In this study, we demonstrate derivation of two primate ES cell lines from somatic cells of an adult female, reproducing our previous report that primate somatic cells can be reprogrammed to the pluripotent state by SCNT. Our results also suggest that SCNT with particular donor somatic cells can be highly efficient, yielding a near threefold increase in the blastocyst formation rate compared with other cells. Blastocyst development of 12.5% observed with the nuclear donor 1, a 9-year-old adult rhesus macaque male, was similar to our previously reported 16% efficiency with the same donor cells [5
]. Culture conditions for nuclear donor skin fibroblasts derived from all three monkeys and SCNT protocols in this study were identical. Moreover, cytogenetic analysis confirmed that all three cell lines possess normal rhesus monkey karyotypes (results not shown). Thus, it is not clear why blastocyst development rates differ dramatically.
A variety of factors in nuclear donor cells have been implicated in reprogramming efficiency after SCNT, including cell type and size, population doubling time, cell cycle, and differentiation status (see for review [34
]). In addition, random genetic and epigenetic changes accumulated in somatic cells in vivo or in vitro also may compromise the developmental potential of donor nuclei [35
]. In the mouse, reprogramming efficiency was inversely correlated with the donor cell differentiation status. For example, cloning efficiency with ES cell nuclei was up to 30 times higher than with commonly used somatic cells, such as cumulus cells and tail tip fibroblasts [36
]. Another potentially confounding influence is donor cell type. Mouse cloning with cumulus cells is usually significantly higher than with tail-tip fibroblasts [37
]. More direct comparisons demonstrated that blastocyst formation rates vary dramatically even if nuclear donor cells were derived from the same tissue and exposed to the same culture conditions [35
We demonstrate here improved ES derivation rates from SCNT embryos associated with plating intact blastocysts as opposed to isolated ICMs onto feeder layers. Previously, the majority of blastocysts was dissected for ICM isolation and only 12% (2/17) of monkey SCNT embryos resulted in ES cell lines while a limited number of intact SCNT blastocysts (n
= 3) did not produce any outgrowth [5
]. In revisiting this issue here, we hypothesized that exposure of SCNT blastocyst to the antibody followed by treatment with complement to selectively destroy TE cells may also affect ICM cell viability. This would be consistent with the poor TE development in SCNT blastocysts compared with IVF-derived counterparts allowing antibody penetration through the TE layer into the blastocoel cavity with access to the ICM. Based on our current SCNT blastocyst formation rate of 43% and ES cell isolation efficiency of 29%, as few as 10 or less primate oocytes could be sufficient to derive one cell line. Thus, the continued systematic optimization of SCNT approaches will likely succeed in the efficient generation of patient-specific ES cells for therapeutic applications.
We further characterized novel SCNT-derived ES cell lines that confirmed their somatic origin and pluripotency. Previously, cytogenetic analysis revealed that one of the two derived ES cell lines, CRES-1, was aneuploid [5
], thus the extent of chromosomal abnormalities in primate SCNT-derived embryos and ES cells was unclear. However, both CRES-3 and -4 exhibited a normal female rhesus macaque chromosomal (42, XX) complement suggesting that cytogenetic aberrations are an unusual attribute of primate SCNT-derived ES cells.
In addition to routine pluripotency tests, we provide evidence that primate ES cells derived by SCNT are capable of differentiating into cell types expressing markers of germ cells. In vitro formation of primordial germ cells and early male and female gametes has been documented from mouse ES cells [16
] and spontaneous or induced differentiation of hESCs can also produce cultures with germ cell-specific gene expression in an appropriate temporal sequence [20
]. However, functional gametes have not yet been produced suggesting that additional studies on appropriate culture conditions for gamete formation are required. Nevertheless, these advances suggest that a patient's somatic cells can be reprogrammed to ES cells, which subsequently can be differentiated into oocytes or sperm. Thus, infertile patients might be able to have children that are genetically their own. Additionally, hESC-derived eggs could be employed in the generation of SCNT-derived ES cells, thereby reducing the ethically problematic demand for donated human eggs.
Although it seems clear that primate SCNT-derived pluripotent cells are identical to ES cells derived from fertilized embryos in terms of transcriptional activity and potential to give rise to diverse cell types, a central question remains; are these cells epigenetically equivalent to ES cells? We found that expression levels of imprinted genes in CRES cell lines were remarkably similar to controls. Methylation analysis of IGF2/H19
ICs demonstrated the presence of both methylated and unmethylated alleles in CRES cell lines, reflecting maintenance of normal differentially methylated patterns. In addition, detailed allele-specific expression based on the sequence polymorphisms demonstrated that both NDN
were expressed monoallelically in CRES cells. Similar to their IVF-derived counterparts, CRES cell lines demonstrated relaxed biallelic expression of IGF2
. This is in agreement with our previous findings that monkey ES cells maintain normal imprinting in NDN
but relaxed imprinting in IGF2
]. Expression of H19
was variable with monoallelic expression in some ORMES cell lines while other analyzed lines expressed both alleles. We also previously showed that monkey IVF produced blastocysts exhibit normal paternal expression of IGF2
suggesting that abnormal biallelic expression of this gene in ES cells is likely acquired during isolation and culture [11
High levels of TERT
expression and significant elongation of telomere length in CRES cells relative to nuclear donor fibroblasts indicated efficient reprogramming of proliferative potential to an early embryonic state. Our study also demonstrated that undifferentiated female CRES cells, similar to their IVF-derived counterparts underwent X-inactivation. X chromosome inactivation is random in the embryonic lineage but in the extra-embryonic lineage the paternal chromosome is preferentially silenced. Thus, reprogramming of female nuclear donor cells could be less efficient due to faulty reactivation and subsequent nonrandom inactivation of the paternal X chromosome that is active in 50% of the somatic cells. Indeed, random X-inactivation observed in the placenta of aborted cloned cattle fetuses suggest that at least some abnormalities in cloning offspring are due to aberrant recapitulation of X chromosome reactivation [42