Liangxue Lai

A Mesenchymal-to-Epithelial Transition Initiates and Is Required for the Nuclear Reprogramming of Mouse Fibroblasts

Epithelial-to-mesenchymal transition (EMT) is a developmental process important for cell fate determination. Fibroblasts, a product of EMT, can be reset into induced pluripotent stem cells (iPSCs) via exogenous transcription factors but the underlying mechanism is unclear. Here we show that the generation of iPSCs from mouse fibroblasts requires a mesenchymal-to-epithelial transition (MET) orchestrated by suppressing pro-EMT signals from the culture medium and activating an epithelial program inside the cells. At the transcriptional level, Sox2/Oct4 suppress the EMT mediator Snail, c-Myc downregulates TGF-beta1 and TGF-beta receptor 2, and Klf4 induces epithelial genes including E-cadherin. Blocking MET impairs the reprogramming of fibroblasts whereas preventing EMT in epithelial cells cultured with serum can produce iPSCs without Klf4 and c-Myc. Our work not only establishes MET as a key cellular mechanism toward induced pluripotency, but also demonstrates iPSC generation as a cooperative process between the defined factors and the extracellular milieu. PAPERCLIP:

Generation of induced pluripotent stem cell lines from tibetan miniature pig

Induced pluripotent stem cell (iPS) technology appears to be a general strategy to generate pluripotent stem cells from any given mammalian species. So far, iPS cells have been reported for mouse, human, rat, and monkey. These four species have also established embryonic stem cell (ESC) lines that serve as the gold standard for pluripotency comparisons. Attempts have been made to generate porcine ESC by various means without success. Here we report the successful generation of pluripotent stem cells from fibroblasts isolated from the Tibetan miniature pig using a modified iPS protocol. The resulting iPS cell lines more closely resemble human ESC than cells from other species, have normal karyotype, stain positive for alkaline phosphatase, express high levels of ESC-like markers (Nanog, Rex1, Lin28, and SSEA4), and can differentiate into teratomas composed of the three germ layers. Because porcine physiology closely resembles human, the iPS cells reported here provide an attractive model to study certain human diseases or assess therapeutic applications of iPS in a large animal model.

Generation of cloned transgenic pigs rich in omega-3 fatty acids

Meat products are generally low in omega-3 (n-3) fatty acids, which are beneficial to human health. We describe the generation of cloned pigs that express a humanized Caenorhabditis elegans gene, fat-1, encoding an n-3 fatty acid desaturase. The hfat-1 transgenic pigs produce high levels of n-3 fatty acids from n-6 analogs, and their tissues have a significantly reduced ratio of n-6/n-3 fatty acids (P < 0.001).

Microrna cluster 302-367 enhances somatic cell reprogramming by accelerating a mesenchymal-to-epithelial transition

MicroRNAs (miRNAs) are emerging critical regulators of cell function that frequently reside in clusters throughout the genome. They influence a myriad of cell functions, including the generation of induced pluripotent stem cells, also termed reprogramming. Here, we have successfully delivered entire miRNA clusters into reprogramming fibroblasts using retroviral vectors. This strategy avoids caveats associated with transient transfection of chemically synthesized miRNA mimics. Overexpression of 2 miRNA clusters, 106a–363 and in particular 302–367, allowed potent increases in induced pluripotent stem cell generation efficiency in mouse fibroblasts using 3 exogenous factors (Sox2, Klf4, and Oct4). Pathway analysis highlighted potential relevant effectors, including mesenchymal-to-epithelial transition, cell cycle, and epigenetic regulators. Further study showed that miRNA cluster 302–367 targeted TGFβ receptor 2, promoted increased E-cadherin expression, and accelerated mesenchymal-to-epithelial changes necessary for colony formation. Our work thus provides an interesting alternative for improving reprogramming using miRNAs and adds new evidence for the emerging relationship between pluripotency and the epithelial phenotype.

Transgenic swine for biomedicine and agriculture

Initial technologies for creating transgenic swine only permitted random integration of the construct. However, by combining the technology for homologous recombination in fetal somatic cells with that of nuclear transfer (NT), it is now possible to create specific modifications to the swine genome. The first such example is that of knocking out a gene that is responsible for hyperacute rejection (HAR) when organs from swine are transferred to primates. Because swine are widely used as models of human diseases, there are opportunities for genetic modification to alter these models or to create additional models of human disease. Unfortunately, some of the offspring resulting from NT have abnormal phenotypes. However, it appears that these abnormal phenotypes are a result of epigenetic modifications and, thus, are not transmitted to the offspring of the clones. Although the technique of producing animals with specific genetic modifications by NT has been achieved, improvements to the NT technique as well as improvements in the culture conditions for somatic cells and the techniques for genetic modification are still needed.

PRODUCTION OF NUCLEAR TRANSFER-DERIVED SWINE THAT EXPRESS THE ENHANCED GREEN FLUORESCENT PROTEIN

The ability to add or delete specific genes in swine will likely provide considerable benefits not just to agriculture but also to medicine, where pigs have potential as models for human disease and as organ donors. Here we have transferred nuclei from a genetically modified fibroblast cell line to porcine oocytes, matured in vitro under defined culture conditions, to create piglets expressing enhanced green fluorescent protein. The nuclear transfer-derived piglets were of normal size, although some mild symptoms of “large offspring syndrome” were evident. These experiments represent a next step towards creating swine with more useful genetic modifications.

Mosaic Gene Expression in Nuclear Transfer-Derived Embryos and the Production of Cloned Transgenic Pigs from Ear-Derived Fibroblasts

Abstract Genetically modified domestic animals have many potential applications ranging from basic research to production agriculture. One of the goals in transgenic animal production schemes is to reliably predict the expression pattern of the foreign gene. Establishing a method to screen genetically modified embryos for transgene expression before transfer to surrogates may improve the likelihood of producing offspring with the desired expression pattern. In order to determine how transgene expression may be regulated in the early embryo, we generated porcine embryos from two distinct genetically modified cell lines by using the nuclear transfer (NT) technique. Both cell lines expressed the enhanced green fluorescent protein (eGFP); the first was a fibroblast cell line derived from the skin of a newborn pig that expressed eGFP, whereas the second was a fetal derived fibroblast cell line into which the eGFP gene was introduced by a retroviral vector. The reconstructed embryos were activated by electrical pulses and cultured in NCSU23. Although the in vitro developmental ability of each group of NT embryos was not different, the eGFP expression pattern was different. All embryos produced from the transduced fetal cell line fluoresced, but only 26% of the embryos generated from the newborn cell line fluoresced, and among those that did express eGFP, more than half had a mosaic expression pattern. This was unexpected because the fetal cell line was not clonally selected, and each cell had potentially different sites of integration. Embryos generated from the newborn cell line were surgically transferred to five surrogate gilts. One gilt delivered four female piglets, all of which expressed eGFP, and all had microsatellites identical to the donor. Here we demonstrate that transgene expression in all the blastomeres of an NT embryo is not uniform. In addition, transgene expression in a genetically manipulated embryo may not be an accurate indicator of expression in the resulting offspring.

Production of cloned pigs by using somatic cells as donors.

NUCLEAR TRANSFER is a procedure by which genetically identical individuals can be created. The applications of these techniques for nuclear transfer will be in agricultural, biomedical and basic research. Based on the sources of donor cells, nuclear transfer can be classified into embryonic cell nuclear transfer and somatic cell nuclear transfer. Offspring from cultured cells were first reported in 1994 (Sims and First, 1994) in cattle. Since this paper showed that cells could be cultured at least 28 days prior to nuclear transfer, it held great promise for genetically modifying the cells prior to nuclear transfer, and thus the possibility of producing animals with specific genetic modifications. This concept is exemplified by the generation of transgenic sheep (Schnieke et al., 1997), pigs (Park et al., 2001), calves (Cibelli et al., 1998a), and gene-targeted sheep (McCreath et al., 2000) and pigs (Lai et al., 2002b), derived from nuclear transfer approaches by using transfected somatic cells. For pigs, somatic cell nuclear transfer has another specific significance, as is it would allow the use of genetic modification procedures to produce tissues and organs from cloned pigs with reduced immunogenicity for use in xenotransplantation (Lai et al., 2002b). However, the efficiency of somatic cell nuclear transfer, when measured as development to term as a proportion of oocytes used, has been very low (1–2%). A number of variables influence the ability to reproduce a specific genotype by cloning. These include species, source of recipient ova, cell type of nuclei donor, treatment of donor cells prior to nuclear transfer, the method of artificial oocyte activation, embryo culture, possible loss of somatic imprinting in the nuclei of reconstructed embryos, failure of adequate reprogramming of the transplanted nucleus, and the techniques employed for nuclear transfer. In pig, there are additional difficulties in that the quality of embryos produced in vitro is low, and at least four good embryos are required to initiate and establish a pregnancy. Procedures for nuclear transfer include the following steps: acquisition of recipient oocytes and donor cells, enucleation (removal of the chromosome from recipient oocytes), insertion of donor nuclei into enucleated oocytes, artificial activation of reconstructed oocytes, and embryo transfer (transfer of the reconstructed embryos into a surrogate).

Dynamic Events Are Differently Mediated by Microfilaments, Microtubules, and Mitogen-Activated Protein Kinase During Porcine Oocyte Maturation and Fertilization In Vitro

Abstract The role of microfilaments, microtubules, and mitogen-activated protein (MAP) kinase in regulation of several important dynamic events of porcine oocyte maturation and fertilization is described. Fluorescently labeled microfilaments, microtubules, and cortical granules were visualized using either epifluorescence microscopy or laser scanning confocal microscopy. Mitogen-activated protein kinase phosphorylation was revealed by Western immunoblotting. We showed that 1) microfilament disruption did not affect meiosis resumption and metaphase I meiotic apparatus formation but inhibited further cell cycle progression (chromosome separation) even though MAP kinase was phosphorylated; 2) cortical granule (CG) migration was driven by microfilaments (but not microtubules), and once the chromosomes and CGs were localized beneath the oolemma their anchorage to the cortex was independent of either microfilaments or microtubules; 3) neither microfilaments nor microtubules were involved in CG exocytosis during oocyte activation; 4) sperm incorporation was mediated by microfilaments, while pronuclear (PN) syngamy was controlled by microtubules rather than microfilaments; 5) spindle microtubule organization was temporally correlated with MAP kinase phosphorylation, while the extensive microtubule organization in the sperm aster that is required for PN apposition and syngamy occurred in the absence of MAP kinase activation; and 6) MAP kinase phosphorylation did not change either when microtubules were disrupted by nocodazole or when cytoplasmic microtubule asters were induced by taxol. The present study suggests that the role of the cytoskeleton during porcine oocyte maturation is similar to that of rodents, while the mechanisms of fertilization in pig resemble those of lower vertebrates.

Expression of Huntington’s disease protein results in apoptotic neurons in the brains of cloned transgenic pigs

Neurodegeneration is a hallmark of many neurological diseases, including Alzheimers, Parkinsons and the polyglutamine diseases, which are all caused by misfolded proteins that accumulate in neuronal cells of the brain. Although apoptosis is believed to contribute to neurodegeneration in these cases, genetic mouse models of these diseases often fail to replicate apoptosis and overt neurodegeneration in the brain. Using nuclear transfer, we generated transgenic Huntingtons disease (HD) pigs that express N-terminal (208 amino acids) mutant huntingtin with an expanded polyglutamine tract (105Q). Postnatal death, dyskinesia and chorea-like movement were observed in some transgenic pigs that express mutant huntingtin. Importantly, the transgenic HD pigs, unlike mice expressing the same transgene, displayed typical apoptotic neurons with DNA fragmentation in their brains. Also, expression of mutant huntingtin resulted in more neurons with activated caspase-3 in transgenic pig brains than that in transgenic mouse brains. Our findings suggest that species differences determine neuropathology and underscore the importance of large mammalian animals for modeling neurological disorders.