Since the initial description of induced pluripotent stem (iPS) cells created by forced expression of four transcription factors in mouse fibroblasts, the technique has been used to generate embryonic stem (ES)-cell-like pluripotent cells from a variety of cell types in other species, including primates and rat. It has become a popular means to reprogram somatic genomes into an embryonic-like pluripotent state, and a preferred alternative to somatic-cell nuclear transfer and somatic-cell fusion with ES cells. However, iPS cell reprogramming remains slow and inefficient. Notably, no live animals have been produced by the most stringent tetraploid complementation assay, indicative of a failure to create fully pluripotent cells. Here we report the generation of several iPS cell lines that are capable of generating viable, fertile live-born progeny by tetraploid complementation. These iPS cells maintain a pluripotent potential that is very close to ES cells generated from in vivo or nuclear transfer embryos. We demonstrate the practicality of using iPS cells as useful tools for the characterization of cellular reprogramming and developmental potency, and confirm that iPS cells can attain true pluripotency that is similar to that of ES cells.

Studies using low-resolution methods to assess gene expression during preimplantation mouse development indicate that changes in gene expression either precede or occur concomitantly with the major morphological transitions, that is, conversion of the oocyte to totipotent 2-cell blastomeres, compaction, and blastocyst formation. Using microarrays, we characterized global changes in gene expression and used Expression Analysis Systematic Explorer (EASE) to identify biological and molecular processes that accompany and likely underlie these transitions. The analysis confirmed previously described processes or events, but more important, EASE revealed new insights. Response to DNA damage and DNA repair genes are overrepresented in the oocyte compared to 1-cell through blastocyst stages and may reflect the oocyte's response to selective pressures to insure genomic integrity; fertilization results in changes in the transcript profile in the 1-cell embryo that are far greater than previously recognized; and genome activation during 2-cell stage may not be as global and promiscuous as previously proposed, but rather far more selective, with genes involved in transcription and RNA processing being preferentially expressed. These results validate this hypothesis-generating approach by identifying genes involved in critical biological processes that can be the subject of a more traditional hypothesis-driven approach.

Zygotic gene activation is essential for development beyond the 2-cell stage in the preimplantation mouse embryo. Based on alpha-amanitin-sensitive BrUTP incorporation, transcription initiates in the 1-cell embryo and a major reprogramming of gene expression driven by newly expressed genes is prominently observed during the 2-cell stage. Superimposed on genome activation is the development of a transcriptionally repressive state that is mediated at the level of chromatin structure. The identity of the genes that are expressed during the 1- and 2-cell stages, however, is poorly described, as are those genes involved in mediating the transcriptionally repressive state. Using the Affymetrix MOE430 mouse GeneChip set, we characterized the set of alpha-amanitin-sensitive genes expressed during the 1- and 2-cell stages, and we used Expression Analysis Systematic Explorer (EASE) and Ingenuity Pathway Analysis (IPA) to identify biological and molecular processes represented by these genes, as well as interactions among them. We find that although the 1-cell embryo is transcriptionally active, we did not detect any transcripts present on the MOE430 GeneChip set to be alpha-amanitin-sensitive. Thus, what the BrUTP incorporation represents remains elusive. About 17% of genes expressed in the 2-cell embryo are alpha-amanitin-sensitive. EASE analysis reveals that genes involved in ribosome biogenesis and assembly, protein synthesis, RNA metabolism and transcription are over-represented, suggesting that genome activation during 2-cell stage may not be as global and promiscuous as previously proposed. IPA implicated Myc and Hdac1 as candidate genes involved in genome activation and the development of the transcriptionally repressive state, respectively.

Immunoprecipitation of mRNA-protein complexes is a method that can be used to study RNA binding protein (RBP)-RNA interactions. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. It can also be used as a second, independent method to verify RBP-mRNA interactions discovered through more universal screening techniques. We describe the immunoprecipitation protocol in practical detail and discuss variations of the method as well as issues associated with it. The procedure takes three days to complete.

-M., Crook, J. M., de Sousa, P. A. et al (2015). Points to consider in the development of seed stocks of pluripotent stem cells for clinical applications: International Stem Cell Banking Initiative (ISCBI). Regenerative Medicine, 10 (2s), 1-44.

Basonuclin is a zinc-finger protein found in abundance in oocytes. It qualifies as a maternal-effect gene because the source of preimplantation embryonic basonuclin is maternal. Using a transgenic-RNAi approach, we knocked down basonuclin specifically in mouse oocytes, which led to female sub-fertility. Basonuclin deficiency in oocytes perturbed both RNA polymerase I-and IImediated transcription, and oocyte morphology was affected (as evidenced by cytoplasmic and cell surface abnormalities). Some of the affected oocytes, however, could still mature to and arrest at metaphase II, and be ovulated. Nevertheless, fertilized basonuclindeficient eggs failed to develop beyond the two-cell stage, and this pre-implantation failure accounted for the sub-fertility phenotype. These results suggest that basonuclin is a new member of the mammalian maternal-effect genes and, interestingly, differs from the previously reported mammalian maternal-effect genes in that it also apparently perturbs oogenesis.

Cellular phenotype is the conglomerate of multiple cellular processes involving gene and protein expression that result in the elaboration of a cell's particular morphology and function. It has been thought that differentiated postmitotic cells have their genomes hard wired, with little ability for phenotypic plasticity. Here we show that transfer of the transcriptome from differentiated rat astrocytes into a nondividing differentiated rat neuron resulted in the conversion of the neuron into a functional astrocyte-like cell in a time-dependent manner. This single-cell study permits high resolution of molecular and functional components that underlie phenotype identity. The RNA population from astrocytes contains RNAs in the appropriate relative abundances that give rise to regulatory RNAs and translated proteins that enable astrocyte identity. When transferred into the postmitotic neuron, the astrocyte RNA population converts 44% of the neuronal host cells into the destination astrocyte-like phenotype. In support of this observation, quantitative measures of cellular morphology, single-cell PCR, single-cell microarray, and single-cell functional analyses have been performed. The host-cell phenotypic changes develop over many weeks and are persistent. We call this process of RNA-induced phenotype changes, transcriptome-induced phenotype remodeling.neuron ͉ transcriptome-induced phenotype remodeling ͉ single cell ͉ Waddington I n multicellular organisms, all cells contain nearly identical copies of the genome but exhibit drastically different phenotypes. Even a single neuron has a set of phenotypic characteristics that distinguish it from other neurons as well as other cell types, such as the nearby astrocytes. Indeed, as Waddington proposed in his classical epigenetic landscape model, genetically predetermined cells can follow any specific permitted trajectories that eventually lead to different cellular phenotypes (1). From this point of view, the genome serves as a repository of dynamic control information whose state can be reprogrammed to match the stable phenotypic states.Emerging evidence has demonstrated the reversibility and flexibility of the cellular phenotype. Gurdon et al. first showed that the ability to obtain fertile adult male and female frogs by injecting endoderm nuclei into enucleated eggs (2). This result not only forms the foundation of the field in nuclear transplantation but also provides evidence that the cytoplasmic components of a differentiated cell can support nuclear reprograming. Generation of induced pluripotent stem (iPS) cells by transfection of transcription factors into dividing fibroblasts (3), followed by cell selection, represents a new strategy to globally revert a mature cell into a different cell type (4-9). The need for redifferentiation of these embryonic stem cell-like-iPS cells into desired cell types adds a layer of complexity that is difficult to control (10, 11). Nevertheless, studies of nuclear reprogramming from genomic and epigenetic modification, as seen from so...

The beta-thalassaemias represent a heterogenous group of diseases resulting from decreased erythroid beta-globin mRNA expression and imbalanced alpha/beta-globin chain synthesis which are manifest clinically by ineffective erythropoiesis and excessive haemolysis. Increasing levels of haemoglobin F (HbF) by pharmacological agents has been proposed to ameliorate the severity of the disease by improving the balance in globin chain synthesis. Hydroxyurea (HU), as an effective agent with low toxicity for activating gamma-globin gene, has been shown to enhance HbF synthesis in experimental animals and in patients with sickle cell anaemia. However, previous trials of HU in beta-thalassaemia patients are ambiguous, with a small number having increased HbF synthesis. In a recent study of HU effects in Chinese beta-thalassaemia patients we unexpectedly found that two unrelated patients with beta-thalassaemia intermedia demonstrated an improvement in the effectiveness of erythropoiesis reflected by an increase in haemoglobin concentration (from 4.1 to 6.3 g/dl, patient 1; from 6.5 to 9.7 g/dl, patient 2) and in red cell volume (from 68 to 104 fl, patient 1; from 68 to 85 fl, patient 2) after a period of excess of 300d of low-dosage HU treatment. These effects, however, appear to be due to increased beta-globin biosynthesis, because the percentage of HbF decreased in each patient as total Hb increased. This was reflected by changes in the beta/alpha ratio (from 0.301 to 0.581, patient 1; from 0.348 to 0.487, patient 2) with minimal changes in gamma-globin biosynthesis. We conclude that in addition to its known effects in stimulating gamma-globin production, hydroxyurea may have a more general role in augmenting globin synthesis, including beta-globin in some thalassaemia intermedia patients who maintain the capacity to express normal beta-globin chains.