Using human induced pluripotent stem cells to treat retinal disease.

The eye is an ideal target for exploiting the potential of human induced pluripotent stem cell (hiPSC) technology in order to understand disease pathways and explore novel therapeutic strategies for inherited retinal disease. The aim of this article is to map the pathway from state-of-the art laboratory-based discoveries to realising the translational potential of this emerging technique. We describe the relevance and routes to establishing hiPSCs in selected models of human retinal disease. Additionally, we define pathways for applying hiPSC technology in treating currently incurable, progressive and blinding retinal disease.

Quantitative evaluation of reference genes for real-time PCR during in vitro maturation of ovine oocytes.

Quantitative reverse transcription PCR (RT-qPCR) is a powerful molecular technique that enables gene expression studies to be performed on extremely small samples such as oocytes and pre-implantation embryos. However, the high sensitivity of this technique requires that to prevent bias in expression data interpretation, the reference genes used for normalization are fully validated before use. Difficulties in choosing a reference gene are further compounded by the variable RNA content of the maturing oocyte. In the present study, we evaluated eight commonly used reference genes such as ACTB, GAPDH, H2AFZ, HPRT1, PPIA, SDHA, TUBB and YWHAZ and for sheep oocyte RT-qPCR before and after in vitro maturation. We have also compared different cDNA priming strategies using random hexamers or oligo-dT. GeNorm analysis of the results identified the most reliable genes for normalization to be SDHA, TUBB and PPIA when oocyte cDNA was made with random hexamers, and YWHAZ, TUBB and SDHA when oligo-dT primers were used (H2AFZ and HPRT1 were excluded from the geNorm analysis). Interestingly, the analysis revealed that the least stable genes were ACTB and GAPDH, which are the conventional 'housekeeping' genes used in many studies. We recommend the use of three reference genes to calculate a normalization factor to accurately quantify transcript abundance in sheep oocytes and these vary with the cDNA priming strategy employed.

Induced pluripotent stem cells: epigenetic memories and practical implications.

Induced pluripotent stem cells (iPSCs) may be obtained by direct reprogramming of different somatic cells to a pluripotent state by forced expression of a handful of transcription factors. It was generally assumed that iPSCs are functionally equivalent to their embryonic stem cell (ESC) counterparts. Recently, a number of research groups have demonstrated that this is not the case, showing that iPSCs retain 'epigenetic memory' of the donor tissue from which they were derived and display skewed differentiation potential. This raises the question whether such cells are fit for experimental, diagnostic or therapeutic purpose. A brief survey of the literature illustrates that differences at both epigenetic and transcriptome level are observed between various pluripotent stem cell populations. Interestingly, iPSC populations with perceived 'anomalies' can be coaxed to a more ESC-like cellular state either by continuous passaging--which attenuates these epigenetic differences--or treatment with small molecules that target the machinery responsible for remodelling the genome. This suggests that the establishment of an epigenetic status approximating an ESC counterpart is largely a passive process. The mechanisms responsible remain to be established. Meanwhile, other areas of reprogramming are rapidly evolving such as, trans-differentiation of one somatic cell type to another by the forced expression of key transcription factors. When it comes to assessing their practical usefulness, the same question will also apply.

Ovine fetal development is more sensitive to perturbation by the presence of serum in embryo culture before rather than after compaction.

The effects on subsequent fetal development of the presence or absence of serum at different times during IVC of ovine zygotes were studied. Zygotes, recovered from superovulated ewes 36h after intrauterine AI using semen from a single sire, were cultured for 5 days in synthetic oviductal fluid (SOF) media supplemented with either BSA and amino acids (SOF-) or with 10% (v/v) steer serum (SOF+). Serum was present or absent during the first two and last 2 days of IVC giving four treatments (SOF-/SOF-; SOF-/SOF+;SOF+/SOF- and SOF+/SOF+). In total, 224 embryos, including 26 in vivo controls, were transferred singly at day 6 post-AI to synchronous recipients and the products of conception recovered at day 125 of gestation. Presence of serum during IVC had a biphasic effect on embryo development. The inclusion of serum during the first 2 days of IVC retarded early embryo development while the inclusion of serum during the last 2 days of IVC produced more blastocysts by day 6. These effects were independent of each other. The presence of serum during the first 2 days of IVC resulted in increased weights of gravid uterus, placenta, fetus, fetal heart and liver. The incidence of fetuses whose total or organ weights were greater than three standard deviations above the corresponding mean weights of control fetuses was also greater when serum was present during the first 2 days of IVC. However, even when serum was absent throughout IVC there was still an infrequent incidence of fetal weights greater than three standard deviations above the mean for control fetuses. These observations provide evidence that it is the early pre-compaction stages of embryo development that are particularly sensitive to perturbations leading to abnormal fetal development.

Zygote donor nitrogen metabolism and in vitro embryo culture perturbs in utero development and IGF2R expression in ovine fetal tissues.

Tests were made of the effects of altering nitrogen metabolism in zygote donor ewes on fetal development and expression of the gene encoding the type II insulin-like growth factor receptor (IGF2R) following the transfer of ovine embryos cultured from these zygotes, either in the absence or presence of serum. Zygotes, recovered from superovulated ewes (32 on a urea supplemented (30 g urea/kg) diet (high N) and 32 on a control diet (low N)) 36 h after intrauterine AI using semen from a single sire, were cultured for 5 days in synthetic oviductal fluid (SOF) media either with BSA and amino acids (SOF-) or with 10% (v/v) steer serum (SOF+). In total, 166 embryos, including 30 in vivo controls, were transferred singly at day 6 post-AI to synchronous recipients and the products of conception recovered at day 125 of gestation. Elevated plasma urea concentrations in zygote donors were associated with accelerated early embryo development, low pregnancy rates (16%) for embryos from the high N, SOF+ treatment, and significantly influenced fetal development and the expression of IGF2R in the fetal heart at day 125 of gestation. Importantly, the culture of sheep zygotes under serum-free conditions led to a high incidence of aberrant conceptus development and IGF2R expression. Consequently, maternal nitrogen metabolism prior to zygote recovery and in vitro culture can influence fetal development and the expression of an imprinted gene following embryo transfer, and these data support the notion that environmental effects on the follicle-enclosed oocyte may contribute to the etiology of the Large Offspring Syndrome.

Modifications to improve the efficiency of zona-free mouse nuclear transfer.

In the present study, some modifications were made to the zona-free nuclear transfer technique in the mouse in order to achieve greater efficiency. Firstly, a 1-h interval was allowed between cumulus removal and zona pellucida digestion. Secondly, acid Tyrode's was selected for zona pellucida removal, because contrary to pronase, it allows embryo survival during parthenogenic activation in the absence of calcium. Even when the exposure time to pronase was reduced to as little as 1 min or washed with fetal calf serum to inhibit the enzyme, the percentage of lysis during activation in the absence of calcium was still very high. Thirdly, electrofusion was performed at room temperature (21 degrees C), instead of 30 degrees C as in our previous experiments. Finally, embryos were cultured in groups of 12-15, instead of individually, using a "well of the wells" system during activation and culture. When compared, parthenogenic activated control embryos showed an increase in the development to blastocyst when cultured in pairs instead of individually. By the end of the experiments and using embryonic stem (ES) cells, there was a significant increase in fusion rate (1.5-fold increase) and in development to morula/blastocyst from cleaved reconstructed embryos (1.5-fold increase) when compared with the results before the modifications. A 2.4-fold increase in overall efficiency was achieved from the oocyte to morula/blastocyst stages.

Development of a zona-free method of nuclear transfer in the mouse.

In the present study, a zona-free nuclear transfer (NT) technique, which had been originally developed in cattle, was modified for the mouse. Steps involved in this approach include removing the zona pellucida and enucleating without a holding pipette; sticking donor cells to the cytoplast before electric pulses are applied to fuse them and culturing reconstructed embryos individually in single droplets, to prevent aggregation. Control zona-free and zona-intact embryos from mated donors showed no significant difference in development to blastocyst, but did show reduced development to term. Removal of the zona pellucida affected the response to activation by strontium in the absence of calcium as a significant proportion of zona-free control oocytes and embryos reconstructed by NT lysed during this treatment. A comparison between cumulus and ES cells as donor cells revealed significant differences in fusion efficiency (58.1 +/- 4.0%, n = 573 vs. 42.9 +/- 2.2%, n = 2064, respectively, p < 0.001), cleavage (77.2 +/- 3.4%, n = 334 vs. 40.8 +/- 2.7%, n = 903, respectively, p < 0.001) but not for development to morula/blastocyst (8.7 +/- 2.1%, n = 334 vs. 13.9 +/- 1.8%, n = 903, respectively, p < 0.1). The stage at which embryo development arrested was also affected by donor cell type. A majority of embryos reconstructed from cumulus cells arrested at two-cell stage, usually with two nuclei, whereas those reconstructed from ES cells arrested at one-cell stage, usually with two pseudo-pronuclei. After transfer of ES cell-derived NT embryos, a viable cloned mouse was produced (3.0% of transferred embryos developed to term). These observations establish that a zona-free cloning approach is possible in the mouse, although further research is required to increase the efficiency.

Cloned mice derived from embryonic stem cell karyoplasts and activated cytoplasts prepared by induced enucleation.

Our objective was to induce enucleation (IE) of activated mouse oocytes to yield cytoplasts capable of supporting development following nuclear transfer. Fluorescence microscopy for microtubules, microfilaments, and DNA was used to evaluate meiotic resumption after ethanol activation and the effect of subsequent transient treatments with 0.4 micro g/ml of demecolcine. Using oocytes from B6D2F1 (C57BL/6 x DBA/2) donors, the success of IE of chromatin into polar bodies (PBs) was dependent on the duration of demecolcine treatment and the time that such treatment was initiated after activation. Similarly, variations in demecolcine treatment altered the proportions of oocytes exhibiting a reversible compartmentalization of chromatin into PBs. Treatment for 15 min begun immediately after activation yielded an optimized IE rate of 21% (n = 80) when oocytes were evaluated after overnight recovery in culture. With this protocol, 30-50% of oocytes were routinely scored as compartmentalized when assessed 90 min postactivation. No oocytes could be scored as such following overnight recovery, with 66% of treated oocytes cleaving to the 2-cell stage (n = 80). Activated cytoplasts were prepared by mechanical removal of PBs from oocytes whose chromatin had undergone IE or compartmentalization. These cytoplasts were compared with mechanically enucleated, metaphase (M) II cytoplasts whose activation was delayed in nuclear transfer experiments using HM-1 embryonic stem cells. Using oocytes from either B6D2F1 or B6CBAF1 (C57BL/6 x CBA) donors, the in vitro development of cloned embryos using activated cytoplasts was consistently inferior to that observed using MII cytoplasts. Live offspring were derived from both oocyte strains using the latter, whereas a single living mouse was cloned from activated B6CBAF1 cytoplasts.

Somatic cell nuclear transfer.

Cloning by nuclear transfer from adult somatic cells is a remarkable demonstration of developmental plasticity. When a nucleus is placed in oocyte cytoplasm, the changes in chromatin structure that govern differentiation can be reversed, and the nucleus can be made to control development to term.

Embryo development and establishment of pregnancy after embryo transfer in pigs: coping with limitations in the availability of viable embryos.

Embryo transfer and pregnancy maintenance strategies in pigs were evaluated with reference to situations in which limited numbers of viable embryos or micromanipulated embryos are available, such as pig cloning. Development of embryos with compromised zona pellucida was compared with development of embryos with intact zona pellucida. Micromanipulation had no effect on blastocyst production rates after development in vivo or in vitro, but development in vivo improved the number of embryos reaching the blastocyst stage. Transfer of embryos with compromised zona pellucida resulted in live piglets. Several hormone treatments to maintain pregnancy were tested in a model in which three embryos were transferred into unmated recipient gilts, compared with transfer of three embryos into mated recipients. None of the hormonal treatments resulted in pregnancy rates of more than 25% at term and no more than 9% of transferred embryos survived, in comparison with 50% of the mated recipients successfully carrying 25% of transferred embryos. Lastly, the developmental potential of parthenogenetic embryos was assessed and 62% of transferred embryos resulted in pregnancies, none of which continued beyond day 55 of gestation. After co-transfer of three fertilized embryos with 55-60 parthenogenetic embryos into each of six recipients, two live piglets were delivered. The results from the present study indicate that transfer of zona pellucida compromised embryos can yield litters of normal piglets. In addition, it was demonstrated in a model system involving the transfer of three fertilized embryos into mature gilts that hormonal pregnancy maintenance strategies support a low proportion of embryos to term. Lastly, the present study shows for the first time a comparably effective but novel alternative for pregnancy maintenance in the pig involving the co-transfer of parthenote embryos.

Deletion of the alpha(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep.

Nuclear transfer offers a cell-based route for producing precise genetic modifications in a range of animal species. Using sheep, we report reproducible targeted gene deletion at two independent loci in fetal fibro-blasts. Vital regions were deleted from the alpha(1,3)galactosyl transferase (GGTA1) gene, which may account for the hyperacute rejection of xenografted organs, and from the prion protein (PrP) gene, which is directly associated with spongiform encephalopathies in humans and animals. Reconstructed embryos were prepared using cultures of targeted or nontargeted donor cells. Eight pregnancies were maintained to term and four PrP-/+ lambs were born. Although three of these perished soon after birth, one survived for 12 days. These data show that lambs carrying targeted gene deletions can be generated by nuclear transfer.

Evaluation of gestational deficiencies in cloned sheep fetuses and placentae.

Sheep fetal development at 35 days of gestation was examined following natural mating, in vitro production (IVP) of fertilized embryos, or somatic cell nuclear transfer (NT). Five crossbred (Blackface x Black Welsh) and four purebred (Black Welsh) fetuses and their associated placentae produced by natural mating were morphologically normal and consistent with each other. From 10 ewes receiving 21 IVP embryos, 17 fetuses (81%) were recovered, and 15 of these (88%) were normal. The NT fetuses were derived from two Black Welsh fetal fibroblast cell lines (BLW1 and 6). Transfer of 21 BLW1 and 22 BLW6 NT embryos into 12 and 11 ewes, respectively, yielded 7 (33%) and 8 (36%) fetuses, respectively. Only three (43%) BLW1 and two (25%) BLW6 NT fetuses were normal, with the rest being developmentally retarded. The NT fetal and placental deficiencies included liver enlargement, dermal hemorrhaging, and lack of placental vascular development reflected by reduced or absent cotyledonary structures. Fibroblasts isolated from normal and abnormal cloned fetuses did not differ in their karyotype from sexually conceived fetuses or nuclear donor cell lines. Our results demonstrate that within the first quarter of gestation, cloned fetuses are characterized by a high incidence of developmental retardation and placental insufficiency. These deficiencies are not linked to gross defects in chromosome number.

Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture.

Manipulation or non-physiological embryo culture environments can lead to defective fetal programming in livestock. Our demonstration of reduced fetal methylation and expression of ovine IGF2R suggests pre-implantation embryo procedures may be vulnerable to epigenetic alterations in imprinted genes. This highlights the potential benefits of epigenetic diagnostic screening in developing embryo procedures.

Gene expression during pre- and peri-implantation embryonic development in pigs.

Embryo technological procedures such as in vitro production and cloning by nuclear transfer are not as advanced in pigs as in cattle and cannot yet be applied under field conditions. The present paper focuses on genome activation in in vivo-derived, in vitro-produced and nuclear transfer pig embryos with special emphasis on the development of embryonic nucleoli, where the ribosomal RNA (rRNA) genes transcribed can be used as markers for genome activity. In addition, contemporary data on gene expression in in vivo-derived pig embryos are reviewed. In in vivo-derived pig embryos, pronounced transcription is initiated at the four-cell stage (the third cell cycle after fertilization), when nucleoli develop. In parallel with the development of the nucleoli as a result of rRNA gene activation, a cascade of other genes is also likely to be transcribed. However, apart from identification of transcripts for the oestrogen receptor at the blastocyst stage, reports on mRNAs resulting from initial transcription of the pig embryonic genome are lacking, in contrast to the situation in cattle and, in particular, mice. More information is available on gene expression during elongation of pig conceptuses, when the genes for steroidogenic enzymes, extracellular matrix receptors, oestrogen receptors, growth factors and their receptors, as well as retinol binding protein and retinoic acid receptors, are expressed. Nucleolus development appears to be disturbed in in vitro-produced pig embryos and in pig embryos reconstructed by nuclear transfer of granulosa cells to enucleated metaphase II oocytes produced by oocyte maturation in vivo or in vitro, which is indicative of disturbances in activation of rRNA genes.