Culture conditions and enzymatic passaging of bovine ESC-like cells.

The goals of the current study were to (1) improve culture conditions and (2) chemical passaging of bovine embryonic stem cell-like (bESC-like) cells. Specifically, the effects of human leukemia inhibitory factor (hLIF), two types of feeders, mouse embryonic fibroblast (MEF) and bovine embryonic fibroblast (BEF), as well as three different enzymatic treatments including Trypsin-EDTA, TrypLE, and Liberase Blendzymes 3 were investigated. The addition of hLIF at 1000 U/mL to the culture medium (41.2 and 36.9%), and the use of either MEF or BEF feeders (40.3 and 38.1%) had no significant effect on the ability of inner cell masses (ICMs) to form primary cell colonies compared to controls. All bESC-like cells were first dissociated mechanically for three passages followed by enzymatic dissociation. The ability to maintain ESC morphology to passage 10 was compared among the three enzymes above. More bESC-like cell lines survived beyond passage 10 when treated with TrypLE compared to Trypson-EDTA (28.8 and 12.6%; p < 0.05), and bESC-like cells differentiated quickly when treated with Liberase Blendzyme 3. The bESC-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as SSEA-1, AP, OCT-4, and Nanog. When removed from feeders, these bESC-like cells formed embryoid bodies (EBs) in a suspension culture. When EBs were cultured on tissue culture plates, they differentiated into various cell types. In summary, we were able to culture bESC-like cells more than 10 passages by enzymatic dissociation, which is important in gene targeting, maintenance, and banking of bESC lines.

Efficient derivation of embryonic stem cells from nuclear transfer and parthenogenetic embryos derived from cryopreserved oocytes.

Deriving histocompatible embryonic stem (ES) cells by somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA) requires fresh oocytes, which prevents their applications in humans. Here, we evaluated the efficiency of deriving ES cells from mature metaphase II (MII) and immature metaphase I (MI) vitrified oocytes, by PA or SCNT, in a mouse model. We successfully generated ES cell lines from PA (MII and MI) and SCNT (MII and MI) blastocysts. These cell lines expressed genes and antigens characteristic of pluripotent ES cells and produced full-term pups upon tetraploid embryo complementation. This study established an animal model for efficient generation of patient-specific ES cell lines using cryopreserved oocytes. This is a major step forward in the application of therapeutic cloning and parthenogenetic technology in human regenerative medicine and will serve as an important alternative to the iPS cell technology in countries/regions where these technologies are permitted.

Engineered heart tissue graft derived from somatic cell nuclear transferred embryonic stem cells improve myocardial performance in infarcted rat heart.

The concept of regenerating diseased myocardium by implanting engineered heart tissue (EHT) is intriguing. Yet it was limited by immune rejection and difficulties to be generated at a size with contractile properties. Somatic cell nuclear transfer is proposed as a practical strategy for generating autologous histocompatible stem (nuclear transferred embryonic stem [NT-ES]) cells to treat diseases. Nevertheless, it is controversial as NT-ES cells may pose risks in their therapeutic application. EHT from NT-ES cell-derived cardiomyocytes was generated through a series of improved techniques in a self-made mould to keep the EHTs from contraction and provide static stretch simultaneously. After 7 days of static and mechanical stretching, respectively, the EHTs were implanted to the infarcted rat heart. Four weeks after transplantation, the suitability of EHT in heart muscle repair after myocardial infarction was evaluated by histological examination, echocardiography and multielectrode array measurement. The results showed that large (thickness/diameter, 2-4 mm/10 mm) spontaneously contracting EHTs was generated successfully. The EHTs, which were derived from NT-ES cells, inte grated and electrically coupled to host myocardium and exerted beneficial effects on the left ventricular function of infarcted rat heart. No teratoma formation was observed in the rat heart implanted with EHTs for 4 weeks. NT-ES cells can be used as a source of seeding cells for cardiac tissue engineering. Large contractile EHT grafts can be constructed in vitro with the ability to survive after implantation and improve myocardial performance of infarcted rat hearts.

Calcium release and development of heat-shocked porcine oocytes after nucleus-ooplasm reconstruction.

We determined the effect of heat shock (HS) on the alterations of development and calcium releasing capacity of nuclear-ooplasmic reconstructed porcine oocytes stimulated by thimerosal. The non-HS (39 degrees C) and the HS2h (41.5 degrees C for 2 h) matured oocytes were enucleated and their spindles/chromosomes were exchanged between these two groups followed by parthenogenetic activation. In the Control group (Csp-Coop), the non-HS spindle (Csp) was transferred to the non-HS ooplasm (Coop). Blastocyst and cleavage rates were higher in both Csp-HSoop (non-HS spindle transferred to the HS ooplasm) and HSsp-Coop (HS spindle transferred to non-HS ooplasm) reconstructed oocytes, but no difference was detected in the average cell number per blastocyst. However, intracellular calcium concentrations ([Ca(2+)](i)) generally declined (p < 0.05) in the reconstructed HS oocytes, with a greater blastocyst rate after parthenogenetic activation. In the present study, time for the completion of spindle transfer in these oocytes was 1-2 h, during which some physiological remodeling or adaptation might have been occurred in the oocytes. Therefore, changes in heat-shock protein70 (HSP70) expression and developmental competence of the HS2h oocytes with 1 or 2 h of recovery time under normal culture temperature (39 degrees C) were examined. The results showed that the expression of HSP70 in the HS2h oocytes was higher (p < 0.05) than those had recovery incubation for 1 h (HC1h) after HS, but the cleavage and blastocyst rates were greater (p < 0.05) in the HC1h group. We demonstrated that a recovery period prior to activation of porcine oocytes and reconstructed oocytes is beneficial to further development. Heat shock to either the karyoplast or the ooplasm enhances embryonic development but reduces intracellular calcium release in the cloned porcine oocytes.

Gene expression profiles of bovine caruncular and intercaruncular endometrium at implantation.

At implantation the endometrium undergoes modifications necessary for its physical interactions with the trophoblast as well as the development of the conceptus. We aim to identify endometrial factors and pathways essential for a successful implantation in the caruncular (C) and the intercaruncular (IC) areas in cattle. Using a 13,257-element bovine oligonucleotide array, we established expression profiles at day 20 of the estrous cycle or pregnancy (implantation), revealing 446 and 1,295 differentially expressed genes (DEG) in C and IC areas, respectively (false discovery rate = 0.08). The impact of the conceptus was higher on the immune response function in C but more prominent on the regulation of metabolism function in IC. The C vs. IC direct comparison revealed 1,177 and 453 DEG in cyclic and pregnant animals respectively (false discovery rate = 0.05), with a major impact of the conceptus on metabolism and cell adhesion. We selected 15 genes including C11ORF34, CXCL12, CXCR4, PLAC8, SCARA5, and NPY and confirmed their differential expression by quantitative RT-PCR. The cellular localization was analyzed by in situ hybridization and, upon pregnancy, showed gene-specific patterns of cell distribution, including a high level of expression in the luminal epithelium for C11ORF34 and MX1. Using primary cultures of bovine endometrial cells, we identified PTN, PLAC8, and CXCL12 as interferon-tau (IFNT) target genes and MSX1 and CXCR7 as IFNT-regulated genes, whereas C11ORF34 was not an IFNT-regulated gene. Our transcriptomic data provide novel molecular insights accounting for the biological functions related to the C or IC endometrial areas and may contribute to the identification of potential biomarkers for normal and perturbed early pregnancy.

Endometrium as an early sensor of in vitro embryo manipulation technologies.

Implantation is crucial for placental development that will subsequently impact fetal growth and pregnancy success with consequences on postnatal health. We postulated that the pattern of genes expressed by the endometrium when the embryo becomes attached to the mother uterus could account for the final outcome of a pregnancy. As a model, we used the bovine species where the embryo becomes progressively and permanently attached to the endometrium from day 20 of gestation onwards. At that stage, we compared the endometrial genes profiles in the presence of an in vivo fertilized embryo (AI) with the endometrial patterns obtained in the presence of nuclear transfer (SCNT) or in vitro fertilized embryos (IVF), both displaying lower and different potentials for term development. Our data provide evidence that the endometrium can be considered as a biological sensor able to fine-tune its physiology in response to the presence of embryos whose development will become altered much later after the implantation process. Compared with AI, numerous biological functions and several canonical pathways with a major impact on metabolism and immune function were found to be significantly altered in the endometrium of SCNT pregnancies at implantation, whereas the differences were less pronounced with IVF embryos. Determining the limits of the endometrial plasticity at the onset of implantation should bring new insights on the contribution of the maternal environment to the development of an embryo and the success of pregnancy.

Nuclear transfer embryonic stem cells provide an in vitro culture model for Parkinson's disease.

Somatic cell nuclear transfer enables the generation of embryonic stem cells (ESCs) that genetically match the donor and can be used to treat disease through cell replacement therapies or to recapitulate patient-specific disease via in vitro differentiation. We performed a "proof-of-principle" study using tail tip fibroblasts from a mouse model of Parkinson's disease (Aphakia) as the donor cell nuclei for nuclear transfer and derived "customized" ESCs for in vitro analysis. Aphakia mice contain deletions in the pitx3 gene and show selective loss of dopamine neurons of the substantia nigra, specifically the neuron population susceptible to degeneration in Parkinson's disease. Using electrofusion nuclear transfer, we produced cloned Aphakia oocytes at rates similar to those for control, cloned oocytes. Aphakia ESCs were isolated and live mice were generated using tetraploid embryo complementation. In vitro differentiation of Aphakia ESCs to dopaminergic neurons revealed significantly fewer TH+ neurons that expressed MAP2, DAT, synaptophysin, VMAT2, and AHD2 compared to control nuclear transfer ESC cultures, supporting a role for Pitx3 in mesodiencephalic dopamine neuron maturation. Taken together, our studies define a customized in vitro ESC culture system used to analyze gene-specific contribution to dopamine neuron generation, maturation, and susceptibility to degeneration.

Nuclear transfer and oocyte cryopreservation.

Somatic cells can be reprogrammed to a totipotent state through nuclear transfer or cloning, because it has been demonstrated that the oocyte has the ability to reprogramme an adult nucleus into an embryonic state that can initiate the development of a new organism. Therapeutic cloning, whereby nuclear transfer is used to derive patient-specific embryonic stem cells, embraces an entire new opportunity for regenerative medicine. However, a key obstacle for human therapeutic cloning is that the source of fresh human oocytes is extremely limited. In the present review, we propose prospective sources of human oocytes by using oocyte cryopreservation, such as an oocyte bank and immature oocytes. We also address some potential issues associated with nuclear transfer when using cryopreserved oocytes. In the future, if the efficacy and efficiency of cryopreserved oocytes are comparable to those of fresh oocytes in human therapeutic cloning, the use of cryopreserved oocytes would be invaluable and generate a great impact to regenerative medicine.

Beneficial effect of young oocytes for rabbit somatic cell nuclear transfer.

This study was designed to examine the effect of the age of rabbit oocytes on the developmental potential of cloned embryos. The metaphase II oocytes used for nuclear transfer (NT) were collected at 10, 12, 14, and 16 h post-hCG injection (hpi). The total number of oocytes collected per donor (21.4-23.7) at 12 to 16 hpi was similar, but significantly higher than that collected at 10 hpi (16.2). Additionally, a significant improvement in blastocyst development was achieved with embryos generated by electrically mediated cell fusion (56.0%), compared to those from nuclear injection (13.1 %) (Experiment 1). Markedly higher blastocyst development (45.8-54.5%) was also achieved with oocytes collected at 10-12 hpi than from those collected 14-16 hpi (8.3-14.3%) (Experiment 2). In Experiment 3, the blastocyst rates of NT embryos derived from oocytes harvested 12 hpi (39.2-42.8 %) were significantly higher than from those collected at 16 hpi (6.8-8.4 %) (p < 0.05), regardless of the donor cell age. Kinase activity assays showed variable changes of activity in rabbit oocytes over the period of 10-16 hpi; however, there was no correlation with preimplantational development (blastocyst rate vs. MPF, R = 0.326; blastocyst rate vs. MAPK, R = -0.131). Embryo transfer of NT embryos utilizing 12 hpi oocytes resulted in one full-term but stillborn, and one live cloned rabbit; thus, an efficiency of 1.7 % (n = 117) (Experiment 4). These results demonstrated that NT utilizing relatively young rabbit oocytes, harvested at 10-12 h after hCG injection, was beneficial for the development of NT embryos.

Expression of X-linked genes in deceased neonates and surviving cloned female piglets.

Animal cloning through somatic cell nuclear transfer (NT) is very inefficient, probably due to insufficient reprogramming of the donor nuclei, which in turn would cause the dysregulation of gene expression. X-Chromosome inactivation (XCI) is a multi-step epigenetic process utilized by mammals to achieve dosage compensation in females. Our aim was to determine if any dysregulation of X-linked genes, which would be indicative of unfaithful reprogramming of donor nuclei, was present in cloned pigs. Real time reverse transcription polymerase chain reaction (RT-PCR) was performed to quantify the transcript levels of five X-linked genes, X inactivation-specific transcript (XIST), TSIX (the reverse spelling of XIST), hypoxanthine guanine phosphoribosyltransferase 1 (HPRT1), glucose-6-phosphate dehydrogenase (G6PD), V-raf murine sarcoma 3,611 viral oncogene homolog 1 (ARAF1), and one autosomal gene, alpha-1 type IV collagen (COL4A1) in major organs of neonatal deceased and surviving female cloned pigs as well as their age-matched control pigs from conventional breeding. Aberrant expression level of these genes was prevalent in the neonatal deceased clones, while it was only moderate in cloned pigs that survived after birth. These results suggest a correlation between the viability of the clones and the normality of their gene expression and provide a possible explanation for the death of a large portion of cloned animals around birth.

17beta-estradiol stimulates proliferation of spermatogonia in experimental cryptorchid mice.

AIM: To investigate whether estrogen stimulates the proliferation of spermatogonia or induces spermatogenesis in cryptorchid mice. METHODS: Mice were surgically rendered cryptorchid, then treated with different doses of 17beta-estradiol (E2) s.c. once a day. Mice were killed at sexual maturity (45 days of age), and histological analysis and immunofluorescence were performed. Serum follicle stimulating hormone (FSH), estradiol, testosterone and luteinizing hormone (LH) were measured. RESULTS: Low doses of E2 had no notable effect on spermatogonia, but at higher doses, E2 stimulated the proliferation of spermatogonia. CONCLUSION: E2 has a dose-related mitogenic effect on spermatogonia.

Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning.

Therapeutic cloning, whereby somatic cell nuclear transfer (SCNT) is used to generate patient-specific embryonic stem cells (ESCs) from blastocysts cloned by nuclear transfer (ntESCs), holds great promise for the treatment of many human diseases. ntESCs have been derived in mice and cattle, but thus far there are no credible reports of human ntESCs. Here we review the recent literature on nuclear reprogramming by SCNT, including studies of gene expression, DNA methylation, chromatin remodeling, genomic imprinting and X chromosome inactivation. Reprogramming of genes expressed in the inner cell mass, from which ntESCs are derived, seems to be highly efficient. Defects in the extraembryonic lineage are probably the major cause of the low success rate of reproductive cloning but are not expected to affect the derivation of ntESCs. We remain optimistic that human therapeutic cloning is achievable and that the derivation of patient-specific ntESC lines will have great potential for regenerative medicine.

Cardiomyocytes.

Derivation of cardiomyocytes from embryonic stem cells would be a boon for treatment of the many millions of people worldwide who suffer significant cardiac tissue damage in a myocardial infarction. Such cells could be used for transplantation, either as loose cells, as organized pieces of cardiac tissue, or even as pieces of organs. Eventual derivation of human embryonic stem cells via somatic cell nuclear cloning would provide cells that not only may replace damaged cardiac tissue, but also would replace tissue without fear that the patient's immune system will reject the implant. Embryonic stem cells can differentiate spontaneously into cardiomyocytes. In vitro differentiation of embryonic stem cells normally requires an initial aggregation step to form structures called embryoid bodies that differentiate into a wide variety of specialized cell types, including cardiomyocytes. This chapter discusses methods of encouraging embryoid body formation, causing pluripotent stem cells to develop into cardiomyocytes, and expanding the numbers of cardiomyocytes so that the cells may achieve functionality in transplantation, all in the mouse model system. Such methods may be adaptable and/or modifiable to produce cardiomyocytes from human embryonic stem cells.

Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer.

Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

Creation of engineered cardiac tissue in vitro from mouse embryonic stem cells.

BACKGROUND: Embryonic stem (ES) cells can terminally differentiate into all types of somatic cells and are considered a promising source of seed cells for tissue engineering. However, despite recent progress in in vitro differentiation and in vivo transplantation methodologies of ES cells, to date, no one has succeeded in using ES cells in tissue engineering for generation of somatic tissues in vitro for potential transplantation therapy. METHODS AND RESULTS: ES-D3 cells were cultured in a slow-turning lateral vessel for mass production of embryoid bodies. The embryoid bodies were then induced to differentiate into cardiomyocytes in a medium supplemented with 1% ascorbic acid. The ES cell-derived cardiomyocytes were then enriched by Percoll gradient centrifugation. The enriched cardiomyocytes were mixed with liquid type I collagen supplemented with Matrigel to construct engineered cardiac tissue (ECT). After in vitro stretching for 7 days, the ECT can beat synchronously and respond to physical and pharmaceutical stimulation. Histological, immunohistochemical, and transmission electron microscopic studies further indicate that the ECTs both structurally and functionally resemble neonatal native cardiac muscle. Markers related to undifferentiated ES cell contamination were not found in reverse transcriptase-polymerase chain reaction analysis of the Percoll-enriched cardiomyocytes. No teratoma formation was observed in the ECTs implanted subcutaneously in nude mice for 4 weeks. CONCLUSIONS: ES cells can be used as a source of seed cells for cardiac tissue engineering. Additional work remains to demonstrate engraftment of the engineered heart tissue in the case of cardiac defects and its functional integrity within the host's remaining healthy cardiac tissue.

Cloning animals by somatic cell nuclear transfer--biological factors.

Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other species, this review will be focused on somatic cell cloning of cattle.