[show abstract][hide abstract] ABSTRACT: Induced pluripotent stem (iPS) cells can be generated from mouse or human fibroblasts by exogenous expression of four factors, Oct4, Sox2, Klf4 and c-Myc, and hold great potential for transplantation therapies and regenerative medicine. However, use of retroviral vectors during iPS cell generation has limited the technique's clinical application due to the potential risks resulting from genome integration of transgenes, including insertional mutations and altered differentiation potentials of the target cells, which may lead to pathologies such as tumorigenesis. Here we review recent progressin generating safer transgene-free or integration-free iPS cells, including the use of non-integrating vectors, excision of vectors after integration, DNA-free delivery of factors and chemical induction of pluripotency.
[show abstract][hide abstract] ABSTRACT: The introduction of double-strand breaks (DSBs) at target sites could greatly enhance homologous recombination, and engineered nucleases, such as zinc finger and transcription activator-like effector nucleases, have been successfully developed for making such breaks. In this study, we present a highly efficient site-specific integration strategy based on homologous recombination and ΦC31 integrase. An attB sequence was introduced at the homologous arm of an insertion targeting vector. DSBs at the target locus and donor were then simultaneously generated by the ΦC31 integrase when co-transfected with the donor vector, consequently stimulating homologous recombination. The results demonstrated that our strategy is feasible and the efficiency at the BF4 target site, which we previously identified in the bovine genome, was as high as 93%. The frequency at another site (BF10) was almost two-fold greater in comparison to the vector without homologous arms. This technology requires no sophisticated nuclease design efforts, and the off-target effect is reduced by ΦC31 integrase compared to the use of engineered nucleases, thereby offering a simple and safe way to effectively express a donor gene at a desired locus. This development has great potential value, especially in transgenesis or gene therapy applications.
Journal of Biotechnology 08/2013; · 3.18 Impact Factor
[show abstract][hide abstract] ABSTRACT: Embryonic stem cells (ESCs) may be useful as a therapeutic source of cells for the production of healthy tissue; however, they are associated with certain challenges including immunorejection as well as ethical issues. Induced pluripotent stem cells (iPSCs) are a promising substitute since a patient's own adult cells would serve as tissue precursors. Ethical concerns prevent a full evaluation of the developmental potency of human ESCs and iPSCs, therefore, mouse iPSC models are required for protocol development and safety assessments. We used a modified culturing protocol to differentiate pluripotent cells from a mouse iPS cell line and two mouse ES cell lines into neurons. Our results indicated that all three pluripotent stem cell lines underwent nearly the same differentiation process when induced to form neurons in vitro. Genomic expression microarray profiling and single-cell RT-qPCR were used to analyze the neural lineage differentiation process, and more than one thousand differentially expressed genes involved in multiple molecular processes relevant to neural development were identified.
International Journal of Molecular Medicine 05/2013; · 1.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human amniotic fluid derived progenitor cells (hAFPCs) may be multipotent and can be considered a potential tool in the field of cell therapy for haemophilia B. Their capacity to express human coagulation factor IX (hFIX) after transduction and their fate after in utero transplantation is unknown. hAFPCs isolated from second trimester pregnancies were assessed for their phenotypic markers, multilineage capacity, and expression of hFIX after transduction. Their engraftment potential was analysed in a mouse model after in utero transplantation at embryonic day 12.5. Immunohistochemistry, fluorescence in situ, ELISA and PCR were used to assess post-transplant chimeras. hAFPCs expressed several pluripotent markers, including NANOG, SOX2, SSEA4 and TRA-1-60, and could differentiate into adipocytes and osteocytes. In vitro, after transduction with hFIX and EGFP cDNAs, constitutive hFIX protein expression and clotting activity were found. Engraftment was achieved in various foetal tissues after in utero transplantation. Safe engraftment without oncogenesis was confirmed, with low donor cell levels, but persistent engraftment, into different organs (liver, heart and lung) through to 12 weeks of age. Transgenic expression of circulating hFIX was detected in recipient mice for up to 12 weeks. hAFPCs can be engrafted long-term in immunocompetent mice after in utero transplantation. Thus, cell transplantation approaches using genetically engineered hAFPCs may prove valuable for the prenatal treatment for haemophilia B.
Cell Biology International 01/2013; · 1.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human induced pluripotent stem (iPS) cells have the ability to differentiate into all somatic cells and to maintain unlimited self-renewal. Therefore, they have great potential in both basic research and clinical therapy for many diseases. To identify potentially universal mechanisms of human somatic cell reprogramming, we studied gene expression changes in three types of cells undergoing reprogramming. The set of 570 genes commonly regulated during induction of iPS cells includes known embryonic stem (ES) cell markers and pluripotency related genes. We also identified novel genes and biological categories which may be related to somatic cell reprogramming. For example, some of the down-regulated genes are predicted targets of the pluripotency microRNA cluster miR302/367, and the proteins from these putative target genes interact with the stem cell pluripotency factor POU5F1 according to our network analysis. Our results identified candidate gene sets to guide research on the mechanisms operating during somatic cell reprogramming.
Journal of Genetics and Genomics 12/2012; 39(12):613-23. · 2.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Domesticated animals cloned by somatic cell nuclear transfer (SCNT) generally have poor developmental competency, with many developmental abnormalities attributed to incomplete reprogramming of the nuclear genome and abnormal expression of genes important for regulation of growth and development. To investigate the molecular mechanism leading to the abnormalities of cloned animals, pathologic and histologic analyses were conducted on seven cloned cattle that were oversized at birth and had cardiac and pulmonary abnormalities. Quantitative reverse transcription (RT)-polymerase chain reaction (PCR) analysis of four imprinted genes IGF2, IGF2R, H19, and GRB10, as well as genes from related regulatory networks, were performed in liver, lung, kidney, and muscle to investigate disruption of expression. Expression of IGFBP2 was not detected in morphologically normal cloned cattle, but was detected in the liver, lung, kidney, and thymus of abnormal calves. Expression levels of IGF1 and imprinted genes IGF2 and H19 were substantially higher in these organs of abnormal cattle. In contrast, expression levels of GRB10, CTSD, and TRPV2 were substantially lower in abnormal cattle. Transcript abundance of IGFBP6 was higher in kidney, but lower in liver and lung. In conclusion, we inferred that altered expression of imprinted and related genes may be closely related to increased birth weight and pathologic changes in abnormal cloned cattle.
[show abstract][hide abstract] ABSTRACT: The dispersion of small weight fraction of carbon nanotube (CNT) in Nylon 6,6 introduces a significant difference in the structure and phase evolution during crystallization at ambient and elevated pressures. In the nanocomposite, the γ-phase is promoted at low crystallization pressure of ∼0.1–25 MPa and is in striking contrast to pure Nylon 6,6, where γ-phase is nucleated only at crystallization pressures exceeding ∼50 MPa. The differences in the behavior of Nylon 6,6 and its nanocomposites is attributed to CNT–polymer interface driven nucleation, which is also responsible for significant reduction in spherulite size and increase in crystallinity. The nanoindentation behavior of the nanocomposite is assessed via nanoscale deformation experiments, which indicated that a significantly higher indentation-force is required for the Nylon 6,6–CNT nanocomposite as compared to Nylon 6,6 to produce a constant displacement. The observed significantly higher modulus and hardness is primarily CNT-induced effect. This observation is of particular relevance to functional devices because they are most likely to experience force in the nanonewton range, which can induce deformation at the micro- and/or nanometer scale.
Materials Science and Engineering: B. 05/2012; 177(9):666–672.
[show abstract][hide abstract] ABSTRACT: The Streptomyces phage φC31 integrase can efficiently target attB-bearing transgenes to endogenous pseudo attP sites within mammalian genomes. To better understand the activity of φC31 integrase in the bovine genome, DNA sequences of 44 integration events were analyzed, and 32 pseudo attP sites were identified. The majority of these sites share a sequence motif that contains inverted repeats and has similarities to wild-type attP site. Genomic DNA flanking these sites typically contained repetitive sequence elements, such as short and long interspersed repetitive elements. These sequence features indicate that DNA sequence recognition plays an important role in guiding φC31-mediated site-specific integration. In addition, BF27 integration hotspot sites were identified in the bovine genome, which accounted for 13.6% of all isolated integration events and mapped to an intron of the deleted in liver cancer 1 (DLC1) gene. Also we found that the pseudo attP sites in the bovine genome had other features in common with those in the human genome. This study represents the first time that the sequence features of pseudo attP sites in the bovine genome were analyzed. We conclude that this site-specific integrase system has great potential for applied modifications of the bovine genome.
Journal of Genetics and Genomics 05/2012; 39(5):217-24. · 2.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: ΦC31 integrase, a site-specific recombinase, can catalyze integration of circular DNA bearing attB site into pseudo attP sites in mammalian genomes. However, the integration efficiency mediated by integrase is relatively low. Our study centered on the investigation of the impact of the position, orientation, and number of attBs in the donor plasmid on the efficiency of ΦC31 integrase system. Donor plasmids bearing various types of attBs (including forward and reverse directions, tandem, and intersperse) and reporter enhanced green fluorescent protein (EGFP) were constructed. The plasmids plus helper plasmid encoding integrase were co-transfected into HeLa cells. After G418 selection, the resistant cell colonies were counted for calculating chromosomal integration frequency. EGFP expression was detected by fluorescence-activated cell sorter and enzyme-linked immunosorbent assay analysis. The results showed that efficiency of integration mediated by integrase accounted for 70% ± 7.1% of total integration events in the transfected HeLa cells. Compared with a forward orientation of attB in donor plasmid, a reverse direction of attB or interspersed attBs showed 1.5- or 2.8-fold increase in integration efficiency, respectively, while tandem attBs in donor plasmids caused a decreased efficiency of integration. We conclude that the adjustment of attB sites in donor plasmids may be of value for gene therapy and routine genetic engineering by using ΦC31 integrase system.
DNA and cell biology 04/2012; 31(7):1335-40. · 2.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The liver is the largest internal organ in mammals, and is important for the maintenance of normal physiological functions of other tissues and organs. Hepatitis, cirrhosis, liver cancer and other chronic liver diseases are serious threats to human health, and these problems are compounded by a scarcity of liver donors for transplantation therapies. Directed differentiation of embryonic stem cells to liver cells is a promising strategy for obtaining hepatocytes that can be used for cell transplantation. In vitro hepatocyte differentiation of embryonic stem cells requires a profound understanding of normal development during embryonic hepatogenesis. Here we provide a simple description of hepatogenesis in vivo and discuss directed differentiation of embryonic stem cells into hepatocytes in vitro.
Protein & Cell 03/2011; 2(3):180-8. · 3.22 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although the therapeutic efficacy of β(654)-thalassaemia treatment using a combination of RNAi and antisense RNA to balance the synthesis of α- and β-globin chains has been demonstrated previously, and the safety of lentiviral delivery remains unclear. Herein, we used the same β(654)-thalassaemia mouse model to develop a therapy involving direct delivery of siRNA and antisense RNA plasmids via intravenous injection to simultaneously knock down α-globin transcript levels and restore correct β-globin splicing. The amount of α-globin mRNAs in siRNA-treated MEL cells decreased significantly, and the properly spliced β-globin mRNA was restored in HeLaβ(654) cells transfected with pcDNA-antisense plasmid. Furthermore, treatment of β(654)-thalassaemic mice with siRNA and antisense RNA plasmids resulted in significant reduction of poikilocytosis and reticulocyte counts in blood samples, decreased nucleated cell populations in bone marrow, and reduced intrasinusoidal extramedullary haematopoiesis loci and iron accumulation in liver. RT-PCR analysis revealed that treatment resulted in down-regulation of α-globin mRNA synthesis by ~50% along with an increase in the presence of normally spliced β-globin transcripts, indicating that the phenotypic changes observed in β(654)-thalassaemic mice following treatment resulted from restoration of the balance of α/β-globin biosynthesis.
International journal of hematology 03/2011; 93(3):301-10. · 1.17 Impact Factor
[show abstract][hide abstract] ABSTRACT: Reconstructed embryos derived from intersubspecies somatic cell nuclear transfer (SCNT) have poorer developmental potential than those from intrasubspecies SCNT. Based on our previous study that Holstein dairy bovine (HD) mitochondrial DNA (mtDNA) haplotype compatibility between donor karyoplast and recipient cytoplast is crucial for SCNT embryo development, we performed intersubspecies SCNT using HD as donor karyoplast and Luxi yellow heifer (LY) as recipient cytoplast according to mtDNA haplotypes determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The results demonstrated that intersubspecies mtDNA homotype SCNT embryos had higher pre- and post-implantation developmental competence than intrasubspecies mtDNA heterotype embryos as well as improved blastocyst reprogramming status, including normal H3K9 dimethylation pattern and promoter hypomethylation of pluripotent genes such as Oct4 and Sox2, suggesting that intersubspecies SCNT using LY oocytes maintains HD cloning efficiency and may reprogram HD nuclei to develop into a normal cloned animal ultimately. Our results indicated that karyoplast-cytoplast interactions and mtDNA haplotype compatibility may affect bovine intersubspecies SCNT efficiency. This study on bovine intersubspecies SCNT is valuable for understanding the mechanisms of mtDNA haplotype compatibility between karyoplast and cytoplast impacting the bovine SCNT efficiency, and provides an alternative and economic resource for HD cloning.
Journal of Genetics and Genomics 01/2011; 38(1):21-8. · 2.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Chromosome abnormalities, especially trisomy of chromosome 21, 13, or 18 as well as sex chromosome aneuploidy, are a well-established cause of pregnancy loss. Cultured cell karyotype analysis and FISH have been considered reliable detectors of fetal abnormality. However, results are usually not available for 3-4 days or more. Multiplex ligation-dependent probe amplification (MLPA) has emerged as an alternative rapid technique for detection of chromosome aneuploidies. However, conventional MLPA does not allow for relative quantification of more than 50 different target sequences in one reaction and does not detect mosaic trisomy. A multiplexed MLPA with more sensitive detection would be useful for fetal genetic screening.
We developed a method of array-based MLPA to rapidly screen for common aneuploidies. We designed 116 universal tag-probes covering chromosomes 13, 18, 21, X, and Y, and 8 control autosomal genes. We performed MLPA and hybridized the products on a 4-well flow-through microarray system. We determined chromosome copy numbers by analyzing the relative signals of the chromosome-specific probes.
In a blind study of 161 peripheral blood and 12 amniotic fluid samples previously karyotyped, 169 of 173 (97.7%) including all the amniotic fluid samples were correctly identified by array-MLPA. Furthermore, we detected two chromosome X monosomy mosaic cases in which the mosaism rates estimated by array-MLPA were basically consistent with the results from karyotyping. Additionally, we identified five Y chromosome abnormalities in which G-banding could not distinguish their origins for four of the five cases.
Our study demonstrates the successful application and strong potential of array-MLPA in clinical diagnosis and prenatal testing for rapid and sensitive chromosomal aneuploidy screening. Furthermore, we have developed a simple and rapid procedure for screening copy numbers on chromosomes 13, 18, 21, X, and Y using array-MLPA.
BMC Medical Genetics 01/2011; 12:68. · 2.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: Somatic cells can now be reprogrammed back to an embryonic stem cell–like pluripotent state by forced expression of external
factors, generating induced pluripotent stem (iPS) cells. These iPS cells resemble embryonic stem cells (ESC) at many different
levels, as measured by multiple in vitro to in vivo assays.In particular, full-term live, viable, and fertile mice were recently
generated from iPS cells through the most stringent test for pluripotency, the tetraploid complementation assay. This demonstration
of the true pluripotency of these iPS cells indicates that they are an ideal alternative to ESCs for future studies and applications
such as in regenerative medicine, disease investigation, and pharmaceutical development.
KeywordsInduced pluripotent stem cells-Chimera generation-Teratoma formation-Germline transmission
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The interaction between the karyoplast and cytoplast plays an important role in the efficiency of somatic cell nuclear transfer (SCNT), but the underlying mechanism remains unclear. It is generally accepted that in nuclear transfer embryos, the reprogramming of gene expression is induced by epigenetic mechanisms and does not involve modifications of DNA sequences. In cattle, oocytes with various mitochondrial DNA (mtDNA) haplotypes usually have different ATP content and can further affect the efficiency of in vitro production of embryos. As mtDNA comes from the recipient oocyte during SCNT and is regulated by genes in the donor nucleus, it is a perfect model to investigate the interaction between donor nuclei and host oocytes in SCNT.
We investigated whether the in vitro development of reconstructed bovine embryos produced by SCNT would be influenced by mtDNA haplotype compatibility between the oocytes and donor cells. Embryos from homotype A-A or B-B showed significantly higher developmental ability at blastocyst stages than the heterotype A-B or B-A combinations. Post-implantation development ability, pregnancy rate up to day 90 of gestation, as well as percent of term births were higher in the homotype SCNT groups than in the heterotype groups. In addition, homotype and heterotype SCNT embryos showed different methylation patterns of histone 3-lysine 9 (H3K9) genome-wide and at pluripotency-related genes (Oct-4, Sox-2, Nanog).
Both histone and DNA methylation show that homotype SCNT blastocysts have a more successful epigenetic asymmetry pattern than heterotype SCNT blastocysts, which indicates more complete nuclear reprogramming. This may result from variability in their epigenetic patterns and responses to nuclear reprogramming. This suggests that the compatibility of mtDNA haplotypes between donor cells and host oocytes can significantly affect the developmental competence of reconstructed embryos in SCNT, and may include an epigenetic mechanism.