Neil Talbot - Academia.edu (original) (raw)

Papers by Neil Talbot

In Vitro Cellular Developmental Biology Animal, 2010

Liver in vitro models are needed to replace animal models for rapid assessment of drug biotransfo... more Liver in vitro models are needed to replace animal models for rapid assessment of drug biotransformation and toxicity. The PICM-19 pig liver stem cell line may fulfill this need since these cells have activities associated with xenobiotic phase I and II metabolism lacking in other liver cell lines. The objective of this study was to characterize phase I and II metabolic functions of a PICM-19 derivative cell line, PICM-19H, compared to the tumor-derived human HepG2 C3A cell line and primary cultures of adult porcine hepatocytes. Following exposure of PICM-19H cells to either 3-methylcholanthrene, rifampicin or phenobarbital, the induced activities of cytochrome P450 (CYP450) isozymes CYP-1A, -2, and-3A were assessed. Relative to adult porcine hepatocytes, PICM-19H cells exhibited 30% and 43%, respectively, of CYP1A and 3A activities, while HepG2 C3A cells exhibited 7% and 0% of those activities. Fluorescent metabolites were extensively conjugated, i.e., 52% and 96% of CYP450-1A and-3A metabolites were released from medium samples following treatment with β-glucuronidase/arylsulfatase. Rifampicin induction of CYP450 isozyme activities was confirmed by conversion of testosterone to 6β-OH-, 2α-OH-and 2β-OH-testosterone, as determined by mass spectrometry. Susceptibility of PICM-19H cells to acetaminophen toxicity was determined; CD 50 was calculated to be 14.9±0.9 mM. Toxicity and bioactivation of aflatoxin B1 was determined in 3-methylcholanthrenetreated cultures and untreated controls; CD 50 were 1.59 μM and 31 μM, respectively. These results demonstrate the potential use of PICM-19H cells in drug biotransformation and toxicity testing and further support their use in extracorporeal artificial liver device technology.

Molecular Reproduction and Development, 2015

World journal of gastroenterology : WJG, Jan 21, 2015

To identify the genes induced and regulated by the MYC protein in generating tumors from liver st... more To identify the genes induced and regulated by the MYC protein in generating tumors from liver stem cells. In this study, we have used an immortal porcine liver stem cell line, PICM-19, to study the role of c-MYC in hepatocarcinogenesis. PICM-19 cells were converted into cancer cells (PICM-19-CSCs) by overexpressing human MYC. To identify MYC-driven differential gene expression, transcriptome sequencing was carried out by RNA sequencing, and genes identified by this method were validated using real-time PCR. In vivo tumorigenicity studies were then conducted by injecting PICM-19-CSCs into the flanks of immunodeficient mice. Our results showed that MYC-overexpressing PICM-19 stem cells formed tumors in immunodeficient mice demonstrating that a single oncogene was sufficient to convert them into cancer cells (PICM-19-CSCs). By using comparative bioinformatics analyses, we have determined that > 1000 genes were differentially expressed between PICM-19 and PICM-19-CSCs. Gene ontology...

Uterine-derived factors are essential for conceptus develop- ment and secretion of the maternal r... more Uterine-derived factors are essential for conceptus develop- ment and secretion of the maternal recognition-of-pregnancy factor, interferon- (IFNT), in ruminant species. The objec- tives of this study were to determine whether fibroblast growth factor-2 (FGF-2) is expressed in the bovine uterus dur- ing early pregnancy in cattle and to determine whether FGF-2 supplementation affects IFNT mRNA and protein abundance in bovine

Veterinary Research, 2004

To characterize further the chemical and biological properties of bovine soluble (bos) CD14, a pa... more To characterize further the chemical and biological properties of bovine soluble (bos) CD14, a panel of ten murine monoclonal antibodies (mAb) reactive with recombinant (r) bosCD14 were produced. A sandwich ELISA, using murine mAb and rabbit polyclonal antibodies reactive with rbosCD14 was developed. All the mAb were reactive by ELISA with baculovirus-derived rbosCD14 and they recognized rbosCD14 (40 kDa) by western blot analysis. The mAb also identified by western blot sCD14 (53 and 58 kDa) in milk and blood and sCD14 (47 kDa) in a lysate of macrophages obtained from involuted bovine mammary gland secretions. Analysis by ELISA of whey samples after intramammary injection of lipopolysaccharide (LPS) (10 µg) revealed increased sCD14 levels between 8 to 48 h after injection. Flow cytometric analysis showed that the mAb bound to macrophages isolated from involuted mammary gland secretions and mouse macrophages but not to swine or horse monocytes. Addition of anti-rbosCD14 mAb to monocytes stimulated with LPS reduced in vitro production of TNF-α. The anti-rbosCD14 antibodies generated in this study will be useful in studying CD14, an accessory molecule that contributes to host innate recognition of bacterial cell wall components in mammary secretions produced during mastitis.

Veterinary Immunology and Immunopathology, 1998

Macrophages were selectively expanded and continuously cultured from adult pig blood. One-half ml... more Macrophages were selectively expanded and continuously cultured from adult pig blood. One-half ml of heparinized adult pig blood was inoculated directly into the medium overlaying a feeder layer of STO mouse fibroblasts. After attachment to the feeder cells for 24 h, the culture was washed several times with the medium to remove most of any unattached blood cells and re-fed. Approximately 7 x 10(4) blood monocytes were initially detected and enumerated by specific binding of DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL). Macrophage outgrowths appeared in the primary culture after 6-7 days. The macrophages grew to relatively high density in 2-3 weeks (2-3 x 10(6) cells/T25 flask), and the culture was passaged on to fresh STO feeder layers to begin secondary culture. Over 2-3 months of culture the macrophage replication produced as many as 1.4 x 10(9) DiI-Ac-LDL-positive cells. The macrophages grew on top of the feeder cells in two forms: either a semi-attached, round morphology, or a closely adherent, flat ameboid morphology with several extended pseudopods. Electron microscopic examination revealed the cells to be uniformly of macrophage character and that 4-5% were giant cells. The macrophages were phagocytic and expressed CD14 on their surfaces. They also reacted positively with pig macrophage-specific monoclonal antibody (mAb), and were negative for reactivity with pig T- and B-cell-specific mAb. This simple method for isolating and propagating macrophages may indicate the replicative capacity of either adult pig blood monocytes or circulating blood stem cells, and it may be useful in providing macrophages for general research, virological assay, adoptive-immunotherapy models, and somatic gene therapy models.

Stem Cell Reviews, 2008

In contrast to differentiated cells, embryonic stem cells (ESC) maintain an undifferentiated stat... more In contrast to differentiated cells, embryonic stem cells (ESC) maintain an undifferentiated state, have the ability to self-renew, and exhibit pluripotency, i.e., they can give rise to most if not all somatic cell types and to the germ cells, egg and sperm. These characteristics make ES cell lines important resources for the advancement of human regenerative medicine, and, if established for domesticated ungulates, would help make possible the improvement of farm animals through their contribution to genetic engineering technology. Combining other genetic engineering technologies, such as somatic cell nuclear transfer with ESC technology may result in synergistic gains in the ability to precisely make and study genetic alterations in mammals. Unfortunately, despite significant advances in our understanding of human and mouse ESC, the derivation of ES cell lines from ungulate species has been unsuccessful. This may result from a lack of understanding of species-specific mechanisms that promote or influence cell pluripotency. Thorough molecular characterizations, including the elucidation of stem cell "marker" signaling cascade hierarchy, species-appropriate pluripotency markers, and pluripotency-associated chromatin alterations in the genomes of ungulate species, should improve the chances of developing efficient, reproducible technologies for the establishment of ES cell lines of economically important species like the pig, cow, goat, sheep and horse.

PROTEOMICS, 2009

Embryo loss during peri-implantation can approach 20% in swine following artificial insemination ... more Embryo loss during peri-implantation can approach 20% in swine following artificial insemination or natural mating and coincides with rapid conceptus elongation. The objective of the present study was to establish a comprehensive profile of the abundant proteins of the pig conceptus at the time prior to implantation and identify stage-specific changes during elongation. The abundant proteins of a homogenous population of gestational day-11 ovoid (0.7-1 cm) and gestational day-12 filamentous (15-20 cm) porcine concepti were compared by extracting proteins from three independent conceptus pools and separating the proteins by 2-DE. Proteins in 305 spots were analyzed by MALDI-TOF or additionally by LC-MS/MS and 275 were positively identified representing 174 distinct proteins. The proteins could be classified into the following functional categories: cell proliferation/differentiation, cytoskeleton, metabolism, and stress response. Based on spot density, 35 proteins associated with cell proliferation, differentiation, apoptosis, and embryo/maternal signaling, were found to be differentially expressed between ovoid and filamentous concepti. A comparison of the protein expression profile with transcriptomic data from pig concepti of the same developmental stages identified similarities and dissimilarities between protein and mRNA expression profiles. This proteomic study helps to elucidate the biological mechanisms underlying the early embryonic development of the pig.

Nature Biotechnology, 2005

Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of... more Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 mg/ml in their milk were produced. In vitro assays demonstrated the milk's ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 mg/ml of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.

Nature Biotechnology, 2005

Molecular Reproduction and Development, 2008

Limited understanding of the importance of known pluripotency factors in pig embryonic stem cells... more Limited understanding of the importance of known pluripotency factors in pig embryonic stem cells (ESC) impedes the establishment and validation of porcine ESC lines. This study evaluated the expression of known mouse ESC and human ESC (hESC) pluripotency markers in in vivo inner cell mass (ICM) and in vitro-cultured undifferentiated porcine epiblast cells isolated from 8-day porcine blastocysts, primary cultures of epiblast-derived neuroprogenitor cells, and endoderm cells. The expression profile of common pluripotency markers (POU domain 5 transcript factor 1, SRY-box containing gene 2, and Nanog homeobox), species-specific markers, ESC-associated factors, and differentiation markers was evaluated. The mRNA of uncultured ICMs, cultured epiblast cells, epiblast-derived neuroprogenitor cells, and endoderm cells was amplified prior to expression analysis of candidate genes by real-time RT-PCR. ESC factors whose expression correlated best with the undifferentiated epiblast state were identified by comparative mRNA expression analysis between porcine epiblast-derived somatic cell lines, fetal fibroblasts, and adult tissues. Across tissue types Nanog homeobox exhibited ubiquitous expression, whereas POU domain 5 transcript factor 1, teratocarcinoma-derived growth factor 1, and RNA exonuclease homolog 1 transcript expression was restricted primarily to undifferentiated epiblasts. Our results suggested that expression of pluripotency markers in undifferentiated pig epiblast cells more closely resembled that observed in hESC. Expression alterations of ESC-associated factors in epiblast cells were also observed during in vitro culture. Our data demonstrate the potential use of some pluripotency factors as markers of porcine epiblast stem cells and indicate that the in vitro environment may influence the cultured epiblast's developmental state.

Molecular Reproduction and Development, 1993

To define better the characteristics of pig and sheep epiblast cells in culture, the cells were t... more To define better the characteristics of pig and sheep epiblast cells in culture, the cells were tested for the presence of alkaline phosphatase (AP), a biochemical marker characteristic of mouse embryonic stem cells. Pig and sheep epiblast cells were positive for AP staining both at isolation from the blastocyst and after primary in vitro culture. The innermost portion of the attendant endoderm surrounding the epiblast was also positive for AP staining during primary culture. AP staining was lost upon differentiation or senescence of the epiblast cells. Also, all differentiated epiblast-derived cell cultures were negative for AP staining, with the exception of neuron-like cultures. Epiblast-like cells were cultured from day 10 (pig) and day 13 (sheep) embryonic discs, and these cells were also AP positive until they differentiated. Trophectoderm-endoderm-like cells from embryonic discs were AP negative or weakly positive. AP is a convenient marker for undifferentiated pig and sheep epiblast cells in culture when used in conjunction with cell morphology analysis.

Molecular Reproduction and Development, 2008

The expression of interferon-tau (IFN-t) is essential for bovine embryo survival in the uterus. A... more The expression of interferon-tau (IFN-t) is essential for bovine embryo survival in the uterus. An evaluation of IFN-t production from somatic cell nuclear transfer (NT)-embryo-derived primary trophectoderm cultures in comparison to trophectoderm cultured from parthenogenote (P) and in vitro matured, fertilized, and cultured (IVP) bovine embryos was performed. In Experiment 1, the success/ failure ratio for primary trophectoderm colony formation was similar for IVP and NT blastocysts [IVP ¼ 155/ 29 (84%); NT 104/25 (81%)], but was decreased (P ¼ .05) for P blastocysts [54/43 (56%)]. Most trophectoderm colonies reached diameters of at least 1 cm within 3-4 weeks, and at this time, 72 hr conditioned cell culture medium was measured for IFN-t concentration by antiviral activity assay. The amount of IFN-t produced by IVP-outgrowths [4311 IU/mL (n ¼ 155)] was greater (P < .05) than that from NT-[626 IU/mL (n ¼ 104)] and P -[1595 IU/ mL (n ¼ 54)] derived trophectoderm. Differential expression of IFN-t was confirmed by immunoblotting. In Experiment 2, colony formation was again similar for IVP and NT blastocysts [IVP ¼ 70/5 (93%); NT 67/ 1 (99%)] and less (P < .05) for P blastocysts [65/ 27 (70%)]. Analysis of trophectoderm colony size after 23 days in culture showed a similar relationship with P-derived colonies being significantly smaller in comparison to IVP and NT colonies. A differential expression of IFN-t was also observed again, but this time as measured over time in culture. Maximal IFN-t production was found at day-14 of primary culture and diminished to a minimum by the 23rd day. Mol.

Molecular Reproduction and Development, 2004

A bovine trophectoderm cell line was established from a parthenogenetic in vitroproduced blastocy... more A bovine trophectoderm cell line was established from a parthenogenetic in vitroproduced blastocyst. To initiate the cell line, 8-day parthenogenetic blastocysts were attached to a feeder layer of STO fibroblasts and primary outgrowths occurred that consisted of trophectoderm, endoderm, and very occasionally epiblast tissue. Any endoderm and epiblast outgrowths were removed from the primary cultures within the first 10 days of culture by dissection. One of the primary trophectoderm cell cultures was chosen for further propagation and was passaged by physical dissociation and replating on STO feeder cells. The cell culture, designated BPT-1, was maintained in T25 flasks and passaged at a 1:3 split ratio for the first 15 passages approximately once every 2 weeks. Thereafter, the cell culture was passaged at 1:10-1:40 split ratios. Transmission electron microscopic examination showed the cells to be a polarized epithelium with apical microvilli, a thin basal lamina, and lateral junctions consisting of tight junctions and desmosomes. Lipid vacuoles and digestive vacuoles were also prominent features of the BPT-1 cells. Metaphase spread analysis at passage 59 indicated a near diploid cell population (2n ¼ 60) with a mode and median of 60 and a mean of 64. BPT-1 cells secreted interferon-tau into the medium as measured by anti-viral assay and Western blot analysis. The cell line provides an in vitro model of parthenogenote trophectoderm whose biological characteristics can be compared to trophectoderm cell lines derived from bovine embryos produced by normal fertilization or nuclear transfer.

Journal of General Virology, 2013

Hepatitis E virus (HEV) causes acute, enterically transmitted hepatitis in human. It is associate... more Hepatitis E virus (HEV) causes acute, enterically transmitted hepatitis in human. It is associated with large epidemics in tropical and subtropical regions where it is endemic or with sporadic cases in non-endemic regions. Unlike other hepatitis viruses, HEV has several animal reservoirs. Phylogenetic studies on HEV human and animal sequences, and the identification of cases of direct transmission from animal to human strongly suggest that HEV is a zoonotic agent. The lack of efficient cell culture models limits studies on molecular and cellular aspects of HEV infection and species barrier crossing. The present study reports on the development of two new in vitro models of HEV replication using a human hepatoma-derived cell line, HepaRG, and a porcine embryonic stem cell-derived cell line, PICM-19. These two cell lines have morphological and functional properties similar to primary hepatocytes. These in vitro culture systems support HEV replication and release of encapsidated RNA. These new models represent a powerful tool for studying the viral replication cycle, species barrier crossing and virulence factors.

In Vitro Cellular & Developmental Biology - Animal, 1993

Pig epiblast cells that had been separated from other early embryonic cells were cultured in vitr... more Pig epiblast cells that had been separated from other early embryonic cells were cultured in vitro. A three-step dissection protocol was used to isolate the epiblast from trophectoderm and primitive endoderm before culturing. Blastocysts collected at 7 to 8 days postestrus were immunodissected to obtain the inner cell mass (ICM) and destroy tropbeetodermal cells. The ICM was cultured for 2 to 3 days on STO feeder cells. The epiblast was then physically dissected free of associated primitive endoderm. Epiblast-derived cells, grown on STO feeders, produced colonies of small cells resembling mouse embryonic stem cells. This primary cell morphology changed as the colonies grew and evolved into three distinct colony types (endodermlike, neural rosette, or complex). Cell cultures derived from these three colony types spontaneously differentiated into numerous specialized cell types in STO co-culture. These included fibroblasts, endodermlike cells, neuronlike cells, pigmented cells, adipogenic cells, contracting muscle cells, dome-forming epithelium, ciliated epithelium, tubule-forming epithelium, and a round amoeboid cell type resembling a plasmacyte after Wright staining. The neuronlike cells, contracting muscle cells, and tubule-forming epithelium had normal karyotypes and displayed finite or undefined life spans upon long-term STO co-culture. The dome-forming epithelium had an indefinite life span in STO co-culture and also retained a normal karyotype. These results demonstrate the in vitro pluripotency of pig epiblast cells and indicate the epiblast can be a source for deriving various specialized cell cultures or cell lines.

In Vitro Cellular Developmental Biology Animal, 2010

Liver in vitro models are needed to replace animal models for rapid assessment of drug biotransfo... more Liver in vitro models are needed to replace animal models for rapid assessment of drug biotransformation and toxicity. The PICM-19 pig liver stem cell line may fulfill this need since these cells have activities associated with xenobiotic phase I and II metabolism lacking in other liver cell lines. The objective of this study was to characterize phase I and II metabolic functions of a PICM-19 derivative cell line, PICM-19H, compared to the tumor-derived human HepG2 C3A cell line and primary cultures of adult porcine hepatocytes. Following exposure of PICM-19H cells to either 3-methylcholanthrene, rifampicin or phenobarbital, the induced activities of cytochrome P450 (CYP450) isozymes CYP-1A, -2, and-3A were assessed. Relative to adult porcine hepatocytes, PICM-19H cells exhibited 30% and 43%, respectively, of CYP1A and 3A activities, while HepG2 C3A cells exhibited 7% and 0% of those activities. Fluorescent metabolites were extensively conjugated, i.e., 52% and 96% of CYP450-1A and-3A metabolites were released from medium samples following treatment with β-glucuronidase/arylsulfatase. Rifampicin induction of CYP450 isozyme activities was confirmed by conversion of testosterone to 6β-OH-, 2α-OH-and 2β-OH-testosterone, as determined by mass spectrometry. Susceptibility of PICM-19H cells to acetaminophen toxicity was determined; CD 50 was calculated to be 14.9±0.9 mM. Toxicity and bioactivation of aflatoxin B1 was determined in 3-methylcholanthrenetreated cultures and untreated controls; CD 50 were 1.59 μM and 31 μM, respectively. These results demonstrate the potential use of PICM-19H cells in drug biotransformation and toxicity testing and further support their use in extracorporeal artificial liver device technology.

Molecular Reproduction and Development, 2015

World journal of gastroenterology : WJG, Jan 21, 2015

To identify the genes induced and regulated by the MYC protein in generating tumors from liver st... more To identify the genes induced and regulated by the MYC protein in generating tumors from liver stem cells. In this study, we have used an immortal porcine liver stem cell line, PICM-19, to study the role of c-MYC in hepatocarcinogenesis. PICM-19 cells were converted into cancer cells (PICM-19-CSCs) by overexpressing human MYC. To identify MYC-driven differential gene expression, transcriptome sequencing was carried out by RNA sequencing, and genes identified by this method were validated using real-time PCR. In vivo tumorigenicity studies were then conducted by injecting PICM-19-CSCs into the flanks of immunodeficient mice. Our results showed that MYC-overexpressing PICM-19 stem cells formed tumors in immunodeficient mice demonstrating that a single oncogene was sufficient to convert them into cancer cells (PICM-19-CSCs). By using comparative bioinformatics analyses, we have determined that > 1000 genes were differentially expressed between PICM-19 and PICM-19-CSCs. Gene ontology...

Uterine-derived factors are essential for conceptus develop- ment and secretion of the maternal r... more Uterine-derived factors are essential for conceptus develop- ment and secretion of the maternal recognition-of-pregnancy factor, interferon- (IFNT), in ruminant species. The objec- tives of this study were to determine whether fibroblast growth factor-2 (FGF-2) is expressed in the bovine uterus dur- ing early pregnancy in cattle and to determine whether FGF-2 supplementation affects IFNT mRNA and protein abundance in bovine

Veterinary Research, 2004

To characterize further the chemical and biological properties of bovine soluble (bos) CD14, a pa... more To characterize further the chemical and biological properties of bovine soluble (bos) CD14, a panel of ten murine monoclonal antibodies (mAb) reactive with recombinant (r) bosCD14 were produced. A sandwich ELISA, using murine mAb and rabbit polyclonal antibodies reactive with rbosCD14 was developed. All the mAb were reactive by ELISA with baculovirus-derived rbosCD14 and they recognized rbosCD14 (40 kDa) by western blot analysis. The mAb also identified by western blot sCD14 (53 and 58 kDa) in milk and blood and sCD14 (47 kDa) in a lysate of macrophages obtained from involuted bovine mammary gland secretions. Analysis by ELISA of whey samples after intramammary injection of lipopolysaccharide (LPS) (10 µg) revealed increased sCD14 levels between 8 to 48 h after injection. Flow cytometric analysis showed that the mAb bound to macrophages isolated from involuted mammary gland secretions and mouse macrophages but not to swine or horse monocytes. Addition of anti-rbosCD14 mAb to monocytes stimulated with LPS reduced in vitro production of TNF-α. The anti-rbosCD14 antibodies generated in this study will be useful in studying CD14, an accessory molecule that contributes to host innate recognition of bacterial cell wall components in mammary secretions produced during mastitis.

Veterinary Immunology and Immunopathology, 1998

Macrophages were selectively expanded and continuously cultured from adult pig blood. One-half ml... more Macrophages were selectively expanded and continuously cultured from adult pig blood. One-half ml of heparinized adult pig blood was inoculated directly into the medium overlaying a feeder layer of STO mouse fibroblasts. After attachment to the feeder cells for 24 h, the culture was washed several times with the medium to remove most of any unattached blood cells and re-fed. Approximately 7 x 10(4) blood monocytes were initially detected and enumerated by specific binding of DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL). Macrophage outgrowths appeared in the primary culture after 6-7 days. The macrophages grew to relatively high density in 2-3 weeks (2-3 x 10(6) cells/T25 flask), and the culture was passaged on to fresh STO feeder layers to begin secondary culture. Over 2-3 months of culture the macrophage replication produced as many as 1.4 x 10(9) DiI-Ac-LDL-positive cells. The macrophages grew on top of the feeder cells in two forms: either a semi-attached, round morphology, or a closely adherent, flat ameboid morphology with several extended pseudopods. Electron microscopic examination revealed the cells to be uniformly of macrophage character and that 4-5% were giant cells. The macrophages were phagocytic and expressed CD14 on their surfaces. They also reacted positively with pig macrophage-specific monoclonal antibody (mAb), and were negative for reactivity with pig T- and B-cell-specific mAb. This simple method for isolating and propagating macrophages may indicate the replicative capacity of either adult pig blood monocytes or circulating blood stem cells, and it may be useful in providing macrophages for general research, virological assay, adoptive-immunotherapy models, and somatic gene therapy models.

Stem Cell Reviews, 2008

In contrast to differentiated cells, embryonic stem cells (ESC) maintain an undifferentiated stat... more In contrast to differentiated cells, embryonic stem cells (ESC) maintain an undifferentiated state, have the ability to self-renew, and exhibit pluripotency, i.e., they can give rise to most if not all somatic cell types and to the germ cells, egg and sperm. These characteristics make ES cell lines important resources for the advancement of human regenerative medicine, and, if established for domesticated ungulates, would help make possible the improvement of farm animals through their contribution to genetic engineering technology. Combining other genetic engineering technologies, such as somatic cell nuclear transfer with ESC technology may result in synergistic gains in the ability to precisely make and study genetic alterations in mammals. Unfortunately, despite significant advances in our understanding of human and mouse ESC, the derivation of ES cell lines from ungulate species has been unsuccessful. This may result from a lack of understanding of species-specific mechanisms that promote or influence cell pluripotency. Thorough molecular characterizations, including the elucidation of stem cell &amp;amp;amp;amp;quot;marker&amp;amp;amp;amp;quot; signaling cascade hierarchy, species-appropriate pluripotency markers, and pluripotency-associated chromatin alterations in the genomes of ungulate species, should improve the chances of developing efficient, reproducible technologies for the establishment of ES cell lines of economically important species like the pig, cow, goat, sheep and horse.

PROTEOMICS, 2009

Embryo loss during peri-implantation can approach 20% in swine following artificial insemination ... more Embryo loss during peri-implantation can approach 20% in swine following artificial insemination or natural mating and coincides with rapid conceptus elongation. The objective of the present study was to establish a comprehensive profile of the abundant proteins of the pig conceptus at the time prior to implantation and identify stage-specific changes during elongation. The abundant proteins of a homogenous population of gestational day-11 ovoid (0.7-1 cm) and gestational day-12 filamentous (15-20 cm) porcine concepti were compared by extracting proteins from three independent conceptus pools and separating the proteins by 2-DE. Proteins in 305 spots were analyzed by MALDI-TOF or additionally by LC-MS/MS and 275 were positively identified representing 174 distinct proteins. The proteins could be classified into the following functional categories: cell proliferation/differentiation, cytoskeleton, metabolism, and stress response. Based on spot density, 35 proteins associated with cell proliferation, differentiation, apoptosis, and embryo/maternal signaling, were found to be differentially expressed between ovoid and filamentous concepti. A comparison of the protein expression profile with transcriptomic data from pig concepti of the same developmental stages identified similarities and dissimilarities between protein and mRNA expression profiles. This proteomic study helps to elucidate the biological mechanisms underlying the early embryonic development of the pig.

Nature Biotechnology, 2005

Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of... more Mastitis, the most consequential disease in dairy cattle, costs the US dairy industry billions of dollars annually. To test the feasibility of protecting animals through genetic engineering, transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 mg/ml in their milk were produced. In vitro assays demonstrated the milk's ability to kill Staphylococcus aureus. Intramammary infusions of S. aureus were administered to three transgenic and ten nontransgenic cows. Increases in milk somatic cells, elevated body temperatures and induced acute phase proteins, each indicative of infection, were observed in all of the nontransgenic cows but in none of the transgenic animals. Protection against S. aureus mastitis appears to be achievable with as little as 3 mg/ml of lysostaphin in milk. Our results indicate that genetic engineering can provide a viable tool for enhancing resistance to disease and improve the well-being of livestock.

Nature Biotechnology, 2005

Molecular Reproduction and Development, 2008

Limited understanding of the importance of known pluripotency factors in pig embryonic stem cells... more Limited understanding of the importance of known pluripotency factors in pig embryonic stem cells (ESC) impedes the establishment and validation of porcine ESC lines. This study evaluated the expression of known mouse ESC and human ESC (hESC) pluripotency markers in in vivo inner cell mass (ICM) and in vitro-cultured undifferentiated porcine epiblast cells isolated from 8-day porcine blastocysts, primary cultures of epiblast-derived neuroprogenitor cells, and endoderm cells. The expression profile of common pluripotency markers (POU domain 5 transcript factor 1, SRY-box containing gene 2, and Nanog homeobox), species-specific markers, ESC-associated factors, and differentiation markers was evaluated. The mRNA of uncultured ICMs, cultured epiblast cells, epiblast-derived neuroprogenitor cells, and endoderm cells was amplified prior to expression analysis of candidate genes by real-time RT-PCR. ESC factors whose expression correlated best with the undifferentiated epiblast state were identified by comparative mRNA expression analysis between porcine epiblast-derived somatic cell lines, fetal fibroblasts, and adult tissues. Across tissue types Nanog homeobox exhibited ubiquitous expression, whereas POU domain 5 transcript factor 1, teratocarcinoma-derived growth factor 1, and RNA exonuclease homolog 1 transcript expression was restricted primarily to undifferentiated epiblasts. Our results suggested that expression of pluripotency markers in undifferentiated pig epiblast cells more closely resembled that observed in hESC. Expression alterations of ESC-associated factors in epiblast cells were also observed during in vitro culture. Our data demonstrate the potential use of some pluripotency factors as markers of porcine epiblast stem cells and indicate that the in vitro environment may influence the cultured epiblast&amp;amp;amp;amp;amp;amp;#39;s developmental state.

Molecular Reproduction and Development, 1993

To define better the characteristics of pig and sheep epiblast cells in culture, the cells were t... more To define better the characteristics of pig and sheep epiblast cells in culture, the cells were tested for the presence of alkaline phosphatase (AP), a biochemical marker characteristic of mouse embryonic stem cells. Pig and sheep epiblast cells were positive for AP staining both at isolation from the blastocyst and after primary in vitro culture. The innermost portion of the attendant endoderm surrounding the epiblast was also positive for AP staining during primary culture. AP staining was lost upon differentiation or senescence of the epiblast cells. Also, all differentiated epiblast-derived cell cultures were negative for AP staining, with the exception of neuron-like cultures. Epiblast-like cells were cultured from day 10 (pig) and day 13 (sheep) embryonic discs, and these cells were also AP positive until they differentiated. Trophectoderm-endoderm-like cells from embryonic discs were AP negative or weakly positive. AP is a convenient marker for undifferentiated pig and sheep epiblast cells in culture when used in conjunction with cell morphology analysis.

Molecular Reproduction and Development, 2008

The expression of interferon-tau (IFN-t) is essential for bovine embryo survival in the uterus. A... more The expression of interferon-tau (IFN-t) is essential for bovine embryo survival in the uterus. An evaluation of IFN-t production from somatic cell nuclear transfer (NT)-embryo-derived primary trophectoderm cultures in comparison to trophectoderm cultured from parthenogenote (P) and in vitro matured, fertilized, and cultured (IVP) bovine embryos was performed. In Experiment 1, the success/ failure ratio for primary trophectoderm colony formation was similar for IVP and NT blastocysts [IVP ¼ 155/ 29 (84%); NT 104/25 (81%)], but was decreased (P ¼ .05) for P blastocysts [54/43 (56%)]. Most trophectoderm colonies reached diameters of at least 1 cm within 3-4 weeks, and at this time, 72 hr conditioned cell culture medium was measured for IFN-t concentration by antiviral activity assay. The amount of IFN-t produced by IVP-outgrowths [4311 IU/mL (n ¼ 155)] was greater (P < .05) than that from NT-[626 IU/mL (n ¼ 104)] and P -[1595 IU/ mL (n ¼ 54)] derived trophectoderm. Differential expression of IFN-t was confirmed by immunoblotting. In Experiment 2, colony formation was again similar for IVP and NT blastocysts [IVP ¼ 70/5 (93%); NT 67/ 1 (99%)] and less (P < .05) for P blastocysts [65/ 27 (70%)]. Analysis of trophectoderm colony size after 23 days in culture showed a similar relationship with P-derived colonies being significantly smaller in comparison to IVP and NT colonies. A differential expression of IFN-t was also observed again, but this time as measured over time in culture. Maximal IFN-t production was found at day-14 of primary culture and diminished to a minimum by the 23rd day. Mol.

Molecular Reproduction and Development, 2004

A bovine trophectoderm cell line was established from a parthenogenetic in vitroproduced blastocy... more A bovine trophectoderm cell line was established from a parthenogenetic in vitroproduced blastocyst. To initiate the cell line, 8-day parthenogenetic blastocysts were attached to a feeder layer of STO fibroblasts and primary outgrowths occurred that consisted of trophectoderm, endoderm, and very occasionally epiblast tissue. Any endoderm and epiblast outgrowths were removed from the primary cultures within the first 10 days of culture by dissection. One of the primary trophectoderm cell cultures was chosen for further propagation and was passaged by physical dissociation and replating on STO feeder cells. The cell culture, designated BPT-1, was maintained in T25 flasks and passaged at a 1:3 split ratio for the first 15 passages approximately once every 2 weeks. Thereafter, the cell culture was passaged at 1:10-1:40 split ratios. Transmission electron microscopic examination showed the cells to be a polarized epithelium with apical microvilli, a thin basal lamina, and lateral junctions consisting of tight junctions and desmosomes. Lipid vacuoles and digestive vacuoles were also prominent features of the BPT-1 cells. Metaphase spread analysis at passage 59 indicated a near diploid cell population (2n ¼ 60) with a mode and median of 60 and a mean of 64. BPT-1 cells secreted interferon-tau into the medium as measured by anti-viral assay and Western blot analysis. The cell line provides an in vitro model of parthenogenote trophectoderm whose biological characteristics can be compared to trophectoderm cell lines derived from bovine embryos produced by normal fertilization or nuclear transfer.

Journal of General Virology, 2013

Hepatitis E virus (HEV) causes acute, enterically transmitted hepatitis in human. It is associate... more Hepatitis E virus (HEV) causes acute, enterically transmitted hepatitis in human. It is associated with large epidemics in tropical and subtropical regions where it is endemic or with sporadic cases in non-endemic regions. Unlike other hepatitis viruses, HEV has several animal reservoirs. Phylogenetic studies on HEV human and animal sequences, and the identification of cases of direct transmission from animal to human strongly suggest that HEV is a zoonotic agent. The lack of efficient cell culture models limits studies on molecular and cellular aspects of HEV infection and species barrier crossing. The present study reports on the development of two new in vitro models of HEV replication using a human hepatoma-derived cell line, HepaRG, and a porcine embryonic stem cell-derived cell line, PICM-19. These two cell lines have morphological and functional properties similar to primary hepatocytes. These in vitro culture systems support HEV replication and release of encapsidated RNA. These new models represent a powerful tool for studying the viral replication cycle, species barrier crossing and virulence factors.

In Vitro Cellular & Developmental Biology - Animal, 1993

Pig epiblast cells that had been separated from other early embryonic cells were cultured in vitr... more Pig epiblast cells that had been separated from other early embryonic cells were cultured in vitro. A three-step dissection protocol was used to isolate the epiblast from trophectoderm and primitive endoderm before culturing. Blastocysts collected at 7 to 8 days postestrus were immunodissected to obtain the inner cell mass (ICM) and destroy tropbeetodermal cells. The ICM was cultured for 2 to 3 days on STO feeder cells. The epiblast was then physically dissected free of associated primitive endoderm. Epiblast-derived cells, grown on STO feeders, produced colonies of small cells resembling mouse embryonic stem cells. This primary cell morphology changed as the colonies grew and evolved into three distinct colony types (endodermlike, neural rosette, or complex). Cell cultures derived from these three colony types spontaneously differentiated into numerous specialized cell types in STO co-culture. These included fibroblasts, endodermlike cells, neuronlike cells, pigmented cells, adipogenic cells, contracting muscle cells, dome-forming epithelium, ciliated epithelium, tubule-forming epithelium, and a round amoeboid cell type resembling a plasmacyte after Wright staining. The neuronlike cells, contracting muscle cells, and tubule-forming epithelium had normal karyotypes and displayed finite or undefined life spans upon long-term STO co-culture. The dome-forming epithelium had an indefinite life span in STO co-culture and also retained a normal karyotype. These results demonstrate the in vitro pluripotency of pig epiblast cells and indicate the epiblast can be a source for deriving various specialized cell cultures or cell lines.