Animal Cloning Research Articles

Culture of somatic cells isolated from frozen-thawed equine semen using fluorescence-assisted cell sorting

Nuclear transfer using somatic cells from frozen semen (FzSC) would allow cloning of animals for which no other genetic material is available. Horses are one of the few species for which cloning is commercially feasible; despite this, there is no information available on the culture of equine FzSC. After preliminary trials on equine FzSC, recovered by density-gradient centrifugation, resulted in no growth, we hypothesized that sperm in the culture system negatively affected cell proliferation. Therefore, we evaluated culture of FzSC isolated using fluorescence-assisted cell sorting. In Exp. 1, sperm were labeled using antibodies to a sperm-specific antigen, SP17, and unlabeled cells were collected. This resulted in high sperm contamination. In Exp. 2, FzSC were labeled using an anti-MHC class I antibody. This resulted in an essentially pure population of FzSC, 13–25% of which were nucleated. Culture yielded no proliferation in any of nine replicates. In Exp. 3, 5 × 103 viable fresh, cultured horse fibroblasts were added to the frozen-thawed, washed semen, then this suspension was labeled and sorted as for Exp. 2. The enriched population had a mean of five sperm per recovered somatic cell; culture yielded formation of monolayers. In conclusion, an essentially pure population of equine FzSC could be obtained using sorting for presence of MHC class I antigens. No equine FzSC grew in culture; however, the proliferation of fibroblasts subjected to the same processing demonstrated that the labeling and sorting methods, and the presence of few sperm in culture, were compatible with cell viability.

31 Overall Goat Cloning Efficiency Under Suboptimal Conditions — A 6-Year Experience

Animal cloning involves a combination of several simple steps that need to be carried out at the highest efficacy to provide acceptable yet low overall cloning efficiency. Oocyte competence is key for proper somatic cell nucleus reprogramming, and technical elements must be refined to minimize interference in the overall outcome. The aim of this study was to compare the progress of a goat-cloning program over a 6-year period in which oocyte donors’ protocols and oocyte and embryo manipulation were continuously refined to cope with success under Brazilian semi-arid conditions. The cloning dataset was divided in 3 periods (P1, 2011-2012; P2, 2013-2014; P3, 2015-2016), using either in vivo- or in vitro-matured goat oocytes for cloning by nuclear transfer (NT) with different cell lines for subsequent transfer to synchronized recipients to produce transgenic liveborn kids. Over time, protocols for recovery of competent oocytes were adapted to existing conditions, also optimizing animal well being and nutritional status to attain better success. Data on total and viable oocytes, oocyte maturation, pregnancy, and cloning efficiency were analysed by the Chi-squared or t-test (P < 0.05). After 111 replicates, viable and matured oocyte rates were improved during the time, but mean number of total oocytes/donor were higher in the second period (25.4 ± 8.9 v. 18.5 ± 10.8 for P1 and 19.5 ± 6.1 for P3), whereas the mean of viable and matured oocytes were similar between P2 (19.4 ± 6.0 and 9.8 ± 4.6) and P3 (18.8 ± 7.0 and 9.6 ± 3.7), but higher than in P1 (12.8 ± 7.6 and 6.0 ± 4.1), respectively. Higher pregnancy rate was achieved in P1 and lower in P2, with both being similar to P3. However, the efficiency based on transferred embryos was similar between periods (Table 1). When compared with total oocytes, birth rate improved from the first to the last period (P1, 0.01%; P2, 0.02%; and P3, 0.09%). Cloning efficiency was measured by the number of transferred embryos compared with matured structures used for cloning, which showed an improvement over time (P1, 34.5%; P2, 43.9%; P3, 60.1%). A 5-fold enhancement was observed in the number of matured oocytes needed to produce a liveborn animal (P1, 2,779/1; P2, 1,892/1; P3, 545/1). Also, the number of donors (P1, 432; P2, 193.5; P3, 56.6) and recipients (P1, 114; P2, 57; P3, 27) needed to produce 1 animal was reduced by 7.6- and 4.2-fold between periods. In conclusion, complex and inefficient procedures such as cloning by NT require technical refinement and adjustments to existing conditions, including animal nutrition and welfare, and cumulative gain of expertise to attain successful results. Table 1.Overall goat cloning efficiency over time.

High rates of colonisation by ampicillin-resistant enterococci in residents of long-term care facilities in Porto, Portugal

This study evaluated the occurrence of enterococci resistant to clinically relevant antibiotics in long-term care facility (LTCF) residents in Porto, Portugal, a region with high rates of multidrug-resistant enterococci in infected patients and healthy carriers. Faecal samples from 48 residents in two LTCFs (2015–2016) were enriched (with/without antibiotics) and plated on Slanetz–Bartley with/without the same antibiotics (ampicillin/vancomycin/linezolid). Two colonies per morphology/sample were selected for susceptibility testing and species identification. Clonality was established by PFGE and MLST. Genes coding for vancomycin resistance (vanA/vanB), virulence and plasmids (replicases) were searched by PCR. A total of 285 isolates were obtained, comprising Enterococcus faecalis, Enterococcus faecium, Enterococcus raffinosus and Enterococcus avium colonising 83%, 77%, 27% and 10% of residents, respectively. Residents from both LTCFs were colonised with vancomycin-resistant E. faecium (VanA-VREfm) (4 residents; 8%) and/or ampicillin-resistant (AmpR) (24 residents; 50%) E. raffinosus, E. faecium and E. avium. Enterococcus faecium previously associated with major human clonal lineages (ST18/ST78) or animal clones (ST393) were identified. Some PFGE types of E. faecium, E. raffinosus and E. avium were shared by residents of both LTCFs. Recent antibiotic exposure was significantly associated with colonisation by AmpR enterococci. Residents from Portuguese LTCFs were colonised with high rates of AmpR enterococci and similar rates of VREfm compared with other EU countries. A high colonisation rate with widespread enterococcal lineages that could be selected by antibiotic consumption in LTCFs was uncovered. These findings suggest that antimicrobial stewardship is warranted in LTCFs, which constitutes a significant challenge in a home-based setting.

Birth of clones of the world\u2019s first cloned dog

Animal cloning has gained popularity as a method to produce genetically identical animals or superior animals for research or industrial uses. However, the long-standing question of whether a cloned animal undergoes an accelerated aging process is yet to be answered. As a step towards answering this question, we compared longevity and health of Snuppy, the world’s first cloned dog, and its somatic cell donor, Tai, a male Afghan hound. Briefly, both Snuppy and Tai were generally healthy until both developed cancer to which they succumbed at the ages of 10 and 12 years, respectively. The longevity of both the donor and the cloned dog was close to the median lifespan of Afghan hounds which is reported to be 11.9 years. Here, we report creation of 4 clones using adipose-derived mesenchymal stem cells from Snuppy as donor cells. Clinical and molecular follow-up of these reclones over their lives will provide us with a unique opportunity to study the health and longevity of cloned animals compared with their cell donors.

Low levels of exosomal-miRNAs in maternal blood are associated with early pregnancy loss in cloned cattle

Nuclear reprogramming mediated by somatic cell nuclear transfer (SCNT) has many applications in medicine. However, animal clones show increased rates of abortion and reduced neonatal viability. Herein, we used exosomal-miRNA profiles as a non-invasive biomarker to identify pathological pregnancies. MiRNAs play important roles in cellular proliferation and differentiation during early mammalian development. Thus, the aim of this study was to identify exosomal-miRNAs in maternal blood at 21 days of gestation that could be used for diagnosis and prognosis during early clone pregnancies in cattle. Out of 40 bovine-specific miRNAs, 27 (67.5%) were with low abundance in the C-EPL (Clone - Early pregnancy loss) group compared with the C-LTP (Clone - Late pregnancy) and AI-LTP (Artificial Insemination - Late pregnancy) groups, which had similar miRNAs levels. Bioinformatics analysis of the predicted target genes demonstrated signaling pathways and functional annotation clusters associated with critical biological processes including cell proliferation, differentiation, apoptosis, angiogenesis and embryonic development. In conclusion, our results demonstrate decreased exosomal-miRNAs in maternal blood at 21 days of gestation in cloned cattle pregnancies that failed to reach term. Furthermore, the predicted target genes regulated by these 27 miRNAs are strongly associated with pregnancy establishment and in utero embryonic development.

Cloning animals for tourism in the year 2070

What role do animals have in a future tourism world – in the year 2070? Contemplating over meat and fish delights on restaurant menus, hunting for sport and leisure purposes, and the presence of safaris and zoos have aspects in common, significantly animals are central to their existence and tourists are key consumers. Importantly, these pastimes, social activates, and leisurely pursuits have their ancestral roots that pre-date modern society. Humans have long been associated with eating meat, hunting animals and holding animals in some form of captivity for recreation and education. Distinctively, this paper explores animal cloning and how it could support the extension and existence of these tourism pursuits in the future, focusing on the year 2070. The paper applies a scenario planning approach as its methodological approach to research. The study contributes innovative and original concepts to the future tourism landscape. Through rigorous analysis and discussions in the fields of animal cloning and its relationship with tourism and tourists in potential future settings, a gap in the future market is presented. This research paper presents three scenarios in which cloning could support future tourism markets, Food tourism 2070: A Japanese restaurant perspective, Sport Hunting and tourism 2070: Designated hunting reserves in South Africa; and Safari-zoo tourism 2070: Into the Wild, cloning for education & conservation in the U.S.A. The paper offers interesting research for the futures field and tourism studies, offering scholars and industry with novel concepts for debating.

Review on Applications of Genetic Engineering And Cloning in Farm animals

Review on Applications of Genetic Engineering And Cloning in Farm animals

ID: 1064 Effects of FSH, cumulus cell morphology and follicular fluid from different follicular sizes on the in vitro maturation of bovine oocytes

The quality of mature oocyte plays a vital role in assisted reproductive technology, as well as animal cloning. Therefore, optimization of the in vitro maturation procedure for oocytes has long been of interests for researchers in the fields of reproduction. In this study, we investigated the effect of different supplement culture factors on in vitro maturation of bovine oocytes such as follicular-stimulating hormone (FSH) (experiment 1), different layers of cumulus cells (CCs) (experiment 2), and follicular fluid (FF) collected from different follicle sizes (experiment 3). With result from experiment 1, bovine oocytes cultured in in vitro maturation (IVM) medium supplemented with FSH reached to higher maturation rate than cultured in the basic one (85.9% and 69.3% respectively). In addition, experiment 2 suggested that, the groups of 3-4 layers and 2-3 layers achieve higher rate of oocyte maturity than group of <1 layers (84.38%; 82.46%; 47.83% respectively). However, the result of experiment 3 show that FF collected from different follicle size did not affect to the maturation rate. In conclusion, FSH and layers of CCs affect to the maturation of bovine oocytes.

Animal cloning applications and issues

Significant progress in the field of biotechnology has allowed for the use of cloning in animals which is being used: to improve genetic makeup, to rescue endangered species, in tissue engineering and to increase farm animal population. Unfortunately, cloning has been met with failure due to a variety of reasons namely early and late abortions, compromised immune systems, circulatory and respiratory problems and a high rate of fetal death. The reasons of these problems are unknown, but may research groups are attempting to understand the underlying molecular and cellular mechanisms involved in cloning efficiency. Atypical epigenetic re-programming appears to be the primary cause of ineffective cloning. Understanding molecular mechanisms involving key regulatory proteins is pivotal in the success of animal cloning. This review shows the current paradigm involving animal cloning efficiency, and also further elucidates applications to improve animal cloning efficiency.

Meaning and Conception of Bioethics and its Realm

Background and Aim : The present era witnesses new biomedical technologies of which the usage has brought about philosophical, moral, religious and social challenges. More questions, challenges and uncertainties in biotechnology have made applied ethics, as a part of its ethics and philosophy, to investigate the issue and find relevant appropriate solutions based on ethical principles and theories. Materials and Methods : The current study aimed at casting light on bioethics and its scope using primary and authentic sources on bioethics and seeking the related keywords in these sources. Ethical Considerations : The principles of research ethics have been observed in studies and citing the primary texts and sources. Findings : Providing an all-inclusive definition of bioethics and its place in different disciplines of ethics, this short discussion tackles the area, realm, and significance of the issues in bioethics. Technologies such as assisted reproductive, in vitro fertilization (IVF), human and animal cloning, obtaining stem cells and using them, euthanasia, and manufacture of human organs and their transplantation are the most challenging matters for which within bioethics, the fundamental answers and strategies against the questions and challenges of their emergence are elaborated. Conclusion : Bioethics is a new area of interest in applied normative ethics which, as an interdisciplinary, examines systematically the moral challenges coming from biological science and medical innovations and organizes the must-to-dos in this connection. The realm of bioethics comprises distinction of consequences of applying modern biomedical technologies against the ethical principles and standards within human life. The aforesaid technologies and advancements that make up issues in bioethics are grouped in four categories: Life preserver technologies at the beginning of life and during it; Life lasting technologies to promote the life quality; Reproductive and esp. cloning technologies; Technologies associated with genetic engineering and gene therapy. Citation: Rahbarpour MR. Meaning and Conception of Bioethics and its Realm. Bioeth Health Law J . 2017; 1(1):1-9.

The Precautionary Principle and Non-scientific Factors in the Regulation of Biotech Foods

In the current climate of rising food insecurity, new technologies such as modern agricultural biotechnologies, i.e. the genetic modification of plants and animals, and the cloning of animals, are identified as one of the main solutions to achieve sustainable intensification. The development of modern agricultural biotechnologies and their derived products have been controversial. Biotech foods are allegedly underpinned by scientific uncertainty. To regulate such technologies and to ensure information and safety, the European Union has adopted a precautionary approach relying on premarket authorisation and mandatory labelling. Despite these provisions, biotech foods continue to attract close scrutiny, in particular from the EU public. Against this background, this opinion explains why I came to research biotech foods, how these foods are currently regulated in the EU and why the existing regimes could be improved to allow for the consideration of consumer preferences and more generally non-scientific factors.

Effects of Trichostatin A and PXD101 on the In Vitro Development of Mouse Somatic Cell Nuclear Transfer Embryos.

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with aberrant epigenetic nuclear reprogramming. It has been demonstrated that treatment with histone deacetylase inhibitors (HDACis) enhances developmental potential of SCNT embryos. Previous studies in many species revealed that treatment of SCNT embryos with trichostatin A (TSA)-an HDACi-significantly enhances the in vitro development of SCNT embryos. In this study, we compared two different SCNT protocols with TSA and investigate, for the first time, the effect of another new HDACi, PXD101 (belinostat), on in vitro development of mouse SCNT embryo. Rates of blastocyst development in mouse SCNT embryos treated with either 5 nM TSA during (6 hours) and after (4 hours) activation (39.1%) or with 50 nM PXD101 during (6 hours) and after (4 hours) activation (40.2%) were significantly higher than those of nontreated SCNT embryos (11.5%) and both treatments also significantly improved the subsequent establishment of NT-ESCs in comparison with the nontreated group (38.1% and 40.9% vs. 11.8%). In conclusion, we optimized the TSA concentration and treatment timing and, for the first time, investigated the effect of PXD101 on mouse development of SCNT embryos and establishment of NT-ESCs.

Transgenic animal production: present scenario-a review

The advent of DNA recombinant technology and the possibility of gene transfer between organisms have opened a wide range of possibilities for improvement of livestock production and human health care products. The recent advances in gene transfer, animal cloning, and assisted reproductive techniques have partly fulfilled the expectation in the field of livestock transgenesis. This paper reviews the recent advances and applications of transgenesis in livestock and their derivative products. Typical agricultural applications include improved carcass composition, wool production, and lactation performance which is enhanced disease resistance and reduced environmental impact. Transgenic farm animals have also become important in human medicine as sources of biologically active proteins, as donors in xenotransplantation, for research in cell and gene therapy and for human disease. modeling. Until recently, pronuclear microinjection of deoxyribonucleic acid (DNA) was the standard method for producing transgenic animals. This technique is now being replaced by more efficient protocols based on somatic nuclear transfer that also permit targeted genetic modifications. Lentiviral vectors and small interfering ribonucleic acid technology are also becoming important tools for transgenesis.

TSA and BIX-01294 Induced Normal DNA and Histone Methylation and Increased Protein Expression in Porcine Somatic Cell Nuclear Transfer Embryos.

The poor efficiency of animal cloning is mainly attributed to the defects in epigenetic reprogramming of donor cells’ chromatins during early embryonic development. Previous studies indicated that inhibition of histone deacetylases or methyltransferase, such as G9A, using Trichostatin A (TSA) or BIX-01294 significantly enhanced the developmental efficiency of porcine somatic cell nuclear transfer (SCNT) embryos. However, potential mechanisms underlying the improved early developmental competence of SCNT embryos exposed to TSA and BIX-01294 are largely unclear. Here we found that 50 nM TSA or 1.0 μM BIX-01294 treatment alone for 24 h significantly elevated the blastocyst rate (P < 0.05), while further improvement was not observed under combined treatment condition. Furthermore, co-treatment or TSA treatment alone significantly reduced H3K9me2 level at the 4-cell stage, which is comparable with that in in vivo and in vitro fertilized counterparts. However, only co-treatment significantly decreased the levels of 5mC and H3K9me2 in trophectoderm lineage and subsequently increased the expression of OCT4 and CDX2 associated with ICM and TE lineage differentiation. Altogether, these results demonstrate that co-treatment of TSA and BIX-01294 enhances the early developmental competence of porcine SCNT embryos via improvements in epigenetic status and protein expression.

Artificial meat and the future of the meat industry

The global population is estimated to plateau at 9 billion by the year 2050; however, projected food-production estimates would supply for only 8 billion people, using the ‘business as usual’ approach. In particular, the meat industry would need to increase production by ~50–73%. In response, there are several different options that have the potential to satisfy demand and increase production. Some of these options require advanced technologies and many may be considered as ‘artificial’ by different consumer groups. Within the meat industry itself, available technologies include selective breeding, agroecology systems, animal cloning and genetic modification. Alternatively, meat proteins can be replaced or substituted with proteins from plants, fungi, algae or insects. Finally, meat products could be produced using in vitro culturing and three-dimensional printing techniques. The protein produced by these techniques can be considered in the following three categories: modified livestock systems, synthetic meat systems, and meat substitutes. In the future, it is likely that meat substitutes will increase market share through competition with low-grade cuts of meat, sausages, ground meat and processed meat. However, synthetic meat systems and meat substitutes have significant barriers to commercialisation and widespread adoption that will affect their presence at least in the high-end premium sector in the market. To meet growing demands for protein, and in the face of growing competition from other sectors, the conventional meat industry must adopt new technologies and farming systems. These must be tailored to the challenges facing the industry and must effectively respond to consumer demands and the changing market place.

Biosidus: Biotecnologia En La Patagonia

The case follows an Argentinean biotech firm as it sets future firm strategy and decides how to fund it. Specifically, the firm is working on a method to produce human growth hormone through the milk of cloned cows. Economic and political uncertainties in Argentina complicate an otherwise challenging strategic decision. Students are required to make a decision based on the conditions present at the time of the case. Excerpt UVA-S-0115 Rev. Aug. 22, 2012 BioSidus: Biotecnologia en la Patagonia In early August 2002, Marcelo Arguelles, son of Antonio Arguelles, the founder of BioSidus, faced a very tough decision concerning the future of the company. BioSidus could no longer implement its 10-year business plan given the current political and economic crises in Argentina. Therefore, Arguelles needed to reevaluate his company's direction along three potential paths. First, should his company focus on its current product base and harvest its existing portfolio of drugs? Second, should it invest in R&D despite adverse market conditions? Third, should it forge a new international partnership to establish a greater market for its current products? BioSidus's latest R&D development involved farm animal cloning techniques that used animals to produce new proteins, which would then be secreted and harvested from their milk. After one unsuccessful attempt, the company anxiously awaited the birth of its second cloned calf. If successful, this new technique would significantly reduce the production costs of proteins, transferring the production line from a multi-million-dollar state-of-the-art facility to a cow (see Exhibit 1 for an illustration of the cloning process). Arguelles was confident that this new technique would revolutionize the industry. He was uncertain, however, of what it would mean for the rest of his company if the cloning was unsuccessful. Faced with a restrictive financial market and weak demand, Arguelles was not sure the company would have enough strength to complete the project. . . .

Distribution of methicillin-resistant coagulase-positive staphylococci (MRCoPS) in a surgical unit and cystotomy operation sites in a veterinary teaching hospital.

This study aimed to investigate the spread of methicillin-resistant coagulase-positive staphylococci (MRCoPS) among veterinary staff, hand-touch sites and surgical tissue during cystotomy operations on cats and dogs that were patients, and to analyze the genetic relatedness and antimicrobial resistance profiles of the isolates. Human and environmental samples were obtained from the nasal passageways of 12 surgeons and veterinary assistants and from 29 hand-touch sites of instruments in operative units and subjected to bacterial isolation and enumeration. Swab samples were collected in triplicate from 29 dogs and three cats at the site of incision, from the incision area, from the peritoneum during surgery and from the peritoneum before suture. MRCoPS were identified by mecA gene detection and characterized by their antibiogram profile, SCCmec type and pulsed-field gel electrophoresis. Twenty-four staphylococci were isolated, derived from one veterinary assistant, 12 operating room floor areas and hand-touch sites, three dogs and one cat. Methicillin-resistant S. pseudintermedius (MRSP) was found on an electric clipper and rebreathing circuits in the operating room. Three dogs were positive for MRSP during surgery, and one methicillin-resistant S. aureus (MRSA) was detected in a cat. All MRCoPS were resistant to doxycycline, erythromycin, clindamycin and enrofloxacin, but no patients developed surgical site infections. According to their genotypic patterns, the clones obtained from the environment and human sources differed from the animal clones. Despite intensive hygienic management, a variety of MRCoPS clones were present within the surgical unit and during surgery.

152 SPECIES-SPECIFIC DIFFERENCES IN THE METHYLATION REPROGRAMMING DURING EARLY PRE-IMPLANTATION DEVELOPMENT

Studies in mouse and human have shown extensive DNA methylation reprogramming in pre-implantation development followed by remethylation from implantation. However, the extent to which such reprogramming is conserved in mammals and the timing of demethylation and remethylation are unknown. As part of a major objective to characterise methylation dynamics in the bovine and porcine species from the oocyte to the blastocyst stage, we aimed here to compare the distribution of methylation at single-base resolution in both species at Day 7.5 of development. The DNA methylation profiles were obtained from individual blastocysts at Day 7.5 [pig: 3 in vivo, 3 in vitro; cow: 3 in vivo, 3 in vitro, 3 inner cell mass (ICM) and 3 trophoectoderm (TE) dissected from in vitro blastocysts] using the post-bisulphite adaptor tagging method and Illumina sequencing. For oocytes, data (GEO: GSE63330) from Schroeder et al. 2015 were analysed. Raw sequences were mapped, methylation calls made using Bismark and data analysis and visualisation was done within the SeqMonk platform. Gene expression profiles from individual blastocysts (3 pig, 3 cow) were obtained by RNA-seq. Annotated mRNA features were quantitated in SeqMonk and these were fed into DESeq2 for differential expression analysis (P &lt; 0.05) as previously reported (Love et al. 2014 Genome Biol. 15, 550). Global methylation levels in whole blastocysts differed substantially between porcine and bovine embryos (in vivo: 12.33 ± 3.6 v. 28.33 ± 3.5%; in vitro: 15.02 ± 3.3 v. 24.41 ± 4.1%). In addition, the distribution of methylation differed: the pattern of cytosine methylated seemed random in the porcine genome, but was highly structured in the bovine genome, with methylation predominantly over gene bodies, resembling the profile previously reported in oocytes (Schroeder et al. 2015 PLoS Genet. 11, e1005442). Regarding correlation analysis, gene expression versus methylation were plotted. It suggested that gene body methylation reflected gene expression pattern in oocytes as well as in bovine blastocysts. Pair-wise comparison of isolated ICM and TE was filtered to require 5% change, and replicate set statistics were applied. This revealed very similar total and regional methylation levels in the 2 compartments, indicating that remethylation does not initiate preferentially in one compartment in bovine pre-implantation embryos. This confirms, from a viewpoint of the genome-wide DNA methylation, what has been observed in mouse for specific genes: the trophoblast-specific DNA methylation occurs after the segregation of the TE and ICM (Nakanishi et al. 2012 Epigenetics 7, 173–183). Our study is the first to provide whole genome methylation profiles from single blastocysts of economically important livestock species. Our data demonstrate that methylation reprogramming in early pre-implantation development is species specific. Knowledge of these specific patterns may have high importance when decisions are taken regarding the use of assisted reproductive technologies, cloning, or generation of transgenic animals. This work was funded by AGL2015–66341-R (MINECO-FEDER), PRX14/00348 (MECD), 19595/EE/14 (F. Séneca).

Hematopoietic Stem and Progenitor Cells from Human Pluripotent Stem Cells Via Transcription Factor Conversion of Hemogenic Endothelium

A variety of tissues can be differentiated from pluripotent stem cells (PSCs) in vitro through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors (TFs). Despite considerable effort, neither approach has yielded functional human hematopoietic stem cells (HSCs). Building upon recent evidence that HSCs derive from definitive hemogenic endothelium (HE), we performed morphogen-directed differentiation of human PSCs into HE followed by screening of 26 candidate HSC-specifying TFs for the capacity to promote multi-lineage hematopoietic engraftment in irradiated immune deficient murine hosts. From genomic PCR of engrafted cells, we recovered seven TFs (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, SPI1) that were sufficient to convert HE into hematopoietic stem and progenitor cells (HSPCs) that engraft GLY-A+ erythrocytes, CD33+ myeloid, CD15+ CD31+ neutrophils, CD19+ IgM+ B and CD3+ T cells in primary and secondary murine recipients for 12-14 weeks. Limiting dilution analysis indicated that the frequency of repopulating cells generated by this method was 1 in 4,707-15,029, lower than the frequency in CD34+ cord blood cells (1 in 1,819-5,173). Functional characterization of terminally differentiated cells demonstrated features of definitive erythropoiesis (expression of adult beta globin and enucleation). Engrafted neutrophils responded to cytokine stimuli by activation of myeloperoxidase. Human IgM and IgG could be detected in the serum of engrafted mice, and titers of ovalbumin specific antibody increased in response to protein immunization, indicating boostable immunity. T-cells responded to PMA/Ionomycin stimuli by activation of IFNγ, and sequencing of the T cell receptor revealed a broad clonotype diversity. Proviral integration analysis demonstrated derivation of myeloid and lymphoid progeny from common clones in secondary animals, indicating generation of self-renewing, multipotential HSC-like cells from PSCs. Mechanistically, the seven TFs induced HOXA target genes (LMO2, SOX4, MEIS1 and ID2); upregulated expression of homing-related genes (CXCR4, VLA5 and S1PR1); and enhanced the endothelial to hematopoietic transition (EHT), as indicated by a 2.4-fold induction of a RUNX1c-reporter. Our combined approach of morphogen-driven differentiation and TF-mediated cell fate conversion produced HSPCs from PSCs that hold promise for modeling hematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. DisclosuresNo relevant conflicts of interest to declare.

28 IMPROVED HATCH RATE AFTER PARTIAL DISSECTION OF ZONA PELLUCIDA IN CLONED PIG EMBRYO

For last 20 years, the efficiency of animal cloning has remained extremely low, despite many attempts to improve it. Although nuclear transfer experiments have been almost optimized, artificial holes are inevitably made in the zona pellucida (ZP) during nuclear transfer experiments, such as enucleation of maternal genome or injection of nuclear donor. Hatching from the ZP is a prerequisite for mammalian embryo implantation, and the condition of the ZP has a lot of influence on hatching. The present studies were performed to investigate the effects of artificial holes in the ZP, because of the nuclear transfer procedure, on hatching of clone embryos in pigs. All experiments were done in triplicate. Statistical analysis was performed using SPSS statistical software (SPSS Inc., Chicago, IL, USA). First, we made a slit in the ZP of porcine parthenote that was identical to the artificial holes of nuclear transfer experiment and compared in vitro development of Day 7 embryos with control group with intact ZP. Of slit blastocysts, 89.9% (80/89) were trapped at a slit, which looked like typical figure-eight shape, and did not complete the hatching process until Day 8, though 68.8% (64/93) of control blastocysts completed the hatching at Day 7. Then, to solve these abnormal hatchings caused by a slit in the ZP, we applied partial zona dissection (PZD) to porcine clone embryos and compared the hatching process with that of conventional clone embryos. Contrary to conventional clone blastocysts that were trapped at slit in the ZP (91.4%; 43/47), 89.5% (60/67) of clone blastocysts in PZD group were preferentially hatched through dissected hole at Day 7. These results suggest that trapping of conventional clone blastocyst in a slit of the ZP could be avoided by PZD. Through this study, we demonstrated that a slit in the ZP would hinder a blastocyst from hatching from the ZP and that partial dissection at the ZP could help clone blastocyst to hatch without trapping at a slit in the ZP. This assisted hatching in clone embryos would be useful for the successful hatching of clone blastocysts with a capacity of full-term development, so that the efficiency of animal cloning might be improved.