We have demonstrated the ability to isolate, in high yield, pluripotent cell lines from monkey parthenotes. These new PESC lines appear similar to existing ESC lines derived from biparental sperm-fertilized embryos with respect to expression of common pluripotency markers and possession of other attributes of primate ESCs, including self-renewal and the capacity to generate cell derivatives representative of all three germ layers in vivo and in vitro [1
]. We found that monkey parthenotes can be produced and cultured to the blastocyst-like stage and used as a source of pluripotent cell lines as efficiently as embryos produced by natural conception or in vitro fertilization. Moreover, the establishment of three separate PESC lines from a single ovarian stimulation cycle in a single animal attests to the feasibility of deriving PESCs as a source of pluripotent cells for the treatment of degenerative diseases in a large population of adult females. Indeed, a recent study demonstrated that human parthenogenetic ESCs can also be generated with high efficiency, where six PESC lines were derived from 44 oocytes donated by four patients [23
Since disruption or inappropriate expression of imprinted genes is associated with severe clinical syndromes and carcinogenesis typically manifested during gestation or early childhood [9
], the potential utility of PESCs will be dependent, in part, on the degree to which epigenetic abnormalities influence differentiation or cellular function in vivo following engraftment. However, although aberrant growth phenotypes have been seen during embryonic and fetal development in chimeras generated with mouse PESCs [20
], this observation may be irrelevant to the normal functional requirements of imprinted genes in differentiated derivatives of ESCs or PESCs in the context of therapeutic engraftment, where these genes may not be expressed or required. Thus, a case can be made that relaxed imprinting need not be detrimental. Indeed, monoparental, parthenogenetic, and androgenetic ESCs in the mouse have been differentiated into transplantable hematopoietic progenitors that were capable of long-term multilineage reconstitution when engrafted into lethally irradiated adult mice [28
]. Moreover, in some cases, postnatal monoparental chimeras were able to alleviate imprinting-related defects [29
Epigenetic instability is seen in the mouse [30
], although human ESCs appear relatively stable, at least at low passage numbers [32
]. In monkey ESCs, we reported normal imprinting in NDN
but relaxed imprinting for IGF2
]. Here, we found that rPESC lines are variable, with the unexpected expression of some imprinted genes that are normally silenced by passage through the maternal germ line. The degree to which abnormal imprinting affects the therapeutic use of ESCs/PESCs must await the outcome of transplantation studies.
The high levels of genomic homozygosity anticipated in diploid, monoparental PESCs represents another safety issue in the context of therapeutic engraftment, since widespread loss of heterozygosity is the most common genetic alteration in human cancers [33
]. However, based on our results here in the rhesus monkey and recent reports in mouse and human PESCs [21
], the majority of loci in PESCs are heterozygous, having undergone meiotic recombination prior to derivation.
Immunological responses to tissue and organ transplants will vary depending on the extent of MHC antigen matching. Our analysis in the monkey provided clear evidence indicating that tight linkage of maternal MHC haplotypes typically leads to either complete homozygosity or heterozygosity of this region in PESCs, depending on whether or not this region was subject to meiotic recombination in the particular oocyte that gave rise to each PESC line. Heterozygous lines that carry haplotypes identical to the egg donors should support autologous transplantation of PESC derivatives with no or limited rejection. Conversely, homozygous PESC lines could also be used for autologous transplantation; however, the presence of only one of the two egg donor MHC haplotypes could incite rejection by natural killer cells [21
]. Therefore, the creation of PESC lines heterozygous at MHC loci is a more attractive outcome for generating fully histocompatible cells. It is also important to note that minor histocompatibility antigens that scattered across the genome could also provoke an immune reaction. Hence, the latest evidence demonstrating that creation of fully matched, genetically identical primate ESCs can be achieved using somatic cell nuclear transfer or direct reprogramming represent major advances in efforts to generate patient-specific cells and tissues for therapeutic purposes [3
Meiotic recombination is an important evolutionary force that plays a vital role in shaping genomes and creating diversity. Recent studies suggest that meiotic recombination events tend to happen in certain regions of the genome, leading to the concept of recombination hot spots [36
]. Direct analysis of fine-scale recombination events is not feasible by pedigree analysis. The only information on recombination in primates comes from single-molecule PCR analysis of sperm. However, recombination frequencies are also sex-dependent. Crossing over is estimated to occur approximately 55 times during meiosis in males and approximately 75 times in females [36
]. We present here a novel system that allows detailed analysis of recombination in the primate female. Each PESC line represents the recombination events that occurred within a single oocyte. Thus, primate PESCs, in combination with powerful genetic and cytological assays, could be valuable in vitro models with which to study increasingly complex aspects of meiosis, chromosome behavior, and recombination. It is generally accepted that exchange during meiotic recombination occurs more frequently at distal telomeric regions of chromosomes, with the frequency of recombination decreasing near the centromeric region. In murine PESC lines, evidence has been presented that the recombination rate is directly proportional to the distance from the centromere [21
]. However, the location of the chromosomal centromeres is not currently known in the rhesus monkey, precluding analysis of recombination frequencies in this study.