (Oh et al 2006, Yin et al 2006, inter-family (Dominko et al 1999, Zhao et al 2007, inter-order (Yang et al 2003, Wen et al 2005, Illmensee et al 2006, and even inter-class iSCNT (Chen et al 2002, Liu et al 2004 nuclear transfer embryos were reported to have Pig-mouse cytoplasmic hybrid embryos developed to the blastocyst stage.…”

“…In view of the contrasting reports and extremely poor developmental efficiency, development of iSCNT embryos, other than those from closely related species, to the blastocyst stage remain elusive. Although, in humans, a single study reported isolation of an ES cell line from blastocyst stage embryos produced by iSCNT using rabbit oocytes (Chen et al 2003b, Chung et al 2009), similar rabbit-human iSCNT embryos produced in a recent study (Chung et al 2009) all resulted in early embryonic arrest.…”

“…Following SCNT, the somatic cell nuclei must undergo extensive reprogramming for successful development of cloned to embryos to blastocyst or beyond. However, it was recently demonstrated that the somatic cell nucleus undergoes only partial or incomplete reprogramming in iSCNT embryos (Chung et al 2009). We hypothesized that using a donor genome that requires less extensive reprogramming than somatic cells may improve the frequency of development to the blastocyst stage or alternatively provide an improved model to study nuclear cytoplasmic interactions between different species.…”

The ups and downs of somatic cell nucleus transfer (SCNT) in humans

“…It is difficult to draw firm conclusions from these papers. In general, embryos produced by NT underwent few cleavage divisions and only exceptionally reached the blastocyst stage [21][22][23][24]. It must be noted, however, that cytoplasts were sourced from oocytes that were either immature at the time of egg retrieval and were subsequently in vitro matured to metaphase II stage in culture, or from oocytes deemed unsuitable for IVF.…”