Zhuo Lv

iPS cells produce viable mice through tetraploid complementation

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.

Activation of the imprinted Dlk1-Dio3 region correlates with pluripotency levels of mouse stem cells

Low reprogramming efficiency and reduced pluripotency have been the two major obstacles in induced pluripotent stem (iPS) cell research. An effective and quick method to assess the pluripotency levels of iPS cells at early stages would significantly increase the success rate of iPS cell generation and promote its applications. We have identified a conserved imprinted region of the mouse genome, the Dlk1-Dio3 region, which was activated in fully pluripotent mouse stem cells but repressed in partially pluripotent cells. The degree of activation of this region was positively correlated with the pluripotency levels of stem cells. A mammalian conserved cluster of microRNAs encoded by this region exhibited significant expression differences between full and partial pluripotent stem cells. Several microRNAs from this cluster potentially target components of the polycomb repressive complex 2 (PRC2) and may form a feedback regulatory loop resulting in the expression of all genes and non-coding RNAs encoded by this region in full pluripotent stem cells. No other genomic regions were found to exhibit such clear expression changes between cell lines with different pluripotency levels; therefore, the Dlk1-Dio3 region may serve as a marker to identify fully pluripotent iPS or embryonic stem cells from partial pluripotent cells. These findings also provide a step forward toward understanding the operating mechanisms during reprogramming to produce iPS cells and can potentially promote the application of iPS cells in regenerative medicine and cancer therapy.

Evaluation of Blastomere Biopsy Using a Mouse Model Indicates the Potential High Risk of Neurodegenerative Disorders in the Offspring

Preimplantation genetic diagnosis (PGD), used in clinical practice, is offered to couples that may suffer from a monogenetic disorder, chromosome aneuploidy, or X-linked disease. However, blastomere biopsy, as an indispensable manipulation during the PGD procedure has not been assessed for its long term health implications. Using a mouse model, we investigated the effect of blastomere biopsy of in vitro cultured four-cell embryos on preimplantation development efficiency, postnatal growth, and physiological and behavioral activity compared with control, non-biopsied embryos. The mice generated after blastomere biopsy showed weight increase and some memory decline compared with the control group. Further protein expression profiles in adult brains were analyzed by a proteomics approach. A total of 36 proteins were identified with significant differences between the biopsied and control groups, and the alterations in expression of most of these proteins have been associated with neurodegenerative diseases. Furthermore hypomyelination of the nerve fibers was observed in the brains of mice in the biopsied group. This study suggested that the nervous system may be sensitive to blastomere biopsy procedures and indicated an increased relative risk of neurodegenerative disorders in the offspring generated following blastomere biopsy. Thus, more studies should be performed to address the possible adverse effects of blastomere biopsy on the development of offspring, and the overall safety of PGD technology should be more rigorously assessed.

Viable Fertile Mice Generated from Fully Pluripotent iPS Cells Derived from Adult Somatic Cells

Previous studies demonstrated that induced pluripotent stem (iPS) cells could produce viable mice through tetraploid complementation, which was thought to be the most stringent test for pluripotency. However, these highly pluripotent iPS cells were previously reported to be generated from fibroblasts of embryonic origin. Achieving fully pluripotent iPS cells from multiple cell types, especially easily accessible adult tissues, will lead to a much greater clinical impact. We successfully generated high-pluripotency iPS cells from adult tail tip fibroblasts (TTF) that resulted in viable, full-term, fertile TTF-iPS animals with no obvious teratoma formation or other developmental abnormalities. Comparison of iPS cells from embryonic origin (MEF), progenitor cells (neural stem cells) or differentiated somatic cells (TTF) reveals that fully pluripotent developmental potential can be reached by each cell type, although with different induction efficiencies. This work provides the means for studying the mechanisms and regulation of direct reprogramming, and has encouraging implications for future clinical applications and therapeutic interventions.

Mice generated from tetraploid complementation competent iPS cells show similar developmental features as those from ES cells but are prone to tumorigenesis.

Mice generated from tetraploid complementation competent iPS cells show similar developmental features as those from ES cells but are prone to tumorigenesis

Production of mice using iPS cells and tetraploid complementation.

Induced pluripotent stem cells (iPSCs) are considered to be an attractive alternative to embryonic stem cells (ESCs) and may provide great potential for clinical applications in regenerative medicine. Although possessing characteristics similar to ESCs, the true pluripotency of these newly studied iPSCs was not known because none of the previously developed iPSCs passed the tetraploid complementation assay, which is regarded as the most stringent test for pluripotency. We have recently shown that by modifying some of the culture conditions for inducing iPSCs, we were able to generate cell lines of high pluripotency, resulting in the production of live-born, fertile animals through tetraploid complementation. In this paper, we describe details of our methods of generating iPS cell lines and subsequently producing full-term live animals through the tetraploid complementation assay; the procedure can be completed within 2 months.

Efficient and rapid generation of induced pluripotent stem cells using an alternative culture medium

Efficient and rapid generation of induced pluripotent stem cells using an alternative culture medium

iPS cells generated without c-Myc have active Dlk1-Dio3 region and are capable of producing full-term mice through tetraploid complementation.

iPS cells generated without c-Myc have active Dlk1-Dio3 region and are capable of producing full-term mice through tetraploid complementation

Decreased cofilin1 expression is important for compaction during early mouse embryo development

Compaction, occurring at the eight-cell stage of mouse development, is the process of cell flattening and polarization by which cellular asymmetry is first established. During this process many molecules and organelles undergo polarized distribution, but the cytoskeletal basis for these distribution specifications remains to be explored. The present study focused on cofilin1, an actin-binding protein that depolymerizes actin filaments. We showed that cofilin1 expression decreased at the compaction stage, and that down-regulation of cofilin1 expression by siRNA microinjection accelerated compaction. Continuous observation using time-lapse video miscroscopy confirmed these findings. That is, the embryonic cells microinjected with anti-cofilin1 antibody exhibit earlier adherence properties compared to uninjected cells. Pronuclear microinjection of a site-directed mutated cofilin1 plasmid, in which cofilin1 is sustained in its active form produced embryos with blastomeres that did not adhere, suggesting that inactivation of cofilin1 is critical for cell flattening and adherence. Fluorescein-phalloidin staining indicated that decreased cofilin1 expression promoted the formation of the apical pole, which is a marker for polarity. Scanning electron microscopy results demonstrated the appearance of microvilli on the outer face of blastomeres in cofilin1 knockdown embryos. Our results suggest that cofilin1 plays an important role in cortical cytoplasmic organization during embryo compaction.

Derivation of embryonic stem cells from Brown Norway rats blastocysts

Knockout Brown Norway (BN) rat could be a useful disease model for human disorders, however, a failure to derive embryonic stem (ES) cells disturbs the further development of the model. In this study, we reported a case of successful derivation of the BN rat ES cells with the derivation efficiency comparable to that of Sprague Dawley (SD) rats. The BN rat ES cells expressed the key transcription factors, and were able to form embryonic bodies (EBs) when being differentiated in vitro. After injecting the BN rat ES cells into the SD rat blastocysts, high-contribution chimeric rats were generated and could survive to their adulthood. Our success in generating pluripotent rat ES cells will benefit the generation of the knockout rats in the future.