Bovine trophectoderm cells induced from bovine fibroblasts with induced pluripotent stem cell reprogramming factors.

Thirteen independent induced bovine trophectroderm (iBT) cell lines were established by reprogramming bovine fetal liver-derived fibroblasts after viral-vector transduction with either six or eight factors, including POU5F1 (OCT4), KLF4, SOX2, MYC, NANOG, LIN28, SV40 large T antigen, and hTERT. Light- and electron-microscopy analysis showed that the iBT cells had epithelial cell morphology typical of bovine trophectoderm cells. Reverse-transcription-PCR assays indicated that all of the cell lines expressed interferon-tau (IFNT) at passages 1 or 2. At later passages (≥ passage 8), however, immunoblot and antiviral activity assays revealed that more than half of the iBT cell lines had stopped expressing IFNT. Messenger RNAs specific to trophectoderm differentiation and function were found in the iBT cell lines, and 2-dimensional-gel analysis for cellular proteins showed an expression pattern similar to that of trophectoderm cell lines derived from bovine blastocysts. Integration of some of the human reprogramming factors, including POU5F1, KLF4, SOX2, MYC, NANOG, and LIN28, were detected by PCR, but their transcription was mostly absent in the iBT cell lines. Gene expression assessment of endogenous bovine reprogramming factor orthologs revealed endogenous bLIN28 and bMYC transcripts in all; bSOX2 and bNANOG in none; and bKLF4 and bPOU5F1 in less than half of the iBT cell lines. These results demonstrate that bovine trophectoderm can be induced via reprogramming factor expression from bovine liver-derived fibroblasts, although other fibroblast populations-e.g., derived from fetal thigh tissue-may produce similar results, albeit at lower frequencies.

Triple-acting Lytic Enzyme Treatment of Drug-Resistant and Intracellular Staphylococcus aureus.

Multi-drug resistant bacteria are a persistent problem in modern health care, food safety and animal health. There is a need for new antimicrobials to replace over used conventional antibiotics. Here we describe engineered triple-acting staphylolytic peptidoglycan hydrolases wherein three unique antimicrobial activities from two parental proteins are combined into a single fusion protein. This effectively reduces the incidence of resistant strain development. The fusion protein reduced colonization by Staphylococcus aureus in a rat nasal colonization model, surpassing the efficacy of either parental protein. Modification of a triple-acting lytic construct with a protein transduction domain significantly enhanced both biofilm eradication and the ability to kill intracellular S. aureus as demonstrated in cultured mammary epithelial cells and in a mouse model of staphylococcal mastitis. Interestingly, the protein transduction domain was not necessary for reducing the intracellular pathogens in cultured osteoblasts or in two mouse models of osteomyelitis, highlighting the vagaries of exactly how protein transduction domains facilitate protein uptake. Bacterial cell wall degrading enzyme antimicrobials can be engineered to enhance their value as potent therapeutics.

Synergistic streptococcal phage λSA2 and B30 endolysins kill streptococci in cow milk and in a mouse model of mastitis.

Bovine mastitis results in billion dollar losses annually in the USA alone. Streptococci are among the most relevant causative agents of this disease. Conventional antibiotic therapy is often unsuccessful and contributes to development of antibiotic resistance. Bacteriophage endolysins represent a new class of antimicrobials against these bacteria. In this work, we characterized the endolysins (lysins) of the streptococcal phages λSA2 and B30 and evaluated their potential as anti-mastitis agents. When tested in vitro against live streptococci, both enzymes exhibited near-optimum lytic activities at ionic strengths, pH, and Ca(2+) concentrations consistent with cow milk. When tested in combination in a checkerboard assay, the lysins were found to exhibit strong synergy. The λSA2 lysin displayed high activity in milk against Streptococcus dysgalactiae (reduction of CFU/ml by 3.5 log units at 100 µg/ml), Streptococcus agalactiae (2 log), and Streptococcus uberis (4 log), whereas the B30 lysin was less effective. In a mouse model of bovine mastitis, both enzymes significantly reduced intramammary concentrations of all three streptococcal species (except for B30 vs. S. dysgalactiae), and the effects on mammary gland wet weights and TNFα concentrations were consistent with these findings. Unexpectedly, the synergistic effect determined for the two enzymes in vitro was not observed in the mouse model. Overall, our results illustrate the potential of endolysins for treatment of Streptococcus-induced bovine mastitis.

Chimeric phage lysins act synergistically with lysostaphin to kill mastitis-causing Staphylococcus aureus in murine mammary glands.

Staphylococci cause bovine mastitis, with Staphylococcus aureus being responsible for the majority of the mastitis-based losses to the dairy industry (up to $2 billion/annum). Treatment is primarily with antibiotics, which are often ineffective and potentially contribute to resistance development. Bacteriophage endolysins (peptidoglycan hydrolases) present a promising source of alternative antimicrobials. Here we evaluated two fusion proteins consisting of the streptococcal λSA2 endolysin endopeptidase domain fused to staphylococcal cell wall binding domains from either lysostaphin (λSA2-E-Lyso-SH3b) or the staphylococcal phage K endolysin, LysK (λSA2-E-LysK-SH3b). We demonstrate killing of 16 different S. aureus mastitis isolates, including penicillin-resistant strains, by both constructs. At 100 µg/ml in processed cow milk, λSA2-E-Lyso-SH3b and λSA2-E-LysK-SH3b reduced the S. aureus bacterial load by 3 and 1 log units within 3 h, respectively, compared to a buffer control. In contrast to λSA2-E-Lyso-SH3b, however, λSA2-E-LysK-SH3b permitted regrowth of the pathogen after 1 h. In a mouse model of mastitis, infusion of 25 µg of λSA2-E-Lyso-SH3b or λSA2-E-LysK-SH3b into mammary glands reduced S. aureus CFU by 0.63 or 0.81 log units, compared to >2 log for lysostaphin. Both chimeras were synergistic with lysostaphin against S. aureus in plate lysis checkerboard assays. When tested in combination in mice, λSA2-E-LysK-SH3b and lysostaphin (12.5 µg each/gland) caused a 3.36-log decrease in CFU. Furthermore, most protein treatments reduced gland wet weights and intramammary tumor necrosis factor alpha (TNF-α) concentrations, which serve as indicators of inflammation. Overall, our animal model results demonstrate the potential of fusion peptidoglycan hydrolases as antimicrobials for the treatment of S. aureus-induced mastitis.

Quantitative and semiquantitative immunoassay of growth factors and cytokines in the conditioned medium of STO and CF-1 mouse feeder cells.

Feeder cells of irradiated mouse fibroblasts are commonly used for, and are generally necessary for, the in vitro maintenance and growth of many fastidious cell types, particularly embryonic stem cells or induced pluripotent stem cells. Quantitative and semiquantitative immunoassays of conditioned media were performed to identify some of the soluble cytokines, chemokines, protein hormones, and cell matrix/adhesion molecules that are elaborated from two commonly used feeder cells, STO and CF-1. Among those quantitatively assayed, the most abundant cytokine proteins expressed by the feeder cells were activin A, hepatocyte growth factor (HGF), insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding protein (IGFBP)-6, macrophage colony-stimulating factor (a.k.a. CSF-1), and pigment epithelium-derived factor (a.k.a. serine protease inhibitor, clade F, member 1). CF-1 cells expressed ten times more activin A than STO cells and also produced larger amounts of interleukin-6 and IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5. Conversely, STO cell produced almost ten times more HGF and five times more stem cell factor (a.k.a. c-kit ligand) than CF-1 cells. Assayed semiquantitatively, relatively large amounts of chemokines were produced by both feeder cells including fractalkine (CX3CL1), interferon-inducible protein 10 (a.k.a. CXCL10 and cytokine-responsive gene-2, CRG-2), monocyte chemotactic protein (MCP)-1 (a.k.a. CCL2 and junctional epithelium chemokine (JE), MCP-5/CCL12), keratinocyte-derived chemokine (a.k.a. CXCL1 and growth-related oncogene alpha, GROα), nephroblastoma overexpressed gene (CCN3, IGFBP-9), stromal cell-derived factor 1 (CXCL12), and serpin E1 (PAI-1). In contrast to one another, STO produced more CXCL16 than CF-1 cells, and CF-1 cell produced more MCP-5 (CCL12), macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1ß (CCL4), pentraxin-3 (TSG-14), and platelet factor-4 (CXCL4) than STO cells. Soluble adhesion molecule, sICAM (ICAM-1, CD54), was expressed by CF-1 cells, but not STO cells, and similarly, the cell matrix-associated molecules endocan (endothelial cell-specific molecule 1), endostatin (collagen XVIII), and matrix metalloproteinase 3 were expressed more by CF-1 cells. Tissue inhibitor of metalloproteinases 1 was robustly expressed by both feeder cells. Other proteins primarily detected from CF-1 cells included retinol-binding protein 4 and FGF21, while STO cells secreted more interferon gamma. Both feeder cells produced no or low amounts of LIF, tumor necrosis factor alpha, vascular endothelial growth factor (VEGF), VEGF-B, prolactin, various interleukins, fibroblast growth factor (FGF)-1, FGF-2, FGF-7, EGF, HB-EGF, and amphiregulin. The results may explain some of the cell growth and maintenance responses by various types of cells co-cultured on STO or CF-1 feeder cells.

Proteomic analysis of the major cellular proteins of bovine trophectoderm cell lines derived from IVP, parthenogenetic and nuclear transfer embryos: Reduced expression of annexins I and II in nuclear transfer-derived cell lines.

Trophectoderm cell lines were established from 8-day in vitro-cultured embryos of cattle derived from fertilization (IVF), somatic cell nuclear transfer (NT), or parthenogenetic activation (P) of in vitro-matured oocytes and from five 8-day-old in vivo (V) embryos. The most abundant cellular proteins of 5 V-, 16 NT-, 12 P-, and 16 IVF-derived cell lines were compared by 2D-gel electrophoresis and mass spectrometry; that is, the unaltered thiourea/urea extract of each cell culture was analyzed. Common protein spots (n=118) were examined, and 95% were identified with significant scores from protein and gene database searches. Of the proteins detected and identified, actin and cytokeratin-8 were found to be the most abundant. Other prominent cellular proteins were metabolic enzymes such as aldose reductase, phosphoglycerate mutase, enolase, triosephosphate isomerase, cytoskeletal interacting proteins transgelin and stratifin, anti-oxidant proteins peroxiredoxin 1 and anti-oxidant protein 2, and the calcium-dependent lipid-binding proteins annexins I and II. In comparative analysis of the 2D-gels, the NT-derived trophectoderm had less annexins I and II in comparison to the IVF- and P-derived trophectoderm. Because annexins I and II are abundant in the placenta and have functions important to the maintenance of placentation, the down-regulation of the annexin genes in the cultured NT trophectoderm may be related to the frequent failures of NT pregnancies.

Comparison of the interferon-tau expression from primary trophectoderm outgrowths derived from IVP, NT, and parthenogenote bovine blastocysts.

The expression of interferon-tau (IFN-tau) is essential for bovine embryo survival in the uterus. An evaluation of IFN-tau 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 = 155/29 (84%); NT 104/25 (81%)], but was decreased (P = .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-tau concentration by antiviral activity assay. The amount of IFN-tau produced by IVP-outgrowths [4311 IU/mL (n = 155)] was greater (P < .05) than that from NT- [626 IU/mL (n = 104)] and P - [1595 IU/mL (n = 54)] derived trophectoderm. Differential expression of IFN-tau was confirmed by immunoblotting. In Experiment 2, colony formation was again similar for IVP and NT blastocysts [IVP = 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-tau was also observed again, but this time as measured over time in culture. Maximal IFN-tau production was found at day-14 of primary culture and diminished to a minimum by the 23rd day.

Establishment of a bovine blastocyst-derived cell line collection for the comparative analysis of embryos created in vivo and by in vitro fertilization, somatic cell nuclear transfer, or parthenogenetic activation.

Tools and methods for analyzing differences in embryos resulting from somatic cell nuclear transfer (NT) in comparison to those derived from normal fertilization are needed to define better the nature of the nuclear reprogramming that occurs after NT. To this end, a collection of bovine blastocyst-derived cell lines was created. In vitro expanded or hatched blastocysts, used as primary culture tissue, were from NT; in vitro maturation, fertilization, and culture (IVF); or parthenogenetic (P) activation. Also, five in vivo-fertilized and developed blastocysts were collected by uterine flushing on the eighth d postfertilization. Whole blastocysts were physically attached to STO feeder layers to initiate all of the cell lines generated. The majority of the cell lines in the collection are trophectoderm, 38 NT-derived, 6 in vivo-derived, 20 IVF-derived, and 13 P-derived. Trophectoderm identity was ascertained by morphology and, in many cases, interferon-tau production. Several visceral endoderm cell lines and putative parietal endoderm cell lines were also established. At approximately 5% efficiency, epiblast masses from NT and IVF blastocysts survived and were isolated in culture. Two epiblast masses were also isolated from P blastocysts. Spontaneous differentiation from the epiblast outgrowths resulted in the establishment of fibroblast cell lines. The use of the trophectoderm cell lines as a comparative in vitro model of bovine trophectoderm and placental function is discussed in relation to NT reprogramming.

Fibroblast growth factor-2 is expressed by the bovine uterus and stimulates interferon-tau production in bovine trophectoderm.

Uterine-derived factors are essential for conceptus development and secretion of the maternal recognition-of-pregnancy factor, interferon-tau (IFNT), in ruminant species. The objectives of this study were to determine whether fibroblast growth factor-2 (FGF-2) is expressed in the bovine uterus during early pregnancy in cattle and to determine whether FGF-2 supplementation affects IFNT mRNA and protein abundance in bovine trophectoderm. FGF-2 mRNA was present in endometrium throughout the estrous cycle and was localized to the luminal and glandular endometrial epithelium at d 17-18 after estrus in pregnant and nonpregnant cows. Immunoreactive FGF-2 protein was detected within the endometrium and in the uterine lumen at d 17-18 after estrus, and concentrations did not differ based on pregnancy status. In a bovine trophectoderm cell line, CT-1, supplementation of medium with at least 1 ng/ml FGF-2 increased the incorporation of [(3)H]thymidine into DNA. Similarly, IFNT secretion from CT-1 cells increased after FGF-2 supplementation (1-100 ng/ml) for 72 h. Abundance of IFNT mRNA in CT-1 cells increased after 24 h exposure to 1, 10, or 100 ng/ml FGF-2. In bovine blastocysts, FGF-2 supplementation did not affect cell number after 72 h of culture but did stimulate IFNT protein concentrations in conditioned medium. In summary, FGF-2 is present in the uterine lumen during early pregnancy and increases IFNT mRNA and protein abundance in trophectoderm. The magnitude by which FGF-2 stimulates IFNT expression suggests that this uterine-derived factor plays an active role in regulating the establishment and maintenance of pregnancy in ruminants.

Isolation and characterization of a bovine visceral endoderm cell line derived from a parthenogenetic blastocyst.

A cell line, BPE-1, was derived from a parthenogenetic 8-d in vitro-produced bovine blastocyst that produced a cell outgrowth on STO feeder cells. The BPE-1 cells resembled visceral endoderm previously cultured from blastocysts produced by in vitro fertilization (IVF). Analysis of the BPE-1 cells demonstrated that they produced serum proteins and were negative for interferon-tau production (a marker of trophectoderm). Transmission electron microscopy revealed that the cells were a polarized epithelium connected by complex junctions resembling tight junctions in conjunction with desmosomes. Rough endoplasmic reticulum was prominent within the cells as were lipid vacuoles. Immunocytochemistry indicated the BPE-1 cells had robust microtubule networks. These cells have been grown for over 2 yr for multiple passages at 1:10 or 1:20 split ratios on STO feeder cells. The BPE-1 cell line presumably arose from embryonic cells that became diploid soon after parthenogenetic activation and development of the early embryo. However, metaphase spreads prepared at passage 41 indicated that the cell population had a hypodiploid (2n = 60) unimodal chromosome content with a mode of 53 and a median and mean of 52. The cell line will be of interest for functional comparisons with bovine endoderm cell lines derived from IVF and nuclear transfer embryos.

Comparison of gene expression in individual preimplantation bovine embryos produced by in vitro fertilisation or somatic cell nuclear transfer.

In vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT) have been implicated in a variety of developmental abnormalities. Aberrant gene expression is likely to account for much of the diminished viability and developmental abnormalities observed. In the present study, the expression of multiple genes in IVF and SCNT bovine blastocyst-stage embryos were evaluated and compared with in vivo-produced embryos. Eleven genes expressed at and following maternal-zygotic transcription transition were evaluated in individual blastocysts by real-time polymerase chain reaction following RNA amplification. A subset of those genes was also evaluated in individual IVF and SCNT eight-cell embryos. A fibroblast-specific gene, expressed by nuclear donor cells, was also evaluated in IVF and SCNT embryos. The observed gene expression pattern at the eight-cell stage was not different between IVF and SCNT embryos (P > 0.05). In vitro fertilisation and SCNT blastocyst expression was lower (P < 0.01) for all genes compared with their in vivo-produced counterparts, except for lactate dehydrogenase isoenzyme A (P < 0.001). The patterns of gene expression of the IVF and SCNT blastocysts were indistinguishable. Neither SCNT eight-cell nor blastocyst-stage embryos expressed the gene used as a fibroblast marker (collagen VIalpha1). For the genes evaluated, the level of expression was influenced more by the environment than by the method used to produce the embryos. These results support the notion that if developmental differences observed in IVF- and SCNT-produced fetuses and neonates are the result of aberrant gene expression during the preimplantation stage, those differences in expression are subtle.

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 micrograms/ml [corrected] 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 micrograms/ml [corrected] 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.

Isolation and characterization of a bovine trophectoderm cell line derived from a parthenogenetic blastocyst.

A bovine trophectoderm cell line was established from a parthenogenetic in vitro-produced 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 = 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.

Comparison of colony-formation efficiency of bovine fetal fibroblast cell lines cultured with low oxygen, hydrocortisone, L-carnosine, bFGF, or different levels of FBS.

A comparison of colony-formation efficiency (CFE) was made between six independent bovine fetal fibroblast (BFF) cell lines used in somatic cell nuclear transfer. Variation in CFE was assessed under different culture conditions. The conditions examined were ambient atmosphere (approximately 20% oxygen) culture versus 5% oxygen culture, three levels of fetal bovine serum (FBS) in the medium (5%, 10% or 20%), and the amendment of 10% FBS medium with basic fibroblast growth factor (1 ng/mL), L-carnosine (20 mM), or hydrocortisone (1 microM). The six BFF cell lines showed significant differences from one another in CFE. No significant difference in CFE was found with reduced oxygen culture. L-Carnosine also had no significant effect on CFE. A FBS concentration of 10% was found to produce the best overall CFE. Hydrocortisone treatment reduced the size of colonies although the number of colonies formed was not affected. Basic FGF increased the size of colonies but the number of colonies formed was not affected. The results showed that different BFF cell lines varied significantly in their CFE. Also, some medium supplements or culture conditions that have shown positive CFE effects on the fibroblasts of other species failed to show significant positive CFE effects on the BFF cell lines tested.

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

The 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.