Guo Qing Tong

Aberrant profile of gene expression in cloned mouse embryos derived from donor cumulus nuclei

Somatic cell nuclear transfer has successfully been used to clone several mammalian species including the mouse, albeit with extremely low efficiency. This study investigated gene expression in cloned mouse embryos derived from cumulus cell donor nuclei, in comparison with in vivo fertilized mouse embryos, at progressive developmental stages. Enucleation was carried out by the conventional puncture method rather than by the piezo-actuated technique, whereas nuclear transfer was achieved by direct cumulus nuclear injection. Embryonic development was monitored from chemically induced activation on day 0 until the blastocyst stage on day 4. Poor developmental competence of cloned embryos was observed, which was confirmed by lower cell counts in cloned blastocysts, compared with the in vivo fertilized controls. Subsequently, real-time polymerase chain reaction was used to analyze and compare embryonic gene expression at the 2-cell, 4-cell, and blastocyst stages, between the experimental and control groups. The results showed reduced expression of the candidate genes in cloned 2-cell stage embryos, as manifested by poor developmental competence, compared with expression in the in vivo fertilized controls. Cloned 4-cell embryos and blastocysts, which had overcome the developmental block at the 2-cell stage, also showed up-regulated and down-regulated expression of several genes, strongly suggesting incomplete nuclear reprogramming. We have therefore demonstrated that aberrant embryonic gene expression is associated with low developmental competence of cloned mouse embryos. To improve the efficiency of somatic cell nuclear transfer, strategies to rectify aberrant gene expression in cloned embryos should be investigated.

Comments about possible use of human embryonic stem cell-derived cardiomyocytes to direct autologous adult stem cells into the cardiomyogenic lineage.

Several studies have shown that cell-transplantation therapy following myocardial infarction has some efficacy in aiding myocardial repair and subsequent recovery of heart function. Large-scale production of human embryonic stem cell-derived cardiomyocytes can potentially provide an abundant supply of donor cells for myocardial transplantation.There are, however, immunological barriers to their use in human clinical therapy.A novel approach would be to look at utilizing human embryonic stem cell-derived cardiomyocytes to reprogram autologous adult stem cells to express cardiomyogenic function, instead of using these directly for transplantation.This could be achieved through a number of novel techniques. Enucleated cytoplasts generated from human embryonic stem cell-derived cardiomyocytes could be fused with autologous adult stem cells to generate cytoplasmic hybrids or cybrids.Adult stem cells could also be temporarily permeabilized and exposed to cytoplasmic extracts derived from these cardiomyocytes. Alternatively, intact cells or enucleated cytoplasts from human embryonic stem cell-derived cardiomyocytes could be co-cultured with adult stem cells in vitro, to provide the cellular contacts and electrical coupling that might enable some degree of trans-differentiation to take place. This review would therefore examine the potential advantages and disadvantages of such a novel approach, in comparison to other more conventional techniques such as the use of exogenous cytokines/growth factors or the use of genetic modulation.

Exposure of mouse cumulus cell nuclei to porcine ooplasmic extract eliminates TATA box protein binding to chromatin, but has no effect on DNA methylation

AbstractPurpose: The low cloning efficiency with SCNT is due to incomplete or partial reprogramming of the donor somatic cell nuclei after microinjection into the enucleated oocyte. A possible solution may be to initiate nuclear reprogramming prior to SCNT. Methods: Pre-exposure of donor somatic cell nuclei to a novel porcine ooplasmic extract prior to microinjection could possibly extend the duration of exposure to ooplamic nuclear reprogramming factors. The effects of the porcine ooplamic extract on two major markers of nuclear preprogramming: (1) TATA box protein binding to chromation and (2) DNA methylation was investigated. Results: The results showed that pre-exposure of mouse cumulus cell nuclei to porcine ooplamic extract drastically reduced TATA box protein binding to chromatin, but had no effect on DNA methylation. Conclusions: Pre-exposure to the porcine ooplasmic extract had some limited effects on nuclear reprogramming. Whether this can lead to enhanced cloning efficiency needs to be further investigated.

Human embryonic stem cell-derived fibroblastic and epitheloid lineages as xeno-free support?

Dear Editor: Human embryonic stem (hES) cells have tremendous potential for application in the newly emerging field of regenerative medicine (Gerecht-Nir and Itskovitz-Eldor, 2004). Recently, the first successful derivation of a hES cell line from cloned human embryos (Hwang et al., 2004), as well as the successful derivation of 17 new hES cell lines from donated in vitro–fertilized embryos (Cowan et al., in press), once again placed hES cells at the forefront of international scientific research. However, a major barrier to the application of hES cells in human clinical therapy is their routine propagation on a feeder layer of mitotically inactivated murine embryonic fibroblasts (Thomson et al., 1998; Reubinoff et al., 2000; Cowan et al., in press). With the advent of newly emerging human diseases that have crossed the species barrier, such as severe acute respiratory syndrome (Normile, 2004) and Nipah virus infection (Chua et al., 2002), there are overriding safety concerns regarding the use of mouse-derived feeder cells for hES propagation. We, therefore, read with great interest the recent studies (Richards et al., 2002, 2003; Amit et al., 2003; Hovatta et al., 2003) that have examined the use of donated human tissues to develop a xeno-free support system for hES cells. It is important to note the various limitations of such an approach. Obviously, the use of fetal tissues originating from human abortuses (Richards et al., 2002, 2003) would raise huge ethical concerns. Donated human adult and neonatal tissues pose much less of an ethical dilemma (Amit et al., 2003; Hovatta et al., 2003). However, there may be problems with their availability for large-scale culture of hES cells. Moreover, the use of donated human tissues would introduce a high degree of variability within the culture milieu, which could confound good-quality control in the laboratory. Although cross-species transfer of pathogens would be eliminated through the use of human-derived feeders, there is still a real risk of contamination with yet unscreened pathogens of newly emerging human diseases. All these would pose significant challenges in the application of hES cells for human clinical therapy. A novel approach around these problems would be to examine the possible use of hES cell–derived fibroblastic and epitheloid lineages as feeder supports. The rationale is that such lineages derived from adult and fetal tissues have already been successfully used for maintaining the undifferentiated state of hES cells during prolonged in vitro culture (Richards et al., 2002, 2003; Amit et al., 2003; Hovatta et al., 2003). Hence, it is likely that similar lineages derived from differentiating hES cells could possibly possess the same ability. Presently, there are as yet no reported studies on screening the various differentiated lineages within embryoid bodies for their ability to act as feeder supports for undifferentiated hES cells. Differentiation of hES cells into the fibroblastic and epitheloid lineages could be enhanced through the use of exogenous cytokines, in particular the various isoforms of fibroblast growth factor (FGF) and epithelial growth factor (EGF) (Fernig et al., 1994; Boonstra et al., 1995; Schuldiner et al., 2000). It is likely that a diverse plethora of fibroblastic and epitheloid lineages could be generated by varying combinations of these isoforms of FGF and EGF. These lineages can then be screened for their ability to act as feeder support for undifferentiated hES cells. Once a particular lineage has been identified, the next major challenge would be to establish well-defined and efficient protocols to generate that lineage from hES cells. Preliminary investigations by the Genome Institute of Singapore have revealed major differences in the transcription profiles and proliferation rates of different hES cell lines, although all of these expressed the common marker genes characteristic of hES cells (B. Lim, pers. comm.). Hence, it may also be necessary to screen differentiated lineages derived from several hES cell lines. The development of such ‘‘autogenic’’ xeno-free feeder support systems for hES cell culture will then provide the stringent levels of quality control and safety standards that enable the application of hES cells in human clinical therapy. We hope this will be achieved in the near future.

Roles of Antiphospholipid Antibodies, Antithyroid Antibodies and Antisperm Antibodies in Female Reproductive Health

Autoantibodies are implicated in various human diseases. In this review, the effects of three different types of autoantibodies (antiphospholipid antibody, antithyroid antibody, and antisperm antibody) on female reproductive health are critically examined. Antiphospholipid antibodies are being detected more frequently in women undergoing in vitro fertilization, but their presence does not appear to influence the outcome of pregnancy, miscarriage, or live birth rates. Available evidence makes it conclusive that antiphospholipid antibodies play negligible roles in recurrent miscarriage. Treatment with aspirin together with low-molecular-weight heparin appears to be effective for recurrent miscarriage. Although antithyroid antibodies are commonly found in women of reproductive age, implantation rates and miscarriage rates are unaffected when women have normal thyroid function. Women with thyroid dysfunction should be treated with L-thyroxine before conception or at early gestational stages to avoid pregnancy complications. Antisperm antibodies are uncommon but are most often associated with ovarian hypofunction. They are believed to cause fertilization failure when found at high titers in seminal plasma, or in the mucosal immune system of women. Intrauterine artificial insemination and in vitro fertilization/embryo transfer are effective treatment modalities for antisperm antibody-related infertility, although this remains a controversial topic in the field of female infertility.