Dongshan Yang

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:

RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency.

Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells.

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.

Generation of PPARγ mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning

Generation of PPARγ mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning

Use of the 2A Peptide for Generation of Multi-Transgenic Pigs through a Single Round of Nuclear Transfer

Multiple genetic modifications in pigs can essentially benefit research on agriculture, human disease and xenotransplantation. Most multi-transgenic pigs have been produced by complex and time-consuming breeding programs using multiple single-transgenic pigs. This study explored the feasibility of producing multi-transgenic pigs using the viral 2A peptide in the light of previous research indicating that it can be utilized for multi-gene transfer in gene therapy and somatic cell reprogramming. A 2A peptide-based double-promoter expression vector that mediated the expression of four fluorescent proteins was constructed and transfected into primary porcine fetal fibroblasts. Cell colonies (54.3%) formed under G418 selection co-expressed the four fluorescent proteins at uniformly high levels. The reconstructed embryos, which were obtained by somatic cell nuclear transfer and confirmed to express the four fluorescent proteins evenly, were transplanted into seven recipient gilts. Eleven piglets were delivered by two gilts, and seven of them co-expressed the four fluorescent proteins at equivalently high levels in various tissues. The fluorescence intensities were directly observed at the nose, hoof and tongue using goggles. The results suggest that the strategy of combining the 2A peptide and double promoters efficiently mediates the co-expression of the four fluorescent proteins in pigs and is hence a promising methodology to generate multi-transgenic pigs by a single nuclear transfer.

Towards an Optimized Culture Medium for the Generation of Mouse Induced Pluripotent Stem Cells

Generation of induced pluripotent stem cells from somatic cells using defined factors has potential relevant applications in regenerative medicine and biology. However, this promising technology remains inefficient and time consuming. We have devised a serum free culture medium termed iSF1 that facilitates the generation of mouse induced pluripotent stem cells. This optimization of the culture medium is sensitive to the presence of Myc in the reprogramming factors. Moreover, we could reprogram meningeal cells using only two factors Oct4/Klf4. Therefore, iSF1 represents a basal medium that may be used for mechanistic studies and testing new reprogramming approaches.

RAG1/2 Knockout Pigs with Severe Combined Immunodeficiency

Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activator-like effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1, 3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG1/2 knockout pigs are promising tools for biomedical and translational research.

Establishment of a Porcine Oct-4 Promoter-Driven EGFP Reporter System for Monitoring Pluripotency of Porcine Stem Cells

Porcine pluripotent cells with the capacity to generate germ line chimeras have not been developed yet. The transcription factor Oct-4 is an important marker of undifferentiating status and a central regulator of pluripotency in cells. Establishment of an Oct-4 promoter-based reporter system, such as that used in mice, will be a useful tool for monitoring the differentiating statuses of porcine cells both in vivo and in vitro. In the present study, we constructed a vector, pOGN2, in which enhanced green fluorescent protein (EGFP) was driven by the porcine Oct-4 promoter. In pigs containing this vector, EGFP was expected to be specifically expressed in pluripotent cells. We delivered the vectors into porcine fetal fibroblasts (PEFs) using liposomes. After transfected PEFs were selected with G418, we established eight cell lines containing the pOGN2 vector. When transgenic cells were used as donor nuclei to make somatic cell nuclear transfer (SCNT) embryos, SCNT embryos derived from four transgenic cell lines expressed green fluorescence. When PEFs with pOGN2 vectors were infected with retroviral vectors encoding the four transcription factors (Oct-4, Sox2, Klf4, and c-Myc), EGFP-expressing iPS cell colonies were observed at day 20. This work lays a foundation that can be used to generate a pig strain with an Oct4-EGFP reporter system, which would be greatly helpful in studying the differentiating and reprogramming mechanisms of pig embryos.

ApoE knockout rabbits: A novel model for the study of human hyperlipidemia

OBJECTIVE Rabbits are one of the best animal models for the study of hyperlipidemia and atherosclerosis. Although many transgenic rabbits have been created, the development of gene knockout (KO) rabbits has been impossible due to the lack of rabbit embryonic stem cells. We along with others recently generated KO rabbits using genome editing techniques. In the current study, we characterized the lipoprotein profiles of apoE KO rabbits on both chow and cholesterol diets and investigated their susceptibility to a diet-induced atherosclerosis. APPROACH AND RESULTS We analyzed plasma lipids and lipoproteins of apoE KO rabbits and compared them with those of wild-type rabbits. On a chow diet, homozygous (but not heterozygous) apoE KO rabbits showed mild hyperlipidemia and, when challenged with a cholesterol diet, they showed greater susceptibility to diet-induced hyperlipidemia than did the wild-type rabbits and their plasma total cholesterol levels were remarkably increased (1070 ± 61 mg/dL in apoE KO vs. 169 ± 79 mg/dL in the wild type, p < 0.001). Hyperlipidemia in apoE KO rabbits was caused by elevated remnant lipoproteins. Interestingly, increased remnant lipoproteins in apoE KO rabbits were predominated by apoB-48 and rich in both apoA-I and apoA-IV contents. Furthermore, apoE KO rabbits developed greater aortic atherosclerosis than wild-type rabbits when fed with a cholesterol diet for 10 weeks. CONCLUSIONS To our knowledge, this is the first report of generating KO rabbits for the study of lipid and lipoprotein metabolism. ApoE KO rabbits should be a useful model for the study of human hyperlipidemia and atherosclerosis.

Species-dependent neuropathology in transgenic SOD1 pigs

Mutations in the human copper/zinc superoxide dismutase 1 (hSOD1) gene cause familial amyotrophic lateral sclerosis (ALS). It remains unknown whether large animal models of ALS mimic more pathological events seen in ALS patients via novel mechanisms. Here, we report the generation of transgenic pigs expressing mutant G93A hSOD1 and showing hind limb motor defects, which are germline transmissible, and motor neuron degeneration in dose- and age-dependent manners. Importantly, in the early disease stage, mutant hSOD1 did not form cytoplasmic inclusions, but showed nuclear accumulation and ubiquitinated nuclear aggregates, as seen in some ALS patient brains, but not in transgenic ALS mouse models. Our findings revealed that SOD1 binds PCBP1, a nuclear poly(rC) binding protein, in pig brain, but not in mouse brain, suggesting that the SOD1-PCBP1 interaction accounts for nuclear SOD1 accumulation and that species-specific targets are key to ALS pathology in large mammals and in humans.