Donor Cell Lines Research Articles

Production and Breeding of Transgenic Cloned Pigs Expressing Human CD73.

ABSTRACTOne of the reasons to causing blood coagulation in the tissue of xenografted organs was known to incompatibility of the blood coagulation and anti-coagulation regulatory system between TG pigs and primates. Thus, overexpression of human CD73 (hCD73) in the pig endothelial cells is considered as a method to reduce coagulopathy after pig-to-non-human-primate xenotransplantation. This study was performed to produce and breed transgenic pigs expressing hCD73 for the studies immune rejection responses and could provide a successful application of xenotransplantation. The transgenic cells were constructed an hCD73 expression vector under control porcine Icam2 promoter (pIcam2-hCD73) and established donor cell lines expressing hCD73. The numbers of transferred reconstructed embryos were 127 ± 18.9. The pregnancy and delivery rate of surrogates were 8/18 (44%) and 3/18 (16%). The total number of delivered cloned pigs were 10 (2 alive, 7 mummy, and 1 died after birth). Among them, three live hCD73-pigs were successfully delivered by Caesarean section, but one was dead after birth. The two hCD73 TG cloned pigs had normal reproductive ability. They mated with wild type (WT) MGH (Massachusetts General Hospital) female sows and produced totally 16 piglets. Among them, 5 piglets were identified as hCD73 TG pigs. In conclusion, we successfully generated the hCD73 transgenic cloned pigs and produced their litters by natural mating. It can be possible to use a mate for the production of multiple transgenic pigs such as α-1,3-galactosyltransferase knock-out /hCD46 for xenotransplantation.

Expression of tumor suppressor REIC/Dkk-3 by a newly improved adenovirus vector with insertion of a hTERT promoter at the 3′-side of the transgene

Reduced expression in immortalized cells (REIC)/Dickkopf-3 (Dkk-3) overexpression, induced using an adenovirus (Ad)-REIC, has been revealed to have a dramatic therapeutic effect on multiple types of cancer. To achieve an improved therapeutic effect from Ad-REIC on cancer, our group previously developed an enhanced gene expression system, the C-TSC cassette [cytomegalovirus (CMV)-RU5' located upstream (C); another promoter unit composed of triple tandem promoters, human telomerase reverse transcriptase (hTERT), simian virus 40 and CMV, located downstream of the cDNA (TSC); plus a polyadenylation (polyA) signal]. When applied to the conventional Ad-REIC, this novel system induced the development of an enhanced product, Ad-C-TSC-REIC, which exhibited a noticeable anticancer effect. However, there were difficulties in terms of Ad-C-TSC-REIC productivity in HEK293 cells, which are a widely used donor cell line for viral production. Productivity of Ad-C-TSC-REIC was significantly reduced compared with the conventional Ad-REIC, as the Ad-C-TSC-REIC had a significantly higher ability to induce apoptotic cell death of not only various types of cancer cell, but also HEK293 cells. The present study aimed to overcome this problem by modifying the C-TSC structure, resulting in an improved candidate: A C-T cassette (C: CMV-RU5' located upstream; T: another promoter unit composed of a single hTERT promoter, located downstream of the cDNA plus a polyA signal), which demonstrated gene expression comparable to that of the C-TSC system. The improved adenovirus REIC/Dkk-3 product with the C-T cassette, named Ad-C-T-REIC, exhibited a higher expression level of REIC/Dkk3, similar to that of Ad-C-TSC-REIC. Notably, the vector mitigated the cell death of donor HEK293 cells, resulting in a higher rate of production of its adenovirus. These results indicated that Ad-C-T-REIC has the potential to be a useful tool for application in cancer gene therapy.

25 PRODUCTION OF TRANSGENIC PIGS WITH CreER-MEDIATED ASTROCYTIC-SPECIFIC RECOMBINATION SYSTEM FOR NEUROLOGICAL DISEASE MODELS

Pigs are one of the most suitable alternative laboratory models than other animals, because they have similar cardiovascular, renal and gastrointestinal organs with those of human. However, in the case of genetically engineered animals, early development of embryos is inhibited by expression of foreign genes, there are many cases of miscarriage or birth early mortality. To overcome these problems, we constructed pig glial fibrillary acidic protein (GFAP) promoter-Cre recombinase fused to a mutated ligand-binding domain of the human oestrogen receptor (CreERT2) and enhanced green fluorescent protein (EGFP)-LoxP transgenes for tamoxifen(TM)-inducible CreERT2-mediated recombination. We then established donor transgenic pig fibroblasts with pGFAP-CreERT2; LCMV-EGFPLoxP transgenes for somatic cell nuclear transfer (SCNT). We produced the SCNT embryos using a Cloud male #5 pGFAP-CreERT2+LCMV-EGFPLoxP donor cell line that was verified in vitro. It was transferred into a surrogate mother and then 5 pGFAP-CreERT2; EGFPLoxP TG piglets were born. By immunofluorescence staining and semi-nested PCR analysis, it was proved that CreER-mediated astrocytic-specific recombination system was operated in some cerebral astrocytic cells after TM-administration to TG pig #4. Additionally, we obtained brain magnetic resonance imaging (MRI) images using 3T-tesla MRI. Brain compartment volume (total brain, grey matter, white matter, cerebellum, brainstem, lateral ventricle, thalamus, midbrain, pons, medulla oblongata, hypophysis) was no significant differences between normal pig and pGFAP-CreERT2; EGFPLoxP transgenic (TG) pig. In summary, we verified the pGFAP promoter-driven CreERT2-LoxP recombination system in TG pig generated by SCNT depending on the TM administration. We suggest that this technology will be a useful tool for studying physiology of astrocytes and generating TG pig model of neurological disease such as Huntington’s disease, Alzheimer’s disease and brain tumour.

Efficient generation of GGTA1-null Diannan miniature pigs using TALENs combined with somatic cell nuclear transfer.

Backgroundα1,3-Galactosyltransferase (GGTA1) is essential for the biosynthesis of glycoproteins and therefore a simple and effective target for disrupting the expression of galactose α-1,3-galactose epitopes, which mediate hyperacute rejection (HAR) in xenotransplantation. Miniature pigs are considered to have the greatest potential as xenotransplantation donors. A GGTA1-knockout (GTKO) miniature pig might mitigate or prevent HAR in xenotransplantation.MethodsTranscription activator-like effector nucleases (TALENs) were designed to target exon 6 of porcine GGTA1 gene. The targeting activity was evaluated using a luciferase SSA recombination assay. Biallelic GTKO cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs following transfection by electroporation with TALEN plasmids. One cell line was selected as donor cell line for somatic cell nuclear transfer (SCNT) for the generation of GTKO pigs. GTKO aborted fetuses, stillborn fetuses and live piglets were obtained. Genotyping of the collected cloned individuals was performed. The Gal expression in the fibroblasts and one piglet was analyzed by fluorescence activated cell sorting (FACS), confocal microscopy, immunohistochemical (IHC) staining and western blotting.ResultsThe luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 17.1-fold higher than those of the control. Three cell lines (3/126) showed GGTA1 biallelic knockout after modification by the TALENs. The GGTA1 biallelic modified C99# cell line enabled high-quality SCNT, as evidenced by the 22.3 % (458/2068) blastocyst developmental rate of the reconstructed embryos. The reconstructed GTKO embryos were subsequently transferred into 18 recipient gilts, of which 12 became pregnant, and six miscarried. Eight aborted fetuses were collected from the gilts that miscarried. One live fetus was obtained from one surrogate by caesarean after 33 d of gestation for genotyping. In total, 12 live and two stillborn piglets were collected from six surrogates by either caesarean or natural birth. Sequencing analyses of the target site confirmed the homozygous GGTA1-null mutation in all fetuses and piglets, consistent with the genotype of the donor cells. Furthermore, FACS, confocal microscopy, IHC and western blotting analyses demonstrated that Gal epitopes were completely absent from the fibroblasts, kidneys and pancreas of one GTKO piglet.ConclusionsTALENs combined with SCNT were successfully used to generate GTKO Diannan miniature piglets.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-016-0212-7) contains supplementary material, which is available to authorized users.

Production of homozygous klotho knockout porcine embryos cloned from genome-edited porcine fibroblasts

Even though klotho deficiency in mice exhibits multiple aging-like phenotypes, studies using large animal models such as pigs, which have many similarities to humans, have been limited due to the absence of cell lines or animal models. The objective of this study was to generate homozygous klotho knockout porcine cell lines and cloned embryos. A CRISPR sgRNA specific for the klotho gene was designed and sgRNA (targeting exon 3 of klotho) and Cas9 RNPs were transfected into porcine fibroblasts. The transfected fibroblasts were then used for single cell colony formation and 9 single cell–derived colonies were established. In a T7 endonuclease I mutation assay, 5 colonies (#3, #4, #5, #7 and #9) were confirmed as mutated. These 5 colonies were subsequently analyzed by deep sequencing for determination of homozygous mutated colonies and 4 (#3, #4, #5 and #9) from 5 colonies contained homozygous modifications. Somatic cell nuclear transfer was performed to generate homozygous klotho knockout cloned embryos by using one homozygous mutation colony (#9); the cleavage and blastocyst formation rates were 72.0% and 8.3%, respectively. Two cloned embryos derived from a homozygous klotho knockout cell line (#9) were subjected to deep sequencing and they showed the same mutation pattern as the donor cell line. In conclusion, we produced homozygous klotho knockout porcine embryos cloned from genome-edited porcine fibroblasts.

Impact of different sources of donor cells upon the nuclear transfer efficiency in Chinese indigenous Meishan pig.

Somatic cell nuclear transfer (SCNT) is currently the most efficient and precise method to generate genetically tailored pig models for both agricultural and biomedical research. However, its efficiency is crucially dependent on the source of nuclear donor cells. In this study, we compared the cloning efficiency by using three lines of donor cells that are derived from fetal, newborn and adult fibroblasts of Chinese indigenous Meishan pig. We showed that cleavage rate and blastocyst formation rate of the reconstructed embryos were not significantly different between the fetal (80.7% and 15.6%) and newborn ear skin (77.5% and 12.3%) fibroblast groups (p>0.05), but in both groups these indices were significantly higher than that found in the adult ear skin (70.5% and 8.8%; p<0.05). Reconstructed embryos derived from fetal, newborn, and adult ear skin fibroblasts were transferred to four surrogates, respectively. For the fetal, newborn, and adult ear skin fibroblasts, the number of pregnancies were two (50.0%), two (50.0%), and one (25.0%), respectively, and the number of deliveries were two (50.0%), one (25.0%), and zero (0.0%), respectively. Seven and two cloned piglets were obtained from the fetal and newborn ear skin fibroblasts respectively, while no piglets were obtained from the adult ear skin fibroblasts. Two cloned piglets from the newborn ear skin fibroblasts died shortly after birth because of neonatal asphyxia caused by dystocia. The birth weights of the piglets derived from the fetal and newborn ear skin fibroblasts were 1230.5 and 1310.0 g, respectively, which were statistically insignificant (p>0.05), but both were significantly higher than that of the control groups (p<0.05). Microsatellite analyses demonstrated that the genotypes of all cloned piglets were identical to their donor cells. Therefore, cloned pigs were successfully produced using two sources of donor cells isolated from the fetal and newborn ear skin fibroblasts of Meishan piglet, and indicating a better cloning efficiency than that obtained from adult fibroblasts. We concluded that the nuclear donor cell lines have significant impact on the developmental competence of cloned embryos as well as on the cloning efficiency of Meishan pig.

Engineering Receptor Expression on Natural Killer Cells Through Trogocytosis.

Trogocytosis is a rapid contact-dependent process by which lymphocytes acquire membrane patches from the target cells ('donor' cells) with which they interact and this phenomenon has been shown to occur in various immune cells. The surface molecules acquired through trogocytosis are functionally incorporated in the 'acceptor' cells transiently. We had previously demonstrated that trogocytosis can be utilized in place of gene transfer to engineer surface receptor expression on NK cells for adoptive immunotherapy applications. In this chapter, we describe detailed protocol for trogocytosis-co-culture of NK cell with the donor cell line, phenotypic assessment of receptor uptake and persistence, and assessment of NK cell function (migration) following receptor acquisition.

25 GENOME EDITING OF SOMATIC CELL NUCLEAR TRANSFER DERIVED ZYGOTES BY CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9 GUIDE RNA INJECTION

Recent applications of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system have greatly improved the efficiency of genome editing in pigs. However, in some cases, genetically modified pig models need an additional modification to improve their application. The objective of this experiment was to determine whether a combination of somatic cell NT (SCNT) by using a previously modified donor cell line and subsequent zygote injection with CRISPR/Cas9 guide RNA to target a second gene would result in embryos and offspring successfully containing both modifications. Fibroblast cell lines were collected from fumarylacetoacetate hydrolase deficient (FAH–/–) fetuses and used as the donor cell line. Somatic cell NT was performed by standard technique. A CRISPR guide RNA specific for recombination activating gene 2 (RAG2) was designed and in vitro transcribed from a synthesised gBlock (IDT) containing a T7 promoter sequence, the CRISPR Guide RNA (20 bp), and 85 bp of tracer RNA. The gBlock was PCR amplified with Q5 polymerase (NEB, Ipswich, MA, USA) and in vitro transcribed with the MEGAshortscript™ T7 Transcription Kit (Life Technologies, Grand Island, NY, USA). Guide RNA (20 ng μL–1) and polyadenylated Cas9 (20 ng μL–1, Sigma, St. Louis, MO, USA) were co-injected into the cytoplasm of SCNT zygotes at 14 to 16 h after fusion and activation. Injected SCNT were then cultured in vitro in PZM3 + 1.69 mM arginine medium (MU1) to Day 5. Three embryo transfers were performed surgically into recipient gilts on Day 4 or 5 of oestrus (50, 62, or 70 embryos per pig) to evaluate in vivo development. The remaining embryos were cultured in MU1 to Day 7 and analysed for the presence of modifications to the RAG2 gene. Embryos were classified as modified if they contained an INDEL as measured by both gel electrophoresis and DNA sequencing of PCR amplicons spanning the targeted exon. The RAG2 modification rate was 83.3% (n = 6), of which 50% (n = 3) of the embryos contained biallelic modifications. All control embryos contained a wild-type RAG2 gene (n = 5). Embryo transfer resulted in a 33.3% pregnancy rate (1/3). The combination of SCNT and CRISPR/Cas9 zygote injection can be a highly efficient tool to successfully create pig embryos with an additional modification. This additional technique further improves the usefulness of already created genetically modified pig models. This study was funded by the National Institutes of Health via U42 OD011140.

An Optimized, Large Scale Generation and Validation of Aspergillus-Specific T Lymphocytes for the Management of Invasive Aspergillosis in Immunocompromised Patients

Invansive aspergillosis (IA) represents a leading cause of morbidity and mortality in patients with hematological malignancies and hematopoietic stem cell (HSC)/solid organ transplant recipients. The limitations of the contemporary antifungal treatment include the lack of efficacy in several cases, the drug-associated toxicity, the emergence of uncommon or drug-resistant molds and the added financial burden in transplant care. Adoptive immunotherapy with Aspergillus-specific T cells (Asp-STs) constitutes an alternative and promising therapeutic approach against IA, however, the complex and costly production limits its broader application. Consequently, and in contrast to the remarkable clinical progress with virus-specific T cells, clinical development of fungus-specific T cell therapy is still in its infancy; only one clinical trial of adoptive immunotherapy for IA has been conducted up to date, showing that recipients of Asp-ST clones after haploidentical HSC transplantation cleared IA more often than conventionally treated patients.We developed and validated a novel, rapid, simplified and minimally laborious process of generating and scaling up functionally active Asp-STs, from a single blood draw, in only 10 days. A total of 40x106 peripheral blood mononuclear cells (PBMCs) derived from 30-40ml of peripheral blood of normal donors were exposed to either an Aspergillus fumigatus lysate (n=10) or a pool of Aspergillus fumigatus overlapping peptides of the glycosidase Crf1, the 1,3-beta-glucanosyltransferase gel1 and the serine hydroxymethyltransferase SHMT, all of which are considered potential immunogenic targets (n=5). To determine the most potent stimulus for the generation of Asp-STs, dual, lysate-derived and peptide-derived, Asp-ST cell lines were produced from four donor samples and compared in terms of antigen specificity. Pulsed PBMCs were cultured in the presence of interleukin 4 (IL-4) and IL-7 for 10 days in G-rex bioreactors.Cell lines produced after stimulation with either Aspergillus lysate or peptide pools had a similar fold-expansion reaching a mean absolute number of 230±81x106 cells (range 131-363x106) and 223±19x106 cells (range 197-244x106), respectively. The cell lines derived from either condition were polyclonal, comprised predominantly of CD4+ cells (75±10% and 74±10%, respectively) and CD8+ cells as well (17±9% and 15±2%, respectively), and expressed central (CD45RA-/CD62L+: 44±20% and 60±5%, respectively) and effector memory markers (CD45RA-/CD62L-: 36±20% and 21±2%, respectively). To address whether these highly expanded T cells are functional and specific against the targeted mold, each cell line was pulsed with its initial stimuli, either Asp lysate or peptide pool, and the secretion of interferon-gamma (IFN-γ) was measured by Elispot. For peptide-derived Asp-STs, specificity was assessed both, collectively for all and individually for each peptide. All lines (5/5) pulsed with peptide pools but only three of 10 cell clones pulsed with Asp lysate presented activity against Asp [lysate: 72±39 Spot forming cells (SFC)/5x105 cells; all 3 peptides: 297±81 SFC/5x105 cells]. Interestingly, not only all donor cell lines derived from pooled peptides elicited strong IFN-γ response, but 4/5 were inducible to stimulation by each peptide separately and 1/5 by Crf1, at comparable levels (Crf1: 109±59 SFC/5x105 cells, Gel1: 113±58 SFC/5x105 cells, SHMT: 121±34 SFC/5x105 cells), indicating that T cells specific for these antigens are commonly present in healthy subjects.To directly compare the stimulatory capacity of Asp lysate versus peptide pools, 4 donor samples were tested for their ability to produce specific clones against both conditions. Interestingly, all 4 donor clones showed response against peptides but only 1 of 4 against the lysate, suggesting that the Asp proteins Crf1, Gel1 and SHMT induce stronger Th1 responses than Asp lysate.In conclusion, we established and validated an optimized, rapid and simple process - which can be easily GMP-adapted- of scaling up Asp-STs, at clinically relevant numbers. Since the effective management of IA in immunocompromised patients still remains a desirable target, our proposal provides a promising therapeutic approach for the management of IA. DisclosuresNo relevant conflicts of interest to declare.

Timing Factors Affecting Blastocyst Development in Equine Somatic Cell Nuclear Transfer

In nuclear transfer (NT), exposure of donor cell chromatin to the ooplast cytoplasm may aid reprogramming; however, the length of exposure feasible is limited by the developmental life span of the oocyte. We examined the effect of duration of nucleus-cytoplasmic exposure before activation and of in vitro maturation (IVM) in equine NT. In experiment 1, 24 h IVM and a delay of 2, 5, or 8 h between reconstruction and activation yielded 4%, 15%, and 11% blastocysts, respectively. In experiment 2, a 5-h activation delay yielded 17% and 22% blastocysts with two donor cell lines. In experiment 3, using a 5-h activation delay, the blastocyst rate was significantly higher using oocytes after 20 h IVM than after 24 h IVM; however, only 28% of oocytes were in metaphase II (MII) at 20 h. In experiment 4, oocytes were denuded of cumulus at 20 h, and those in metaphase I (MI) were returned to culture for 3 h (20+3H treatment); blastocyst rates were 30% and 27%, respectively (8-h and 5-h delay to activation, respectively). Four live foals resulted from the transfer of 17 blastocysts (24%) produced using MII oocytes and a 5- or 8-h activation delay. Use of equine oocytes immediately after reaching MII, combined with a longer delay from reconstruction to activation, increased developmental competence after equine NT.

33 TELOMERASE ACTIVITIES IN CLONED BEAGLE DOGS

Telomerase is important ribonucleoprotein for restoring telomere length from its own RNA template. Regarding cloned animals derived from somatic cell nuclear transfer (SCNT), interesting questions have been raised about whether the cloning process restores cellular telomerase activity undergone by their donor cells. The present study was conducted to determine the effects of cloning on telomerase activity in the dog and normality of telomerase activity in cloned dogs. Focusing our attention on differences in telomerase activity depending on the age, we analysed telomerase activity in dogs produced by natural breeding of various ages. Comparison of the telomerase activities of cloned dogs and those of dogs produced by natural breeding was also performed. For SCNT, 2 cell donors, 7- and 9-year-old beagles, were used and donor cells were isolated from ear skin. After establishing donor cell lines, the enucleated canine in vivo-matured oocytes and the cells were injected and fused by electrofusion. After 30 days from embryo transfer, pregnancy diagnosis was performed and 7 cloned dogs were produced on the due date. For standardization of telomerase activity in beagles produced by natural breeding, blood of total 14 dogs at each age (10 months, 20 months, 5, 7, and 8 years old) were collected and telomerase activity was measured by the telomeric repeat amplification protocol (TRAP) assay. Telomerase activity measurements of at least 6 replications in each dog were performed. For statistical analysis, one-way ANOVA with Dunn's Multiple Comparison Test was used. Significant differences in telomerase activity were observed between the blood of cloned and donor dogs. It was shown that mean telomerase activities were decreased according to biological aging with significances. Mean telomerase activities in 10 cloned dogs were higher than those of a donor dog. Cloned dogs also showed similar levels of telomerase activities as their age-matched natural bred dogs, suggesting that they are within the variation in normal dogs. These observations indicate that the cloning process restores the telomerase activity in the dog. Thus, complex regulation of telomerase activity during nuclear reprogramming may regulate and be involved in telomerase activity in cloned dogs. It remains to be determined whether telomere length is correlated with telomerase activity and if it accurately reflects the physiological age of cloned dogs.This study was supported by IPET (#311062–04–2-SB010), RDA (PJ008975022013), Research Institute for Veterinary Science, the BK21 program, Nestle Purina Korea, and TS Corporation.

Immunology of Hematopoietic Stem Cell Transplant

Hematopoietic stem cell transplantation (HSCT) is a procedure in which infusion of hematopoietic stem cells is used to reestablish hematopoietic function in patients with damaged or defective bone marrow or immune systems. Early and late complications following allogeneic HSCT include acute and chronic graft-versus-host disease (GVHD), donor rejection, graft failure, relapse of primary malignancy, conditioning-related toxicity, immunodeficiency and infections. Immunology has a central role in allogeneic hematopoietic cell transplantation. Any appreciation of the immunological mechanism involved in engraftment, GVHD, the development of tolerance, immune reconstitution, and the control of malignancy requires some understanding of the immunologic basis for immune reactions provoked by grafting tissue from one individual to another. In the future it should be possible to learn what gene(s) must be activated and which must be repressed to force stem cells into division without maturation; to engineer a mechanism into the cells that stops proliferation and sets the stage for amplification; to search if there could be a universal donor cell line, neatly packaged and stabilized in sealed vials and distributed by the pharmaceutical industry; to modify the transplanted cells in such a way that they have a proliferative advantage over those of the host and to deliver the lethal blow against the neoplasm, perhaps the cells that are infused will be engineered in such a way as to be able to distinguish between normal host cells and tumor.

Age-Related Effects on the Potency of Human Adipose-Derived Stem Cells: Creation and Evaluation of Superlots and Implications for Musculoskeletal Tissue Engineering Applications

Human adipose-derived stem cells (hASC) are now a prevalent source of adult stem cells for studies in tissue engineering and regenerative medicine. However, researchers utilizing hASC in their investigations often encounter high levels of donor-to-donor variability in hASC differentiation potential. Because of this, conducting studies with this primary cell type can require extensive resources to generate statistically significant data. We present a method to generate pooled donor cell populations, termed "superlots," containing cell populations derived from four to five age-clustered donors. The goal of generating these superlots was to 1) increase experimental throughput, 2) to utilize assay resources more efficiently, and 3) to begin to establish global hASC differentiation behaviors that may be associated with donor age. With our superlot approach, we have validated that pooled donor cell populations exhibit proliferative activity representing the combined behavior of each individual donor cell line. Further, the superlots also exhibit differentiation levels roughly approximating the average combined differentiation levels of each individual donor cell line. We established that high donor-to-donor variability exists between the pre-, peri-, and postmenopausal age groupings and that proliferation and differentiation characteristics can vary widely, independent of age. Interestingly, we did observe that cell lines derived from postmenopausal donors demonstrated a relatively high proclivity for osteogenic differentiation and a relatively lowered proclivity for adipogenic differentiation as compared with cells derived from pre- and perimenopausal donors. In general, superlots effectively represented the average differentiation behavior of each of their contributing cell populations and could provide a powerful tool for increasing experimental throughput to more efficiently utilize resources when studying hASC differentiation.

First cloned swamp buffalo produced from adult ear fibroblast cell

The world’s first cloned swamp buffalo (Bubalus bubalis) derived from adult ear skin fibroblast has been reported. Donor fibroblast cells were produced from biopsies taken from adult male ear skin and in vitro matured oocytes obtained from a slaughterhouse were used as cytoplasts. A total of 39 blastocysts and 19 morulae fresh embryos were transferred into 12 recipient buffaloes. Progesterone assays indicated establishment of pregnancy in 10 of the 12 buffaloes (83.3%) after 45 days, with six animals still pregnant at 3 months. One recipient maintained pregnancy to term and naturally delivered a 40 kg male calf after 326 days of gestation. DNA analysis showed that the cloned calf was genetically identical to the donor cells. Genotype analyses, using 12 buffalo microsatellite markers, confirmed that the cloned calf was derived from the donor cell lines. In conclusion, the present study reports, for the first time, the establishment of pregnancy and birth of the first cloned Thai swamp buffalo derived from adult ear skin fibroblast cells.

221 SOMATIC CELL GOAT CLONING USING ALLOGENEIC OR SYNGENEIC TRANSGENIC CELL LINES

The aim of this study was to compare the efficiency of goat cloning by using cell lineages from distinct transgenic backgrounds. Primary fibroblast skin cell cultures from 2 females (allogeneic), transgenic for the human lysozyme gene (hLZ), were established following standard procedures. Cells from one hLZ genotype were used for the establishment of 2 double transgenic syngeneic cell lines by cell transfection (Nucleofector®, Lonza, Germany) with transgene cassettes containing either the human glucocerebrosidase gene (hGC) and neomycin resistance gene, or the human lactoferrin gene (hLF) with no selection gene. The hGC-transfected hLZ cells were antibiotic-selected (G418, Sigma-Aldrich, St. Louis, MO, USA) until the isolation of positive cell colonies, whereas hLF-transfected hLZ cells were seeded onto 100-mm culture plates (100 cells/plate) to allow colony outgrowth from individual cells. Isolated colonies were screened by PCR using specific primers for each transgene (hGC or hLF) and for hLZ and GAPDH (controls). Positive cells from one hLZ-hGC and one hLZ-hLF colony were used for cloning at passage 9, whereas hLZ cells from the other genotype were at passage 4. Cells were synchronized by high confluence and 24 h of serum starvation. Goat cloning was performed according to standard procedures (Feltrin et al. 2012 Reprod. Fertil. Dev. 25, 163). Briefly, cumulus-oocyte complexes from abattoir ovaries were in vitro-matured for 20 h. Oocyte enucleation and hLZ, hLZ-hGC, or hLZ-hLF donor cell insertion were done by micromanipulation. Reconstructed structures were fused by two 1.2-KV cm–1 DC pulses for 20 μs. Cloned embryos were cultured for 1 h in cytochalasin B and then activated in ionomycin/6-DMAP. After 12 h of in vitro culture in G-1™ medium (Vitrolife, USA), 1-cell stage embryos were transferred into the oviduct of synchronous females (Keefer et al. 2002 Biol. Reprod. 66, 199-203). Pregnancy diagnosis was performed by ultrasonography on Day 30, with weekly monitoring afterwards. Preliminary data from 6 replicates were analysed by the chi-square test (P &lt; 0.05). Maturation rate and survival after enucleation were 42.8% (610/1425) and 72.9% (291/399), respectively. A total of 271 structures were reconstructed using the 3 donor cell lines. Fusion rates did not differ between hLZ (59.5%), hLZ-hGC (47.5%), and hLZ-hLF (48.5%) groups. A total of 68 hLZ, 92 hLZ-hGC, and 39 hLZ-hLF-derived embryos were transferred to 5, 7, and 3 recipients, respectively. No pregnancies were detected with the use of hLZ and hLZ-hLF cells. However, 3 pregnancies (one nonviable) were detected on Day 30 with hLZ-hGC cells (42.9%), with both viable pregnancies lost on Days 40 and 130 of gestation. Molecular analyses confirmed both concepti as transgenic clones from the hLZ-hGC cell line. In summary, antibiotic selection of positive colonies was effective at maintaining cell viability, with a positive response when used for cloning. Replications are in progress to evaluate the effect of cell colony isolation from individual cells (e.g. hLZ-hLF cells) on cell viability over time and on cloning outcome.

THE USE OF PROTOPLAST FUSION AND TRANSFORMATION FOR THE PRODUCTION OF ASYMMETRIC SOMATIC HYBRIDS

ABSTRACT In asymmetric hybridization experiments, a Nicotiana paniculata cell line transformed with the Agrobacterium tumefaciens strain B6S3 was used as the donor plant material. This cell line exhibits two dominant marker genes, hormone-independent growth behavior and synthesis of octopine, as a result of the incorporation of T-DNA from Agrobacterium. The recipient cell line was the cnx-68 N. tabacum cell line which has two recessive markers: nitrate reductase deficiency and presence of an intracellular calcium-oxalate druse. Isolated protoplasts of the donor cell line N. paniculata B6S3 were mitotically inactivated by X-rays and fused with protoplasts of the cell line cnx-68. Asymmetric somatic hybrids were selected on hormone-free agar medium supplemented with 50 mM KClO3. Eighteen cell lines survived the selection treatment; seven of them exhibited the two dominant and the two recessive markers. In a second experiment, the marker gene hygromycin resistance was introduced into Brassica nigra by using ...

Abstract 1573: An inducible transgenic porcine model for human cancer.

Abstract Common rodent-based models have limitations in terms of modeling human cancers. Given that pigs share many genetic and physiological similarities with humans, we investigated the potential of developing genetic porcine models of cancer. In this regard, we previously reported that activation of oncogenes like Ras in conjunction with inhibiting tumor suppressor pathways like p53 were required, in part, to convert normal porcine cells to a tumorigenic state. Based on this, we chose to generate transgenic pigs that can be induced to express oncogenic Kras and dominant-negative p53. First, porcine Kras and p53 wild-type genes were cloned, sequenced and aligned with porcine, human and murine homologues to identify porcine-specific mutation sites corresponding to those commonly found in human cancers. Porcine Kras mutation occurs at the 12th glycine (G) to aspartic acid (D), whereas p53 arginine (R) at 167th position was mutated to histidine (H). KrasG12D and p53R167H mutants were linked by internal ribosome entry sites (IRES) for their simultaneous expression and then inserted into a vector following the LoxP-polyA(STOP)-LoxP sequence (LSL). Porcine fetal fibroblasts were transfected in vitro with this vector construct and infected by adenovirus (Ad) vectors encoding Cre recombinase (Ad-Cre-GFP), which deletes the LSL sequence and permits transgene expression, or control Ad vectors without the inserted Cre transgene (Ad-GFP). Cre recombinase-mediated KrasG12D and p53R167H expression was significantly induced in porcine fibroblasts transfected with Ad-Cre-GFP virus compared with Ad-GFP control, which provides an in vitro proof of functional test of the “oncopig” construct. We then transfected porcine fibroblasts with the aforementioned “oncopig” construct to produce donor cell lines for nuclear transfer cloning. Transgenic fibroblast cell lines generated from the cloned pig were subjected to a wound assay through which we observed a statistically significant difference of in vitro migration capability between Ad-Cre-GFP cells and Ad-GFP control cells. In a migration time of 24h, the number of cells in the wound area for the Ad-Cre-GFP cells was 184 as for the Ad-GFP cells was only 67 (p-value ≤ 0.01). A statistically significant difference was also observed between the cell cycle length for these cell lines by flow cytometry. Ad-Cre-GFP cells went through a greater number of cell divisions compared with Ad-GFP cells. Within a 73h time period, Ad-Cre-GFP cells divided twice as many times than Ad-GFP cells (p-value ≤ 0.01). Present results demonstrate that induction of the transgenes in these porcine cells triggered a transformed phenotype. In the future, these cells will be tested for growth in soft agar, tumor growth in mice, and then the pigs will be monitored for tumor incidence following site-specific transgene induction. Such an approach could provide a porcine model to cancer etiology and the development of anti-cancer therapy. Citation Format: Fernanda M. Rodrigues, Wenping Hu, Laurie Rund, Ying Liang, Christopher Counter, Lawrence Schook. An inducible transgenic porcine model for human cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1573. doi:10.1158/1538-7445.AM2013-1573

Impact of Dose Intensification of FluBu2 to FluBu4 on Transplant Related Mortality, Relapse, and Survival After Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia in Remission

Methods: A retrospective review of the patients who relapsed following an allogeneic HCT for a myeloid malignancy at Mayo Clinic in Arizona was performed. The BM sample closest in time prior to relapse was identified and the morphologic, flow cytometric, karyotypic, FISH, PCR and chimerism results were reviewed. Results: Out of 187 patients with a myeloid malignancy, 40 (31 AML, 4 MPN blast crisis, 4 MDS, 1 CML) relapsed at a median of 113 days (range 22-820 days) after HCT. Thirty percent of the relapses occurred after a myeloablative HCT. Donor type was equally distributed between matched related and mismatched/ matched unrelated donors. A cytogenetic and molecular marker with the diagnosis was identifiable in 22/40 and 6/40 patients respectively. 18/40 patients had residual disease present at HCT. 13/40 patients showed an early relapse within the first 90 days. In the remaining patients, time between the last BM and relapse ranged from 38-184 days (median 69). Abnormal results were found only in 9/27 patients in the preceding BM and included abnormal cytogenetic clone identical to diagnosis in 2 patients; abnormal RT-PCR for BCR/ABL in 1, cytogenetic abnormality in donor cell line in 3, and decreased chimerisms in unsorted marrow cells in 3 patients. Conclusions: A 3-month monitoring schedule of BM evaluation using the currently available tools had low sensitivity to predict relapse. Findings that may justify intervention were rare (less than 10% of patients), and were restricted to abnormal cytogenetic and abnormal RT-PCR results. The clinical significance of cytogenetic abnormalities observed in donor cells is not clear. Our findings emphasize that until the optimal surveillance technique and schedule can be defined and standardized, more frequent use of pre-emptive therapy in patients at high risk of relapse may be of benefit.

Nuclear Donor Cell Lines Considerably Influence Cloning Efficiency and the Incidence of Large Offspring Syndrome in Bovine Somatic Cell Nuclear Transfer

Total five ear skin fibroblast lines (named F1, F2, F3, F4 and F5) from different newborn Holstein cows have been used as nuclear donor cells for producing cloned cows by somatic cell nuclear transfer (SCNT). The effects of these cell lines on both in vitro and in vivo developmental rates of cloned embryos, post-natal survivability and incidence of large offspring syndrome (LOS) were examined in this study. We found that the different cell lines possessed the same capacity to support pre-implantation development of cloned embryos, the cleavage and blastocyst formation rates ranged from 80.2±0.9 to 84.5±2.5% and 28.5±0.9 to 33.3±1.4%, respectively. However, their capacities to support the in vivo development of SCNT embryos showed significant differences (p<0.05). The pregnancy rates at 90 and 240day were significantly lower in groups F2 (4.9% and 3.3%) and F3 (5.4% and 5.4%) compared to groups F1 (23.3% and 16.3%), F4 (25.7% and 18.6%) and F5 (25.9% and 19.8%) (p<0.05). The cloning efficiency was significantly higher in group F5 than those in group F1, F2, F3 and F4 (9.3% vs 4.1%, 1.2%, 2.0% and 5.0%, respectively, p<0.05). Moreover, large offspring syndrome (LOS) incidence in group F5 was significantly lower than those in other groups (p<0.05). All cloned offspring from cell line F1, F2, F3 and F4 showed LOS and gestation length delay, while all cloned offspring from F5 showed normal birthweight and gestation length. We concluded that the nuclear donor cell lines have significant impact on the in vivo development of cloned embryos and the incidence of LOS in cloned calves.

Strategy for the creation of clinical grade hESC line banks that HLA‐match a target population

Here, we describe a pre-derivation embryo haplotyping strategy that we developed in order to maximize the efficiency and minimize the costs of establishing banks of clinical grade hESC lines in which human leukocyte antigen (HLA) haplotypes match a significant proportion of the population. Using whole genome amplification followed by medium resolution HLA typing using PCR amplification with sequence-specific primers (PCR-SSP), we have typed the parents, embryos and hESC lines from three families as well as our eight clinical grade hESC lines and shown that this technical approach is rapid, reliable and accurate. By employing this pre-derivation strategy where, based on HLA match, embryos are selected for a GMP route on day 3–4 of development, we would have drastically reduced our cGMP laboratory running costs.