Chunjing Feng

Androgenetic haploid embryonic stem cells produce live transgenic mice

Haploids and double haploids are important resources for studying recessive traits and have large impacts on crop breeding, but natural haploids are rare in animals. Mammalian haploids are restricted to germline cells and are occasionally found in tumours with massive chromosome loss. Recent success in establishing haploid embryonic stem (ES) cells in medaka fish and mice raised the possibility of using engineered mammalian haploid cells in genetic studies. However, the availability and functional characterization of mammalian haploid ES cells are still limited. Here we show that mouse androgenetic haploid ES (ahES) cell lines can be established by transferring sperm into an enucleated oocyte. The ahES cells maintain haploidy and stable growth over 30 passages, express pluripotent markers, possess the ability to differentiate into all three germ layers in vitro and in vivo, and contribute to germlines of chimaeras when injected into blastocysts. Although epigenetically distinct from sperm cells, the ahES cells can produce viable and fertile progenies after intracytoplasmic injection into mature oocytes. The oocyte-injection procedure can also produce viable transgenic mice from genetically engineered ahES cells. Our findings show the developmental pluripotency of androgenentic haploids and provide a new tool to quickly produce genetic models for recessive traits. They may also shed new light on assisted reproduction.

Genetic modification and screening in rat using haploid embryonic stem cells.

The rat is an important animal model in biomedical research, but practical limitations to genetic manipulation have restricted the application of genetic analysis. Here we report the derivation of rat androgenetic haploid embryonic stem cells (RahESCs) as a tool to facilitate such studies. Our approach is based on removal of the maternal pronucleus from zygotes to generate androgenetic embryos followed by derivation of ESCs. The resulting RahESCs have 21 chromosomes, express pluripotency markers, differentiate into three germ layer cells, and contribute to the germline. Homozygous mutations can be introduced by both large-scale gene trapping and precise gene targeting via homologous recombination or the CRISPR-Cas system. RahESCs can also produce fertile rats after intracytoplasmic injection into oocytes and are therefore able to transmit genetic modifications to offspring. Overall, RahESCs represent a practical tool for functional genetic studies and production of transgenic lines in rat.

One-step generation of p53 gene biallelic mutant Cynomolgus monkey via the CRISPR/Cas system

One-step generation of p53 gene biallelic mutant Cynomolgus monkey via the CRISPR/Cas system

Successful generation of cloned mice using nuclear transfer from induced pluripotent stem cells

Successful generation of cloned mice using nuclear transfer from induced pluripotent stem cells

Efficient generation of mouse ESCs-like pig induced pluripotent stem cells

Dear Editor, Porcine induced pluripotency stem cells (piPSCs) are promised in basic research, animal husbandry and regenerative medicine. However, the efficiency of the piPSCs induction has been low and the generated piPSCs varied in cell morphology and cell characteristics. Here we report a novel approach to improve efficiency of piPSCs generation. The induced piPSCs are dome-shaped mouse embryonic stem cells (ESCs)-like and display molecular properties of mouse ESCs. Electroporation study reveals that mouse ESCslike status facilitates genetic manipulating of piPSCs. Importantly, we demonstrate that the domed piPSC colonies are more suitable as donor cells for nuclear transfer (NT) to generate reconstructed embryos than those flattened piPSCs. The potential applications of the newly generated piPSCs in ungulate pluripotent research are discussed.

Induced Pluripotent Stem–Induced Cells Show Better Constitutive Heterochromatin Remodeling and Developmental Potential After Nuclear Transfer Than Their Parental Cells

Recently, reprogramming of somatic cells from a differentiated to pluripotent state by overexpression of specific external transcription factors has been accomplished. It has been widely speculated that an undifferentiated state may make donor cells more efficient for nuclear transfer. To test this hypothesis, we derived induced pluripotent stem cells (iPS cells) from several somatic cell lines: mouse embryonic fibroblast (MEF), adult tail tip fibroblast (TTF), and brain neural stem cells (NSCs). Three dimensional (3D)-fluorescent in situ hybridization (FISH) and quantitative-FISH (Q-FISH) were then used to evaluate constitutive (pericentric and telomeric) heterochromatin organization in these iPS cells and in their parental differentiated cells. Here, we show that important nuclear remodeling and telomeres rejuvenation occur in these iPS cells regardless of their parental origin. When we used these cells as donors for nuclear transfer, we produced live-born cloned mice at much higher rates with the iPS-induced cells than with the parental cell lines. Interestingly, we noticed that developmental potential after nuclear transfer could be correlated with telomere length of the donor cells. Altogether, our findings suggest that constitutive heterochromatin organization from differentiated somatic cells can be reprogrammed to the pluripotent state by induction of iPS cells, which in turn support nuclear transfer procedure quite efficiently.

Tex101 is essential for male fertility by affecting sperm migration into the oviduct in mice

Dear Editor, Sperm transport in the female genital tract is physiologically important for mammalian fertilization. The female reproductive system contains multiple natural selective barriers, such as successful uterotubal junction (UTJ) migration and zona pellucida (ZP) binding, to ensure sperm with normal motility and morphology to transmit into oviduct for fertilization (Yanagimachi, 1994; Ikawa et al., 2010). Tex101 is a glycosylphosphatidyl inositol (GPI)-anchored glycoprotein identified as a molecular marker of germ cells (Kurita et al., 2001). Although there have been indications that the malfunction of Tex101 may affect male fertility (Yin et al., 2009), little is known about its exact physiological function and the underlying molecular mechanisms. Recently, a study showed that Tex101 gene knockout sperm were unable to pass through UTJ or bind to ZP, which led to male infertility (Fujihara et al., 2013). Here, we independently generated Tex101 knockout mice and confirmed the infertile phenotype caused by UTJ migration defect. We also found that Tex101 knockout sperm lost the adhesive ability to the surface of female genital tract. Several members of a disintegrin and metalloprotease (ADAM) transmembrane protein family with cell adhesion ability, including ADAM3, ADAM4, ADAM5, and ADAM6, were lost in Tex101 knockout epididymal sperm. These observations may shed new light on the diagnosis of male infertility and development of contraceptive methods in human. High abundant Tex101 protein was only detected in the testis of male mice (Supplementary Figure S1A). To investigate the function of Tex101 in vivo, we generated Tex101 gene knockout mice (Supplementary Figure S1). During the 2-year observation period, neither Tex101 heterozygous mutant (Tex101+/2 ) nor Tex101 homozygous mutant (Tex101 ) mice (over 30 mice per group) showed any overt developmental abnormalities. However, although with normal mating ability, male Tex101 mice could not produce offspring, which confirmed the infertile defect of Tex101 deletion (Supplementary Table S1) (Fujihara et al., 2013). We next characterized the defects of Tex101 sperm causing male infertility. The histology and weight of testis from wildtype (Tex101+/+ ) and Tex101 male mice exhibited no identifiable difference (Supplementary Figure S2). In addition, no difference in sperm count, sperm viability, or motility parameters was observed (Supplementary Table S2). However, none of oocytes from females mated with Tex101 mice was fertilized at 18 h after mating plug formation (Supplementary Figure S3), suggesting that sperm from Tex101 mice were either unable to reach the fertilization place or unable to fertilize the oocytes. We then counted sperm collected from the oviducts of mated females. Large amounts of sperm were found in female mice mated with Tex101+/+ males (323 + 84, n 1⁄4 8), yet no sperm (0, n 1⁄4 24) was recovered from females mated with Tex101 males (Figure 1A). Similarly, sperm were only observed in the UTJ lumen of female mice mated with Tex101+/+ males but not those mated with Tex101 males (Supplementary Figure S4). These results demonstrated that Tex101 sperm were unable to pass through the UTJ of female genital tract. However, Tex101 sperm still fertilized oocytes (Figure 1B) at a lower rate compared with Tex101+/+ sperm (Figure 1C, 40% vs. 58%, P 1⁄4 0.048) in in vitro fertilization (IVF) assays. Moreover, among 24 in-tubal inseminated (ITI) female mice, four were successfully pregnant and produced 12 healthy offspring, indicating that Tex101 sperm were still capable to fertilize oocytes in vivo when the UTJ transportation was avoided (Figure 1D, E, and Supplementary Table S3). In contrast, in intra-uterine insemination (IUI) assays, no offspring was produced in the Tex101 group (Supplementary Table S3), further confirming that the male infertility defect of Tex101 mice was primarily caused by the UTJ migration defect of sperm. We noticed that Tex101 sperm seldom bound to dissected epithelium and ZP in the computer-assisted sperm analysis and IVF experiments. To further assess the membrane adhesive ability of Tex101 sperm, different cells inside the female genital tract, including the epithelium of UTJ and isthmus oviduct, cumulus cells, and oocytes, were dissected out and incubated separately in vitro with Tex101+/+ and Tex101 sperm. After incubation for 30 min, Tex101+/+ sperm adhered to all types of epithelium cells robustly, whereas Tex101 sperm were rarely attached (Figure 1F and G). These results demonstrated that sperm of Tex101 mice had lost their adhesive ability, thus failed to bind to the surface of cells in female genital tract. To investigate the functional mechanisms of Tex101, we used mass spectrometry to characterize the differentially expressed proteins between Tex101+/+ and Tex101 cauda epididymal sperm. A total of 30 proteins were identified with .1.5-fold expression changes, including two ADAM protein family members, ADAM5 and ADAM6 (Supplementary Table S4). Previous studies showed that ADAM3 but not other ADAM proteins played a key role in causing the infertile phenotypes (Ikawa et al., 2010; Fujihara et al., 2013); therefore, we detected the expression of all ADAM family proteins with predominant expression in testis by western blot. All examined proteins had no observable expression difference in testicular sperm between Tex101+/+ and Tex101 mice. However, in cauda doi:10.1093/jmcb/mjt031 Journal of Molecular Cell Biology (2013), 5, 345–347 | 345 Published online August 22, 2013

Pluripotency of Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells can be generated by forced expression of four pluripotency factors in somatic cells. This has received much attention in recent years since it may offer us a promising donor cell source for cell transplantation therapy. There has been great progress in iPS cell research in the past few years. However, several issues need to be further addressed in the near future before the clinical application of iPS cells, like the immunogenicity of iPS cells, the variability of differentiation potential and most importantly tumor formation of the iPS derivative cells. Here, we review recent progress in research into the pluripotency of iPS cells.

Generation of Transgenic Rats through Induced Pluripotent Stem Cells

Background: Rat induced pluripotent stem cells (riPSCs) failed to produce transgenic rats. Results: We found that an optimized induction medium improved the efficiency of iPSC generation from rat somatic cells. The riPSCs could successfully generate transgenic rats. Conclusion: We could generate high quality riPSCs that could be used to produce transgenic rats. Significance: RiPSCs can be used as a novel tool in genetic and genomic studies of the rat. The rat is an important animal model for human disease research. Using inhibitors of glycogen synthase kinase 3 and MAPK signaling pathways, rat embryonic stem cells and rat induced pluripotent stem cells (riPSCs) have been derived. However, unlike rat embryonic stem cells, germ line competent riPSCs have only been derived from Wistar rats at low efficiency. Here, we found that an optimized induction medium containing knock-out serum replacement and vitamin C improved the rate and efficiency of riPSCs generation from Dark Agouti rat fibroblasts and Sertoli cells. riPSCs maintained an undifferentiated status for >30 passages and could differentiate into various cells types including germ cells when injected into rat blastocysts. Moreover, transgenic riPSCs could be generated through the PiggyBac transposon, which could be used to generate transgenic rats through germ line transmission. riPSCs can be used as a novel tool in genetic and genomic studies of the rat.

Accreditation of Biosafe Clinical-Grade Human Embryonic Stem Cells According to Chinese Regulations

Summary Human embryonic stem cells (hESCs) are promising in regenerative medicine. Although several hESC-based clinical trials are under way, a widely accepted standard of clinical-grade cells remains obscure. To attain a completely xeno-free clinical-grade cell line, the system must be free of xenogenic components, the cells must have a comprehensive set of functions, and good manufacturing practice conditions must be used. In this study, following these criteria, we successfully derived two hESC lines, which were thereby considered “clinical-grade embryonic stem cells”. In addition to the primary capacity for pluripotency, these two cell lines were efficiently differentiated into various types of clinical-grade progeny. Importantly, the cells were recognized by the National Institutes for Food and Drug Control of China for further eligible accreditation. These data indicate that we have established completely xeno-free clinical-grade hESC lines and their derivatives, which will be valuable for the foundation of an international standard for clinical-grade cells for therapy.