Neil C. Talbot

Genetically enhanced cows resist intramammary Staphylococcus aureus infection

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 milks 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.

Bovine Blastocyst-Derived Trophectoderm and Endoderm Cell Cultures: Interferon Tau and Transferrin Expression as Respective In Vitro Markers

Abstract Continuous cultures of bovine trophectoderm (CT-1 and CT-5) and bovine endoderm (CE-1 and CE-2) were initiated and maintained on STO feeder cells. CT-1 and CT-5 were derived from the culture of intact, 10- to 11-day in vitro-produced blastocysts. CE-1 and CE-2 were derived from the culture of immunodissected inner cell masses of 7- to 8-day in vitro-produced blastocysts. The cultures were routinely passaged by physical dissociation. Although morphologically distinct, the trophectoderm and endoderm both grew as cell sheets of polarized epithelium (dome formations) composed of approximately cuboidal cells. Both cell types, particularly the endoderm, grew on top of the feeder cells for the most part. Trophectoderm cultures grew faster, relative to endoderm, in large, rapidly extending colonies of initially flat cells with little or no visible lipid. The endoderm, in contrast, grew more slowly as tightly knit colonies with numerous lipid vacuoles in the cells at the colony centers. Ultrastructure analysis revealed that both cell types were connected by desmosomes and tight junctional areas, although these were more extensive in the trophectoderm. Endoderm was particularly rich in rough endoplasmic reticulum and Golgi apparatus indicative of cells engaged in high protein production and secretion. Interferon tau expression was specific to trophectoderm cultures, as demonstrated by reverse transcription-polymerase chain reaction, Western blot, and antiviral activity; and this property may act as a marker for this cell type. Serum protein production specific to endoderm cultures was demonstrated by Western blot; this attribute may be a useful marker for this cell type. This simple coculture method for the in vitro propagation of bovine trophectoderm and endoderm provides a system for assessing their biology in vitro.

CULTURING THE EPIBLAST CELLS OF THE PIG BLASTOCYST

SummaryPig 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 trophectodermal 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.

Cell Donor Influences Success of Producing Cattle by Somatic Cell Nuclear Transfer

Abstract To assess sources of variation in nuclear transfer efficiency, bovine fetal fibroblasts (BFF), harvested from six Jersey fetuses, were cultured under various conditions. After transfection, frozen-thawed lung or muscle BFF donor cells were initially cultured in DMEM in 5% CO2 and air and some were transferred to MEM, with 5% or 20% O2 or 0.5% or 10% serum and G418 for 2–3 wk. Selected clonal transfected fibroblasts were fused to enucleated oocytes. Fused couplets (n = 4007), activated with ionomycin and 6-dimethylaminopurine, yielded 927 blastocysts, and 650 were transferred to 330 recipients. Fusion rate was influenced by oxygen tension in a fetus-dependent manner (P < 0.001). Blastocyst development was influenced in a number of ways. Hip fibroblast generated more blastocysts when cultured in MEM (P < 0.001). The influence of serum concentration was fetus dependent (P < 0.001) and exposing fibroblast to low oxygen was detrimental to blastocyst development (P < 0.001). Cells from two of the six fetuses produced embryos that maintained pregnancies to term, resulting in eight viable calves. Pregnancy rates 56 days after transfer for the two productive donor fetuses, was at least double that of other recipients and may provide a fitness indicator of BFF cell sources for nuclear transfer. We conclude that a significant component in determining somatic cell nuclear transfer success is the source of the nuclear donor cells.

The Pursuit of ES Cell Lines of Domesticated Ungulates

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.

Identification of pig primordial germ cells by immunocytochemistry and lectin binding

Monoclonal antibodies anti‐SSEA‐1 and EMA‐1, and the lectins DBA and LTA, bound to the surface of large, round cells randomly distributed in the 26‐day pig genital ridge. Other antibodies, SSEA‐3, SSEA‐4, TRA‐1‐60, and TRA‐1‐81, did not react with any cells in the pig genital ridge. SSEA‐1‐positive cells displayed pseudopods and appeared to migrate from the dorsal mesentery of the hindgut (18‐day) to the primordium of the gonad (day 23) and entered the genital ridge by 26 days. The number of SSEA‐1‐positive cells associated with the dorsal mesentery and genital ridge markedly increased from the 18‐day to the 26‐day pig embryo. It was concluded that the SSEA‐1‐positive cells were primordial germ cells (PGCs). Using these markers and alkaline phosphatase histochemistry, pig PGCs derived from the 26‐day genital ridge showed no proliferation when grown in STO co‐culture in the presence of human LIF, bFGF and SCF. Mol. Reprod. Dev. 46:567–580, 1997. Published 1997 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

New models of hepatitis E virus replication in human and porcine hepatocyte cell lines.

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.

Spontaneous differentiation of porcine and bovine embryonic stem cells (Epiblast) into astrocytes or neurons

SummaryThe culture of porcine or bovine epiblasts, i.e., embryonic stem cells, on STO feeder cells resulted in their spontaneous differentiation into multiple cell types that were subsequently isolated as separate cell lines. Some of these cell lines were “neuron-like” in morphology. Immunofluorescent analysis of two porcine epiblast-derived cell lines demonstrated that the cells were positive for the expression of vimentin and the glial fibrillary acidic protein (GFAP). Because of their stellate morphology and lack of neurofilament expression, it is possible that the cells are type 2 astrocytes. Similar analysis of a bovine epiblast-derived cell line showed that the cells were positive for vimentin but that they did not express GFAP. However, a few cells within the population expressed neurofilaments and alpha-internexin. It is possible that the bovine cells are neural precursor cells. The results confirm and extend the demonstrated in vitro pluripotency of porcine and bovine epiblast cultures and provide evidence for an in vitro model of embryonic neuroectoderm development.

COLONY ISOLATION AND SECONDARY CULTURE OF FETAL PORCINE HEPATOCYTES ON STO FEEDER CELLS

SummaryThe secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.

Ultrastructure of the embryonic stem cells of the 8-day pig blastocyst before and after in vitro manipulation: Development of junctional apparatus and the lethal effects of PBS mediated cell–cell dissociation

Ultrastructural examination of 8‐day hatched pig blastocysts (large and small), their cultured inner cell mass (ICM), and cultured epiblast tissue (embryonic stem cells) was undertaken to assess the development of epiblast cell junctions and cytoskeletal elements. In small blastocysts, epiblast cells had no desmosomes or tight junction (TJ) connections and few organized microfilament bundles, whereas in large blastocysts the epiblast cells were connected by TJ and desmosomes with associated microfilaments. ICM isolation by immunodissection damaged the endoderm cells beneath the trophectoderm cells but did not appear to damage the epiblast cells or their associated endoderm cells. Epiblast cells in cultured ICMs were similar in character to those in the intact large blastocyst except that perinuclear microfilaments were observed. Isolated pig epiblasts, cultured for ∼36 hr on STO feeder layers, formed a monolayer whose cells were connected by TJ, adherens junctions and desmosomes with prominent microfilament bundles running parallel to the apical cytoplasmic membranes. Perinuclear microfilaments were a consistent feature in the ∼36 hr cultured epiblast cells. A feature characteristic of differentiation into notochordal cells, i.e., a solitary cilium, was also observed in the cultured epiblast. Exposure of the cultured epiblast cells to Ca++‐Mg++‐free phosphate buffered saline (PBS) for 5–10 min resulted in extensive cell blebbing and lysis. The results may indicate that pig epiblast cells could be more easily dissociated from early blastocysts (∼400 μm in diameter) if immunodissection damage to the ICM can be avoided. It may be difficult, however, to establish them as embryonic stem cell lines because the cultured pig epiblast cells were easily lysed by standard cell–cell dissociation methods. Anat Rec 264:101–113, 2001.