Trophoblast stem cells derived from nuclear transfer embryos: phenotypically unique, bad neighbors, or poor communicators?

N uclear transfer is an effective reprogramming strategy that can redirect the utilization of nuclear instructions and ultimately phenotypes (1). When oocytes are used as the host, a somatic cell nucleus can be reprogrammed to activate the earliest stages of embryonic development (2, 3). Nuclear transfer embryos can develop until the stage when they are ready to implant into the uterus or shortly thereafter and then most degenerate. Very few nuclear transfer embryos successfully progress to term. Those that do mature are characterized by abnormalities in placentation, which in the mouse is placentomegaly (ref. 4 and Fig. 1). Recent reports, including one appearing in this issue of PNAS (5), investigate the etiology of this placentation defect in the mouse and arrive at somewhat different conclusions (5, 6). In the mouse, a population of stem cells can be isolated from embryonic day 3.5 blastocysts, or somewhat later from a structure referred to as extraembryonic ectoderm, and expanded in vitro (7). These cells are called trophoblast stem (TS) cells. They exhibit the capacity to self-renew and differentiate into each of the trophoblast cell lineages existing in the placenta (7). The latter point is most convincingly demonstrated by the reintroduction of marked TS cells into a blastocyst and observation of the developmental capabilities of these cells after blastocyst transfer to a pseudopregnant mouse. In this issue of PNAS, Oda et al. (5) investigated this important cell population in embryos generated by either nuclear transfer or fertilization. TS cell lines were readily established from both types of embryos and shown to possess similar capacities to differentiate (Fig. 1). Gene expression profiles and epigenetic marks, such as DNA methylation, did not differ between the nuclear transfer TS (ntTS) cells and TS cells derived from embryos generated by fertilization. Comparison of the ntTS and TS cell lines was extensive and led to the conclusion that placentomegaly associated with nuclear transfer was not caused by defects intrinsic to the trophoblast lineage. A recent report from Rielland et al. (6) requires some reconsideration of this conclusion. Those scientists also investigated the derivation of TS cells from embryos produced by nuclear transfer and fertilization, but in contrast to Oda et al. (5) they show that ntTS cell lines were easier to establish and depended less on exogenous growth factors than were TS cell lines arising from fertilized embryos (6). Such observations are consistent with earlier tetraploid complementation experiments suggesting that ntTS cells have an in vivo growth advantage over TS cells derived from fertilized embryos (8, 9). Rielland et al. proposed that placentomegaly associated with nuclear transfer embryos could be caused by a number of factors, including the unique growth characteristics of ntTS cells. How do we reconcile these apparently disparate findings and how do they impact our understanding of nuclear transfer-associated placentomegaly? Experimental protocols for the reports by Oda et al. (5) and Rielland et al. (6) were not identical. Differences existed in the donor nuclei [cumulus cell versus embryonic stem (ES) cell] and the genetic background of the mice used in the nuclear transfer experiments. Such procedural variations may influence efficiencies in TS cell derivation but would not appear to be factors in the development of placentomegaly with nuclear transfer embryos. Insights into the etiology of nuclear transfer-associated placentomegaly have also been derived from tetraploid recombination experiments (8, 9). These experiments are based on (i) the ability to produce one-cell tetraploid embryos from two-cell embryos by electrofusion, (ii) the competence of embryos generated by aggregating tetraploid and euploid embryonic cells to generate live offspring, and (iii) the developmental restriction of tetraploid embryonic cells within the newly formed chimeric embryos to extraembry-