Here, we report our attempts to overcome the logistical and scientific obstacles impeding the production of patient-specific stem cell lines by nuclear transfer. While it has been difficult to recruit “altruistic” oocyte donors24
we did succeed in sourcing more than 400 normally fertilized eggs (zygotes) for our nuclear transfer studies.
Development after nuclear transfer was normal through the cleavage stages, but, unlike IVF controls, all cleaving nuclear transfer cells arrested before or at the time of compaction with severe transcriptional abnormalities. An identical phenotype could be induced by inhibition of transcription in IVF controls, but not by the deliberate induction of karyotypic abnormalities, suggesting that transcriptional defects are more proximally responsible for the developmental arrest.
Our findings are not the trivial result of a small sample size. Instead our results of more than 160 nuclear transfer experiments and more than 200 control manipulations indicate that there is a robust species-specific blockade to reprogramming that must be overcome before human stem cell lines can be derived. Our work and those of others suggests that the developmental arrest we observed is not simply the result of using zygotes for nuclear transfer as a temporally similar arrest is commonly observed after nuclear transfer into to human unfertilized oocytes18,20,25
. Although a single group has reported efficient transcriptional reprogramming after human nuclear transfer20
, they compared somatic cells to nuclear transfer samples and their results are therefore confounded by the presence of maternal mRNAs, which were not appropriately accounted for by their analyses. Another group has generated a single blastocyst after transfer of embryonic stem cell nuclei into human oocytes17
, but development arrested when fibroblasts were transferred using identical methods18
. This suggests that development and activation of the transferred genome depend on the epigenetic state of the injected nucleus.
In contrast to human zygotes, when we performed nuclear transfer into mouse zygotes, we found that reprogramming was essentially complete within hours and indeed within a single cell cycle. This result also points to a fundamental difference between reprogramming after nuclear transfer and iPS reprogramming: at least in animals, nuclear transfer mediates an immediate transition from a somatic to a pluripotent gene expression pattern, while reprogramming by defined factors seems to be a gradual process, requiring days or weeks37,38
. It is interesting to consider that this could explain why stem cells generated by nuclear transfer are indistinguishable44
from stem cells derived from fertilized blastocysts, while in mouse16,45
iPS cell reprogramming may at times be less complete.
Incomplete reprogramming and developmental defects after somatic cell nuclear transfer have been described in other vertebrate species36,49-54
. For example, in bovine nuclear transfer embryos, 3.8% of genes were found to be incompletely reprogrammed53
. This is comparable to the results reported here with mouse zygotes, where the majority of transcripts (>91%) are normally expressed within 24 hours after nuclear transfer. In contrast, the transcriptional defects after nuclear transfer into human zygotes, extend far beyond incomplete reprogramming of a few genes: the majority of transcripts, or more than 88%, were not normally expressed. Surprisingly, even genes that were active in the somatic donor cell were not normally expressed, suggesting a failure to properly activate the transferred somatic cell genome.
The species-specific differences in transcription after nuclear transfer might be due to a property of the human egg, or of the human somatic cells. Our results suggest that investigating the requirements for transcriptional activation of the donor cell genome may help to overcome the developmental arrest commonly observed after human nuclear transfer.