Progress towards human primordial germ cell specification in vitro.

Primordial germ cells (PGCs) have long been considered the link between one generation and the next. PGC specification begins in the early embryo as a result of a highly orchestrated combination of transcriptional and epigenetic mechanisms. Understanding the molecular events that lead to proper PGC development will facilitate the development of new treatments for human infertility as well as species conservation. This article describes the latest, most relevant findings about the mechanisms of PGC formation, emphasizing human PGC. It also discusses our own laboratory's progress in using transdifferentiation protocols to derive human PGCs (hPGCs). Our preliminary results arose from our pursuit of a sequential hPGC induction strategy that starts with the repression of lineage-specific factors in the somatic cell, followed by the reactivation of germ cell-related genes using specific master regulators, which can indeed reactivate germ cell-specific genes in somatic cells. While it is still premature to assume that fully functional human gametes can be obtained in a dish, our results, together with those recently published by others, provide strong evidence that generating their precursors, PGCs, is within reach.

Human therapeutic cloning.

Somatic cell nuclear 'reprogramming' in livestock species is now routine in many laboratories. Here, Robert Lanza, Jose Cibelli and Michael West discuss how these techniques may soon be used to clone genetically matched cells and tissues for transplantation into patients suffering from a wide range of disorders that result from tissue loss or dysfunction.

Somatic cell cloned transgenic bovine neurons for transplantation in parkinsonian rats.

Parkinson's disease symptoms can be improved by transplanting fetal dopamine cells into the putamen of parkinsonian patients. Because the supply of human donor tissue is limited and variable, an alternative and genetically modifiable non-human source of tissue would be valuable. We have generated cloned transgenic bovine embryos, 42% of which developed beyond 40 days. Dopamine cells collected from the ventral mesencephalon of the cloned fetuses 42 to 50 days post-conception survived transplantation into immunosuppressed parkinsonian rats and cells from cloned and wild-type embryos improved motor performance. Somatic cell cloning can efficiently produce transgenic animal tissue for treating parkinsonism.

Extension of cell life-span and telomere length in animals cloned from senescent somatic cells.

The potential of cloning depends in part on whether the procedure can reverse cellular aging and restore somatic cells to a phenotypically youthful state. Here, we report the birth of six healthy cloned calves derived from populations of senescent donor somatic cells. Nuclear transfer extended the replicative life-span of senescent cells (zero to four population doublings remaining) to greater than 90 population doublings. Early population doubling level complementary DNA-1 (EPC-1, an age-dependent gene) expression in cells from the cloned animals was 3.5- to 5-fold higher than that in cells from age-matched (5 to 10 months old) controls. Southern blot and flow cytometric analyses indicated that the telomeres were also extended beyond those of newborn (<2 weeks old) and age-matched control animals. The ability to regenerate animals and cells may have important implications for medicine and the study of mammalian aging.

Cloned transgenic calves produced from nonquiescent fetal fibroblasts.

An efficient system for genetic modification and large-scale cloning of cattle is of importance for agriculture, biotechnology, and human medicine. Here, actively dividing fetal fibroblasts were genetically modified with a marker gene, a clonal line was selected, and the cells were fused to enucleated mature oocytes. Out of 28 embryos transferred to 11 recipient cows, three healthy, identical, transgenic calves were generated. Furthermore, the life-span of near senescent fibroblasts could be extended by nuclear transfer, as indicated by population doublings in fibroblast lines derived from a 40-day-old fetal clone. With the ability to extend the life-span of these primary cultured cells, this system would be useful for inducing complex genetic modifications in cattle.

Zebrafish macroH2A variants have distinct embryo localization and function.

Mouse and cell-based studies have shown that macroH2A histone variants predominantly associate with heterochromatin. Functional studies found that macroH2As are involved in gene repression, inhibiting the acquisition of pluripotency and preserving cell differentiation. However, only a few studies have analysed the role of macroH2A during early embryo development. We report the development of transgenic zebrafish lines expressing macroH2A isoforms (mH2A1 and mH2A2) fusion proteins (with GFP) under identified endogenous promoters. We found that mH2A1 and mH2A2 have different spatial and temporal expression patterns during embryonic development. mH2A1 is expressed mostly in the extraembryonic Yolk Syncytial Layer (YSL) starting before shield stage and decreasing once morphogenesis is completed. mH2A2 expression lags behind mH2A1, becoming evident at 24 hpf, within the whole body of the embryo proper. Our ChIP-seq analysis showed that mH2A1 and mH2A2 bind to different DNA regions, changing dramatically after gastrulation. We further analysed RNA-seq data and showed that there is not a general/unspecific repressing function of mH2A1 or mH2A2 associated with heterochromatin but a fine regulation depending on cell types and stage of development. mH2A1 downregulates DNA expression in specific cells and embryo stages and its effect is independent of heterochromatin formation but it is correlated with nucleus quiescence instead. Whereas mH2A2 DNA association correlates with upregulation of differentially expressed genes between 75% epiboly and 24 hpf stages. Our data provide information for underlying molecules that participate in crucial early developmental events, and open new venues to explore mH2A related mechanisms that involve cell proliferation, differentiation, migration and metabolism.

Prospects for the use of nuclear transfer in human transplantation.

The successful application of nuclear transfer techniques to a range of mammalian species has brought the possibility of human therapeutic cloning significantly closer. The objective of therapeutic cloning is to produce pluripotent stem cells that carry the nuclear genome of the patient and then induce them to differentiate into replacement cells, such as cardiomyocytes to replace damaged heart tissue or insulin-producing beta cells for patients with diabetes. Although cloning would eliminate the critical problem of immune incompatibility, there is also the task of reconstituting the cells into more complex tissues and organs in vitro. In the review, we discuss recent progress that has been made in this field as well as the inherent dangers and scientific challenges that remain before these techniques can be used to harness genetically matched cells and tissues for human transplantation.

Transgenic bovine chimeric offspring produced from somatic cell-derived stem-like cells.

We have developed a method, using nuclear transplantation, to produce transgenic embryonic stem (ES)-like cells from fetal bovine fibroblasts. These cells, when reintroduced into preimplantation embryos, differentiated into derivatives from the three embryonic germ layers, ectoderm, mesoderm, and endoderm, in 5-month-old animals. Six out of seven (86%) calves born were found to be chimeric for at least one tissue. These experiments demonstrate that somatic cells can be genetically modified and then de-differentiated by nuclear transfer into ES-like cells, opening the possibility of using them in differentiation studies and human cell therapy.

Oocyte spontaneous activation in different rat strains.

Oocyte spontaneous activation (OSA) has been reported to occur during in vitro culture of ovulated rat oocytes. The objective of this study was to compare the rate of oocyte spontaneous activation and the level of maturation promoting factor (MPF) activity in oocytes from different strains. Twelve strains were selected from two commercial sources. Females were superovulated and oocytes collected 17 h after hCG injection. Denuded oocytes were cultured in M16 medium under oil at 37 degrees C and 5% CO(2) in air. The proportion of activated oocytes was determined after 6 h of in vitro culture. Data were compared by analysis of variance (ANOVA), considering each animal as an experimental unit. MPF activity was determined in oocytes from the different strains at 0, 1.5, and 3 h after oocyte collection. The log ratio of the MPF activity at 1.5 and 3 h relative to 0 hours for each animal was analyzed by ANOVA. While significant (p < 0.01) differences were observed between strains in the rate of OSA, there were no differences between strains in the level of MPF during the time points measured (p > 0.3).

Cloning: new breakthroughs leading to commercial opportunities.

Research on cloning animals, again, came to the forefront of public attention in 1997. Most scientists involved in biomedical and agricultural research have emphasized the benefits, of which there are many, of cloning to the public. Basic studies on nuclear transfer have and will continue to contribute to our understanding of how genomic activation and cell cycle synchrony affect nuclear reprogramming and cloning efficiencies, specifically. Also, more basic information on actual mechanisms and specific factors in the oocyte causing nuclear reprogramming is forthcoming. As new molecular approaches in functional genomics are combined with nuclear transfer experiments, new genes involved in nuclear reprogramming will be found. The commercial potentials of products stemming from discoveries in cloning are vast. Cloning will be a more efficient, faster and more useful way of making transgenic fetuses for cell therapies, adult animals for protein production and organs for xenotransplantation. Clearly there are new opportunities in animal cloning technology that will produce many benefits to society.

Clinical and pathologic features of cloned transgenic calves and fetuses (13 case studies).

The neonatal abnormalities, treatments and outcomes in a group of 13 cloned transgenic calves and fetuses that progressed into the third trimester of pregnancy are described. From these 13 fetuses, 8 calves were born live, 4 stillborn fetuses were recovered from 3 cows that died 7 d to 2 mo before term, and 1 aborted fetus was recovered at 8 mo gestation. All fetuses and calves were derived from the same male fetal Holstein fibroblast cell line transfected with a beta-galactosidase marker gene. Six calves were delivered by Cesarian section and two by vaginal delivery between 278 and 288 d of gestation. Birth weights ranged from 44 to 58.6 kg. Five of the 8 live born calves were judged to be normal within 4 h of birth based on clinical signs and blood gas measurements. One of these 5 calves died at 6 wk of age from a suspected dilated cardiomyopathy. Three of the 8 calves were diagnosed with neonatal respiratory distress immediately following birth, one of which died (at 4 d of age) as a result of pulmonary surfactant deficiency coupled with pulmonary hypertension and elevated systemic venous pressures. Similar findings of chronic pulmonary hypertension were also observed in 2 of 5 fetuses. Placental edema was present in both calves that later died and in the 2 fetuses with cardiopulmonary abnormalities. Hydrallantois occurred with or without placental edema in 6 cows, and only 1 calf from this group survived. The 6 cows without hydrallantois or placental edema produced 5 live calves and 1 aborted fetus. The cardiopulmonary abnormalities observed in the calves and fetuses occurred in utero in conjunction with placental abnormalities, and it is likely that the cloning technique and/or in vitro embryo culture conditions contributed to these abnormalities, although the mechanism remains to be determined.

Reprogramming mammalian somatic cells.

Somatic cell nuclear transfer (SCNT), the technique commonly known as cloning, permits transformation of a somatic cell into an undifferentiated zygote with the potential to develop into a newborn animal (i.e., a clone). In somatic cells, chromatin is programmed to repress most genes and express some, depending on the tissue. It is evident that the enucleated oocyte provides the environment in which embryonic genes in a somatic cell can be expressed. This process is controlled by a series of epigenetic modifications, generally referred to as "nuclear reprogramming," which are thought to involve the removal of reversible epigenetic changes acquired during cell differentiation. A similar process is thought to occur by overexpression of key transcription factors to generate induced pluripotent stem cells (iPSCs), bypassing the need for SCNT. Despite its obvious scientific and medical importance, and the great number of studies addressing the subject, the molecular basis of reprogramming in both reprogramming strategies is largely unknown. The present review focuses on the cellular and molecular events that occur during nuclear reprogramming in the context of SCNT and the various approaches currently being used to improve nuclear reprogramming. A better understanding of the reprogramming mechanism will have a direct impact on the efficiency of current SCNT procedures, as well as iPSC derivation.

Placental anomalies in a viable cloned calf.

Placental anomalies are associated with a high mortality rate in mammalian cloning programs. In this report, we detail the very unusual occurrence of a grossly abnormal placenta that supported a viable cloned calf to term. The placenta was recovered intact 3 h following birth, and its weight was within normal limits (4.3 kg). The chorioallantois of the cloned transgenic female Holstein calf contained only 26 cotyledons. Twelve of these were enlarged and functional. Six were poorly developed, and eight were degenerating. The 12 functional cotyledons ranged in diameter from 8 to 20 cm. The nongravid horn had six rudimentary (<5 cm in diameter) cotyledons and eight cotyledons that remained as oval, mineralized plaques. Despite the reduction in number of placentomes, there was no adventitial placentation. Although this report documents observations from a single case, it does show that a morphologically deficient placenta was able to support development to term and resulted in a viable calf.

Evaluation of developmental competence, nuclear and ooplasmic maturation of calf oocytes.

In this study we evaluated nuclear and ooplasmic maturation of prepuberal calf oocytes to determine a possible cause for their low developmental competency. Calf oocytes resumed meiosis and arrested at the MII stage at rates similar to that of adult animals; however, zygotes derived from calf oocytes cleaved and developed at significantly lower rates. Ooplasmic maturation was assessed during oocyte maturation and fertilization. Transmission electron microscopy revealed that a majority of calf oocytes exhibited some delay in organelle migration and redistribution following maturation. Immunofluorescence microscopy showed that following IVF, a higher percentage of calf oocytes had abnormal chromatin and microtubule configurations than those of adult cattle. These anomalies were characterized by delayed formation of sperm aster and asynchronous pronuclear formation. Microfluorometry was used to characterize the Ca2+ responses of calf oocytes to the addition of agonists or after IVF. The addition of thimerosal demonstrated the presence of Ca2+ stores in calf oocytes. Injection of near threshold concentrations of inositol 1,4,5-trisphosphate (InsP3), used to test the sensitivity of the InsP3R, released significantly less Ca2+ in calf than in cow oocytes, whereas higher concentrations of InsP3 (500 microM) released maximal [Ca2+]i in both oocytes. These results suggested that the Ca2+ content of intracellular stores was similar, but the sensitivity of the InsP3R may be different. Following insemination, calf oocytes exhibiting [Ca2+]i oscillations displayed comparable amplitude and intervals to cow oocytes; however, a significantly higher number of fertilized calf oocytes failed to show oscillations. Our findings suggest that the low developmental competence of calf oocytes can be attributed, at least in part, to incomplete or delayed ooplasmic maturation.

Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer.

Approximately 100 species become extinct a day. Despite increasing interest in using cloning to rescue endangered species, successful interspecies nuclear transfer has not been previously described, and only a few reports of in vitro embryo formation exist. Here we show that interspecies nuclear transfer can be used to clone an endangered species with normal karyotypic and phenotypic development through implantation and the late stages of fetal growth. Somatic cells from a gaur bull (Bos gaurus), a large wild ox on the verge of extinction, (Species Survival Plan < 100 animals) were electrofused with enucleated oocytes from domestic cows. Twelve percent of the reconstructed oocytes developed to the blastocyst stage, and 18% of these embryos developed to the fetal stage when transferred to surrogate mothers. Three of the fetuses were electively removed at days 46 to 54 of gestation, and two continued gestation longer than 180 (ongoing) and 200 days, respectively. Microsatellite marker and cytogenetic analyses confirmed that the nuclear genome of the cloned animals was gaurus in origin. The gaur nuclei were shown to direct normal fetal development, with differentiation into complex tissue and organs, even though the mitochondrial DNA (mtDNA) within all the tissue types evaluated was derived exclusively from the recipient bovine oocytes. These results suggest that somatic cell cloning methods could be used to restore endangered, or even extinct, species and populations.