Elizabeth W. Uhl

The Cell Surface Glycosphingolipids SSEA‐3 and SSEA‐4 Are Not Essential for Human ESC Pluripotency

Pluripotent cells can be isolated from the human blastocyst and maintained in culture as self‐renewing, undifferentiated, human ESCs (hESCs). These cells are a valuable model of human development in vitro and are the focus of substantial research aimed at generating differentiated populations for cellular therapies. The extracellular markers that have been used to characterize hESCs are primarily carbohydrate epitopes on proteoglycans or sphingolipids, such as stage‐specific embryonic antigen (SSEA)‐3 and ‐4. The expression of SSEA‐3 and ‐4 is tightly regulated during preimplantation development and on hESCs. Although this might imply a molecular function in undifferentiated cells, it has not yet been tested experimentally. We used inhibitors of sphingolipid and glycosphingolipid (GSL) biosynthesis to block the generation of SSEA‐3 and ‐4 in hESCs. Depletion of these antigens and their precursors was confirmed using immunostaining, flow cytometry, and tandem mass spectroscopy. Transcriptional analysis, immunostaining, and differentiation in vitro and in teratomas indicated that other properties of pluripotency were not noticeably affected by GSL depletion. These experiments demonstrated that the GSLs recognized as SSEA‐3 and ‐4 do not play critical functional roles in maintaining the pluripotency of hESCs, but instead suggested roles for this class of molecules during cellular differentiation.

Characterization of a New NIH‐Registered Variant Human Embryonic Stem Cell Line, BG01V: A Tool for Human Embryonic Stem Cell Research

Human embryonic stem cells (hESCs) offer a renewable source of a wide range of cell types for use in research and cell‐based therapies. Characterizing these cells provides important information about their current state and affords relevant details for subsequent manipulations. For example, identifying genes expressed during culture, as well as their temporal expression order after passaging and conditions influencing the formation of all three germ layers may be helpful for the production of functional beta islet cells used in treating type I diabetes. Although several hESC lines have demonstrated karyotypic instability during extended time in culture, select variant lines exhibit characteristics similar to their normal parental lines. Such variant lines may be excellent tools and abundant sources of cells for pilot studies and in vitro differentiation research in which chromosome number is not a concern, similar to the role currently played by embryonal carcinoma cell lines. It is crucial that the cells be surveyed at a genetic and proteomic level during extensive propagation, expansion, and manipulation in vitro. Here we describe a comprehensive characterization of the variant hESC line BG01V, which was derived from the karyotypically normal, parental hESC line BG01. Our characterization process employs cytogenetic analysis, short tandem repeat and HLA typing, mitochondrial DNA sequencing, gene expression analysis using quantitative reverse transcription‐polymerase chain reaction and microarray, assessment of telomerase activity, methylation analysis, and immunophenotyping and teratoma formation, in addition to screening for bacterial, fungal, mycoplasma, and human pathogen contamination.

Brief Report: Chimeric Pigs Produced from Induced Pluripotent Stem Cells Demonstrate Germline Transmission and No Evidence of Tumor Formation in Young Pigs

The recent development of porcine induced pluripotent stem cells (piPSCs) capable of generating chimeric animals, a feat not previously accomplished with embryonic stem cells or iPSCs in a species outside of rodents, has opened the doors for in‐depth study of iPSC tumorigenicity, autologous transplantation, and other key aspects to safely move iPSC therapies to the clinic. The study of iPSC tumorigenicity is critical as previous research in the mouse showed that iPSC‐derived chimeras possessed large numbers of tumors, rising significant concerns about the safety of iPSC therapies. Additionally, piPSCs capable of generating germline chimeras could revolutionize the transgenic animal field by enabling complex genetic manipulations (e.g., knockout or knockin of genes) to produce biomedically important large animal models or improve livestock production. In this study, we demonstrate for the first time in a nonrodent species germline transmission of iPSCs with the live birth of a transgenic piglet that possessed genome integration of the human POU5F1 and NANOG genes. In addition, gross and histological examination of necropsied porcine chimeras at 2, 7, and 9 months showed that these animals lacked tumor formation and demonstrated normal development. Tissue samples positive for human POU5F1 DNA showed no C‐MYC gene expression, further implicating C‐MYC as a cause of tumorigenicity. The development of germline‐competent porcine iPSCs that do not produce tumors in young chimeric animals presents an attractive and powerful translational model to study the efficacy and safety of stem cell therapies and perhaps to efficiently produce complex transgenic animals. STEM CELLS 2011;29:1640–1643

Advances in FIV vaccine technology

Advances in vaccine technology are occurring in the molecular techniques used to develop vaccines and in the assessment of vaccine efficacy, allowing more complete characterization of vaccine-induced immunity correlating to protection. FIV vaccine development has closely mirrored and occasionally surpassed the development of HIV-1 vaccine, leading to first licensed technology. This review will discuss technological advances in vaccine designs, challenge infection assessment, and characterization of vaccine immunity in the context of the protection detected with prototype and commercial dual-subtype FIV vaccines and in relation to HIV-1.

Identification of altered MicroRNA expression in canine lymphoid cell lines and cases of B- and T-Cell lymphomas†

Canine lymphoma is a common spontaneous tumor with many similarities to human lymphoma, and thus has potential to be an important animal model of lymphomagenesis. This study determined that microRNA (miRNA) expression in canine tumors can be assessed using a commercially available human cancer miRNA qPCR array. miRNA expression in six different canine lymphoid cell lines and in naturally occurring canine B‐ and T‐cell lymphomas was compared using RNA harvested from normal canine peripheral blood mononuclear cells (PBMC) and normal lymph nodes (LN) as controls. We found that false discovery rate (FDR) correction for multiple testing after quantile normalization controlled for variation across arrays and that they were the best methods for normalization and statistical analysis. Increases in miRNAs known to upregulate oncogenes (miR19a+b, miR17‐5p) and decreased expression of miRNAs with tumor suppressor functions (miR‐203, miR‐218, and miR‐181a) also seen in human lymphoid malignancies were observed. However, there were few similarities between canine groups. The results of this study indicate that the use of both PBMC and LN cells as controls provides different, but potentially equally important targets for further analysis. Our findings of miRNA dysregulation in canine lymphoid cell lines and clinical cases of lymphoma emphasize the potential of canine lymphoma as an important spontaneous, large animal model of human B‐ and T‐cell lymphomas.

Cardiomyopathy and Right-sided Congestive Heart Failure in a Red-tailed Hawk (Buteo jamaicensis)

Abstract A 15-year-old female red-tailed hawk (Buteo jamaicensis) was evaluated because of dyspnea, anorexia, and coelomic distension. Diagnostic imaging results confirmed severe coelomic effusion and revealed a markedly dilated right ventricle. The diagnosis was right-sided congestive heart failure. Results of measurements of vitamin E, selenium, lead, zinc, and cardiac troponin levels were normal or nondiagnostic. The hawk was treated with furosemide, antifungal and antimicrobial agents, and supplemental fluids and oxygen, but euthanasia was elected because of the poor prognosis and the practical difficulties associated with intensive case management. To our knowledge, this is the first described case of cardiomyopathy and congestive heart failure in a captive red-tailed hawk.

Mouse Models as Predictors of Human Responses: Evolutionary Medicine

Mice offer a number of advantages and are extensively used to model human diseases and drug responses. Selective breeding and genetic manipulation of mice have made many different genotypes and phenotypes available for research. However, in many cases, mouse models have failed to be predictive. Important sources of the prediction problem have been the failure to consider the evolutionary basis for species differences, especially in drug metabolism, and disease definitions that do not reflect the complexity of gene expression underlying disease phenotypes. Incorporating evolutionary insights into mouse models allow for unique opportunities to characterize the effects of diet, different gene expression profiles, and microbiomics underlying human drug responses and disease phenotypes.

FIV-infected cats respond to short-term rHuG-CSF treatment which results in anti-G-CSF neutralizing antibody production that inactivates drug activity.

Abstract The hematological and virological effects of recombinant human granulocyte colony-stimulating factor (rHuG-CSF) were evaluated in feline immunodeficiency virus (FIV)-infected cats. Six age-matched, FIV-infected cats used in this cross-over study were injected subcutaneously with 5μg/kg of rHuG-CSF daily for 3 weeks, while six control cats received a placebo. Five of six rHuG-CSF-treated cats had significant increases in neutrophil counts that peaked on days 11–21 of treatment. All rHuG-CSF-treated cats exhibited an increase in myeloid:erythroid ratios of the bone marrow cells without significant changes in lymphocyte, CD4 counts, CD4/CD8 ratios, RBC counts, FIV antibody titers, and FIV loads in peripheral blood, and without clinical and hematological toxicities. Five of six rHuG-CSF-treated cats developed antibodies to rHuG-CSF by 14–21 days of treatment, which correlated with decreasing neutrophil counts and increasing neutralizing antibodies to rHuG-CSF. Three cats re-treated with rHuG-CSF rapidly developed neutralizing antibodies to rHuG-CSF, while one cat also developed neutralizing antibodies to recombinant feline G-CSF (rFeG-CSF). Overall, rHuG-CSF treatment increased neutrophil counts in FIV-infected cats without affecting the infection status of cats. However, long-term use of rHuG-CSF is not recommended in cats because of the neutralizing antibody production to rHuG-CSF that affects the drug activity. In addition, a preliminary finding suggests that repeated treatment cycle can also induce cross-neutralizing antibodies to rFeG-CSF, which may potentially affect the homeostasis of endogenous FeG-CSF.

Treatment of intestinal pythiosis in a dog with a combination of marginal excision, chemotherapy, and immunotherapy

CASE DESCRIPTION A 1.5-year-old mixed-breed dog was examined because of a 1-month history of anorexia, vomiting, diarrhea, and weight loss. CLINICAL FINDINGS The dog was very thin on physical examination (body condition score, 3/9). Results of all diagnostic tests were within reference limits except intestinal thickening and lymphadenopathy were identified on abdominal ultrasound examination. During exploratory laparotomy, thickening at the ileocecal-colic junction and within the transverse colon and mesenteric lymphadenopathy were identified, and the ileocecal-colic junction was resected. Histopathologic evaluation of the ileocecal-colic junction and full-thickness biopsy specimens from other sites as well as results of a serum ELISA were diagnostic for gastrointestinal Pythium insidiosum infection. TREATMENT AND OUTCOME Pythiosis was initially treated medically with administration of itraconazole and terbinafine by mouth, but the colonic lesion was progressive with this regimen. Two months after diagnosis, a subtotal colectomy was performed; marginal excision (0.6 cm) was obtained at the aboral margin. The dog was treated with 3 doses of a pythiosis vaccine beginning approximately 2 weeks after surgery and was continued on itraconazole and terbinafine for 5 months. Parenteral and enteral nutrition as well as considerable general supportive care were required postoperatively. Six months after treatment, the dog had a normal serum ELISA titer. Two years after treatment, the dog had returned to preoperative weight and was clinically normal. CLINICAL RELEVANCE This patient had an unusually positive therapeutic response to chronic, extensive, marginally excised gastrointestinal pythiosis.

IN OVO AND IN VITRO SUSCEPTIBILITY OF AMERICAN ALLIGATORS (ALLIGATOR MISSISSIPPIENSIS) TO AVIAN INFLUENZA VIRUS INFECTION

Abstract Avian influenza has emerged as one of the most ubiquitous viruses within our biosphere. Wild aquatic birds are believed to be the primary reservoir of all influenza viruses; however, the spillover of H5N1 highly pathogenic avian influenza (HPAI) and the recent swine-origin pandemic H1N1 viruses have sparked increased interest in identifying and understanding which and how many species can be infected. Moreover, novel influenza virus sequences were recently isolated from New World bats. Crocodilians have a slow rate of molecular evolution and are the sister group to birds; thus they are a logical reptilian group to explore susceptibility to influenza virus infection and they provide a link between birds and mammals. A primary American alligator (Alligator mississippiensis) cell line, and embryos, were infected with four, low pathogenic avian influenza (LPAI) strains to assess susceptibility to infection. Embryonated alligator eggs supported virus replication, as evidenced by the influenza virus M gene and infectious virus detected in allantoic fluid and by virus antigen staining in embryo tissues. Primary alligator cells were also inoculated with the LPAI viruses and showed susceptibility based upon antigen staining; however, the requirement for trypsin to support replication in cell culture limited replication. To assess influenza virus replication in culture, primary alligator cells were inoculated with H1N1 human influenza or H5N1 HPAI viruses that replicate independent of trypsin. Both viruses replicated efficiently in culture, even at the 30 C temperature preferred by the alligator cells. This research demonstrates the ability of wild-type influenza viruses to infect and replicate within two crocodilian substrates and suggests the need for further research to assess crocodilians as a species potentially susceptible to influenza virus infection.