In the mid-1990’s, Dr. James Thomson of the Wisconsin National Primate Research Center reported the isolation of the first embryonic stem cell (ESC) lines from rhesus macaque blastocysts (Thomson et al., 1995
). These primate ESCs were maintained in an undifferentiated state while cultured on feeder layers but retained the potential to differentiate into cells representing all three germ cell layers (endoderm, mesoderm, and ectoderm) (Thomson et al., 1995
; Thomson et al., 1996
). Prior to the establishment of rhesus ESCs, only mouse ESCs were available (Martin, 1981
). Primate ESCs provided an exciting model for better understanding human development and disease in vitro
and in vivo
. As one would expect, following protocols and markers developed for monkey ESCs, studies were soon carried out with human embryos that culminated in the successful derivation of human ESCs (Thomson et al., 1998
Coupling of ESC research with SCNT technology also catalyzed the development of new strategies for reprogramming somatic cells and deriving patient-matched pluripotent cells (Byrne et al., 2007
). The ability to derive pluripotent cells that are genetically identical to individual patients, holds tremendous potential to produce cells that will not be rejected by the patient’s immune system and that may help prevent numerous degenerative diseases (Drukker and Benvenisty, 2004
; McKay, 2000
). We employed a modified SCNT approach to produce blastocysts from adult rhesus macaque skin fibroblasts and established two ESC lines from these cloned embryos, designated as CRES-1 and CRES-2 (Byrne et al., 2007
). These CRES cell lines are genetically identical to each other in terms of nuclear DNA, since nuclear donor cells employed were from the same adult male. However, these cell lines contain different mitochondrial genomes since oocytes from two different females were used for SCNT. Characterization of these novel SCNT-derived ESC lines confirmed their pluripotency and origin from somatic cells (Byrne et al., 2007
As for the efficiency of this approach, in our initial study we used a total of 304 oocytes to derive two CRES cell lines; less than a 1% derivation efficiency rate. While the low efficiency rates still leave room for improvement, these results suggest that nuclear reprogramming by SCNT can support derivation of ESCs in higher primates, including humans. In a more recent study, we used adult rhesus female skin fibroblasts for SCNT and derived two additional ESC lines (CRES-3 and CRES-4) (Sparman et al., 2009
). Further optimizations and improvements in SCNT resulted in a nearly three-fold higher blastocyst development rate (43%) and ESC derivation rate (29%) as compared to the previous outcomes (Byrne et al., 2007
). Of particular interest, the oocytes that gave rise to CRES-3 and -4 were all recovered from only one rhesus female, subjected to a single controlled ovarian stimulation. These findings reveal that it is now possible to derive a primate stem cell line from as few as 10 oocytes, or less, and that it may be economically and technically feasible to derive patient-matched ESCs for tissue replacement therapy.
The nonhuman primate is clearly an attractive research model for SCNT because of its remarkable similarity to humans, genetically and from a reproductive standpoint. The benefits of producing rhesus monkeys by reproductive cloning for biomedical research are tremendous. For instance, the production of genetically identical monkeys would significantly reduce the number of animals utilized in biomedical research. Another advantage is that we can carry out a variety of genetic manipulations with cultured nuclear donor cells, including gene targeting. Reproductive cloning with such cells would allow for the production of genetically modified primates, including gene knock-out models, to study gene function and human diseases.
At present, the production of live primate offspring following SCNT has yet to be accomplished (Mitalipov et al., 2002
; Simerly et al., 2003
). We summarize here our recent unpublished efforts in embryo transfer using rhesus blastocysts produced by SCNT with adult monkey skin cells expressing GFP (). A total of 5 pregnancies were established following transfer of 67 embryos into 10 recipients ( and ). Only one pregnancy resulted in a live fetus that possessed a fetal heartbeat, detected by ultrasonographic scans, while other pregnancies contained sacs without a fetus (). Unfortunately, this pregnancy failed to go to term and was aborted at day 81 of gestation. To determine if there was an abnormal phenotype in the aborted cloned pregnancy, we recovered fetal tissues and carried out histological and molecular analyses. The SCNT origin of the fetus was confirmed by both GFP-specific PCR and direct observation of GFP expression (). Macroscopically, results demonstrated poor placental development with fewer chorionic villi compared to controls (). Microscopically, the fetal tissue also lacked ectodermal components (). These observations suggest possible insufficient development of the placental component that ultimately impaired the development of the resulting fetus.
Ultrasound images of the SCNT pregnancy
Analysis of aborted SCNT fetal tissues