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.     

Cell allocation to the inner cell mass and the trophectoderm in bovine embryos cultured in two different media

Data from other laboratories have shown that speed of bovine blastocyst development is higher when Ménézo B2 is used for coculture compared to TCM199. It was our purpose to investigate whether this early blastocyst formation was also indicative of embryo quality by studying the allocation of inner cells in embryos generated by B2-coculture and by TCM199-coculture. For this purpose, a differential staining technique was used. General embryo development was similar for TCM199- and B2-embryos expressed as rate of cleavage at day 3 and morula-blastocyst formation at day 8 (P > 0.05), but significantly different when expressed as number of eight-cell stages at day 3 and expanded or hatched blastocysts at day 8 (P < 0.01). B2-embryos cultured until day 5, 6, and 7 post insemination, had total cell numbers of 24, 65, and 109 respectively, which was significantly higher than the cell number of TCM199 embryos cultured over the same time period (18, 41, and 71 respectively, P < 0.001). Morphological differentiation was significantly more advanced for B2-embryos at day 7 and 8 (P < 0.0001 and P < 0.001, respectively). First presumptive inner cells appeared in eight- to 16-cell stages at day 3. Because the determination of inner cells by differential staining is depending upon the presence of functional tight junctions, we concluded that the establishment of the tight junction seal in B2-embryos differed from that in TCM199-embryos: Inner cells appeared 0.56 cell cycle later in B2-embryos (P < 0.001) and a larger variation existed in the number of ICM-cells in B2-blastocysts (P < 0.001). The higher total cell number of B2-expanded blastocysts was mainly acquired by trophectoderm growth (P < 0.06). These data indicate that the apparent better quality of B2-embryos (faster cleavage, earlier blastocyst formation) is not reflected in a reliable number of inner cells of B2-blastocysts. © 1996 Wiley-Liss, Inc.     

Global gene expression analysis of bovine blastocysts produced by multiple methods

Reproductive efficiency using somatic cell nuclear transfer (SCNT) technology remains suboptimal. Of the various efforts to improve the efficiency, chromatin transfer (CT) and clone-clone aggregation (NTagg) have been reported to produce live cloned animals. To better understand the molecular mechanisms of somatic cell reprogramming during SCNT and assess the various SCNT methods on the molecular level, we performed gene expression analysis on bovine blastocysts produced via standard nuclear transfer (NT), CT, NTagg, in vitro fertilization (IVF), and artificial insemination (AI), as well as on somatic donor cells, using bovine genome arrays. The expression profiles of SCNT (NT, CT, NTagg) embryos were compared with IVF and AI embryos as well as donor cells. NT and CT embryos have indistinguishable gene expression patterns. In comparison to IVF or AI embryos, the number of differentially expressed genes in NTagg embryos is significantly higher than in NT and CT embryos. Genes that were differentially expressed between all the SCNT embryos and IVF or AI embryos are identified. Compared to AI embryos, more than half of the genes found deregulated between SCNT and AI embryos appear to be the result of in vitro culture alone. The results indicate that although SCNT methods have altered differentiated somatic nuclei gene expression to more closely resemble that of embryonic nuclei, combination of insufficient reprogramming and in vitro culture condition compromise the developmental potential of SCNT embryos. This is the first set of comprehensive data for analyzing the molecular impact of various nuclear transfer methods on bovine pre-implantation embryos.     

Isolation and characterization of epithelial cells from bovine pancreatic duct

Summary Epithelial cells derived from bovine pancreatic duct have been grown continuously in culture for 30 weeks (approximately 90 doublings of the cell population). The cells were grown in Eagle's minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 0.1 mM nonessential amino acids, and antibiotics. In confluent cultures, the cells are multilayered and form circular structures. When tested at various passages, the cells neither formed colonies in soft agar nor produced tumors after inoculation into athymic, nude mice. Hydrocortisone (1 and 5 μg per ml) and insulin (1,5 and 10 μg per ml) had no effect on the growth of the cells. β-Retinyl acetate inhibited growth rate and cell yield at a concentration of 5 μg per ml but was not growth-inhibitory at lower concentrations. By electron microscopy the cells have numerous mitochondria, Golgi and microvilli. Mucous droplets were observed in a small proportion of the cells. Desmosome-like structures and occluding junctions were observed more frequently between cells that had been transferred as aggregates than between cells transferred as single cells. Cytochemical studies indicated that some cells produce PAS positive granules that were not removed after treatment of the cultures with diastase. Eleven cell clones were isolated from the mass culture. The growth rates of the clones are different as well as the period of time in which the clones can be propagated in vitro. This work was supported in part by Y01 CP 60204 and N01 CP 43237.     

A serum-free culture system for efficient in vitro production of bovine blastocysts with improved viability after freezing and thawing

The aim of this study was to evaluate whether two completely serum-free media (IVMD101 and IVD101) could improve the yield and quality of bovine blastocysts from in vitro matured and fertilized oocytes. The media were evaluated in the presence (IVMD101) or absence (IVD101) of bovine cumulus/granulosa cell (BCGC) cocultures. The proportion of embryos developing to the blastocyst stage in IVMD101 medium with BCGC cocultures (36.5%) and IVD101 medium without BCGC cocultures (37.1%) was significantly higher than in serum-supplemented medium (TCM199 + 5% calf serum) with BCGC cocultures (25.1%). Furthermore, the mean cell numbers per blastocyst on Day 7 developed in IVMD101 medium (179.5 cells) and IVD101 medium (177.1 cells) were greater than in the serum-supplemented medium (145.7 cells). The survival rates of blastocysts derived in IVMD101 medium (73.3%) and IVD101 medium (60.0%) based on hatching after 72 h of post-thaw culture were superior to that of blastocysts derived in the serum-supplemented medium (48.1%). Under microscopic observation, bovine blastocysts derived in the serum-supplemented medium showed abundant lipid droplets, largely into the trophectoderm cells. This morphological difference may partly explain the sensitivity of serum-derived embryos after freezing and thawing. In conclusion, these new serum-free culture media are useful, not only to study the mechanisms of early embryogenesis, but also for mass production of good quality embryos for embryo transfer, cloning and transgenesis. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of oxidative stress and inhibitors of the pentose phosphate pathway on sexually dimorphic production of IFN-tau by bovine blastocysts

Bovine interferon-tau (IFN-tau), the anti-luteolytic factor secreted by conceptuses of pecoran ruminants, is a product of autosomal genes, yet in vitro produced (IVP) female expanded blastocysts (EB) secrete about twice as much IFN-tau as males. Two possible explanations have been tested here. One is that embryos of one sex are differentially susceptible to oxidative stress. The second is that female EB produce more IFN-tau because pentose-phosphate pathway (PPP) activity is elevated as a result of delayed X-chromosome inactivation. IVP bovine zygotes were cultured to the 8-cell stage and placed under conditions designed either to promote oxidative stress (+/-H2O2; 20 vs. 5% O2), or to inhibit glucose 6-phosphate dehydrogenase (G6PDH) activity (addition of dehydroepiandrosterone, DHEA or 6-aminonicotinamide, 6-AN to the medium). At day 8, blastocysts were cultured individually for a further 48 hr to assess IFN-tau production, and embryo sex determined retrospectively. Blastocyst numbers were reduced (P < 0.05) and their continued development impaired (P < 0.05) in presence of H2O2 (200 microM) and 20% O2, but neither IFN-tau production nor sexually dimorphic expression of IFN-tau were affected. IFN-tau production was reduced, particularly in females (P < 0.05), and sexual dimorphic differences in production were lost in the presence of both DHEA (100 microM) and 6-AN (1 microM). In the case of 6-AN, these effects were achieved without a significant decline in blastocyst developmental progression, quality, or cell number. The data suggest that the higher production of IFN-tau by female EB is an indirect outcome of the increased activity of the oxidative arm of the PPP pathway.     

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

Summary A cell line, BPE-1, was derived from a parthegogenetic 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 growth 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. Disclaimer: Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Isolation and characterization of embryonic stem-like cells derived from in vivo-produced cat blastocysts

Embryonic stem (ES)-like cells were isolated from in vivo-produced cat embryos. Total of 101 blastocysts were collected from female cats. The inner cell mass (ICM) were mechanically isolated and cultured on mitomycin-C-treated cat embryonic fibroblast feeder layers in medium supplemented with knockouttrade mark Serum Replacement (KSR-medium) or fetal bovine serum (FBS-medium). Putative ES-like cell colonies developed in both KSR- and FBS-medium conditions, but formed domed and flat colonies, respectively. ICM cell attachment and ES-like cell colony formation were significantly higher in KSR-medium, but subsequent cell proliferation was significantly lower than in FBS-medium. For passaging, 32 and 18 colonies in KSR- and FBS-medium were separated by enzymatic dissociation or mechanical disaggregation. Enzymatic dissociation resulted in cell differentiation; however, mechanical disaggregation generated cells that remained undifferentiated over more than four passages and yielded two cat ES-like cell lines that continued to grow for up to eight passages in FBS-medium. These cells had typical stem cell morphology, expressed high levels of alkaline phosphatase activity, and were positive for the ES cell-markers Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-3, and SSEA-4. These cells formed embryoid bodies (EBs) in suspension culture after extended suspension culture. When simple EBs were cultured on tissue culture plates, they differentiated into several cell types, including epithelium-like and neuron-like cells. In addition, EBs were positive for mesoderm marker, desmin. After prolonged in vitro culture, some colonies spontaneously differentiated into beating myocardiocytes, and were positive for alpha-actinin. These observations indicate that cat ES-like cells were successfully isolated and characterized from in vivo-produced blastocysts.     

Morphological development,cell number,and allocation of cells to trophectoderm and inner cell mass of in vitro fertilized and parthenogenetically developed buffalo embryos: The effect of IGF-I

The morphology and number of cells in the trophectoderm (TE) and inner cell mass (ICM) of buffalo blastocysts derived from in vitro fertilization and cultured in the presence or absence of insulin-like growth factor-I (IGF-I) were analyzed by differential fluorochrome staining technique. The total cell number (TCN), TE number, and ICM cell number were significantly higher in blastocysts developed in vitro in the presence of IGF-I as compared to blastocysts developed without IGF-I (P < 0.01). It was observed that the buffalo blastocyst took 5–9 days postfertilization to develop in vitro. In order to correlate the time required for blastocyst development and the allocation of cells to TE and ICM, blastocysts were designated as fast (developing on or before day 7) or slow (developing after day 7). The TCN, TE, and ICM cells of fast-developing blastocysts cultured in the presence of IGF-I were significantly higher than slow-developing blastocysts (P < 0.01). The blastocysts developed on day 6 had a mean total cell number 118.6 ± 21.4, which significantly decreased to 85.6 ± 17.4, 62.0 ± 14.5, and 17.0 ± 4.0 on days 7, 8, and 9, respectively (P < 0.05). Normal development of buffalo embryo showed that, on average, embryos reached compact morula stage at the earliest between days 4.5–5.5. Blastocysts developed, at the earliest, between days 5.0–6.0, and it took them, on average, 6.5 days to hatch from the zona pellucida. TCN, TE, and ICM increased three times from morula to blastocyst; however, the proportion of ICM to TCN remained the same, in both embryonic stages. TE approximately doubled in hatched blastocysts, as compared to unhatched blastocysts (P < 0.05). However, ICM cells were decreased. The time required for development of parthenogenetic blastocysts was observed to be greater as compared to in vitro fertilized (IVF) blastocysts. The total cell number of parthenogenetic blastocysts was 100.8 ± 11.3, including 59.2 ± 8.4 cells of TE and 42.1 ± 6.9 cells of ICM. © 1996 Wiley-Liss, Inc.     

Antigenic and ultrastructural markers associated with urothelial cytodifferentiation in primary explant outgrowths of mouse bladder.

The purpose of this study was to establish an in vitro culture model that closely resembles whole mouse urothelial tissue. Primary explant cultures of mouse bladder were established on porous membrane supports and explant outgrowths were analysed for morphology and the presence of antigenic and ultrastructural markers associated with urothelial cytodifferentiation. When examined at the ultrastructural level, the cultured urothelium was polarized and organized as a multilayered epithelium. Differentiation was found to increase from the porous membrane towards the surface and from the explant towards the periphery of the culture. Scanning and transmission electron microscopical analysis of the most superficially-located cells revealed four successive differentiation stages: cells with microvilli, cells with ropy microridges, cells with rounded microridges, and highly-differentiated cells with asymmetric unit membrane (AUM) plaques forming rigid microridges and fusiform vesicles. The more highly-differentiated cells were numerous at the periphery of the culture, but rare close to the explant. Epithelial organization was stabilized by well developed cell junctions. Immunolabeling demonstrated that superficial urothelial cells in culture: (1) develop tight junctions, E-cadherin adherens junctions and abundant desmosomes and (2) express uroplakins and cytokeratin 20 (CK 20). Using a culture model of primary explant outgrowth we have shown that non-differentiated mouse urothelial cells growing on a porous membrane show a high level of de novo differentiation.     

Isolation, culture, and characterization of embryonic cell lines from vitrified sheep blastocysts

This study was conducted to isolate, to culture, and to characterize embryonic cell lines from in vitro produced vitrified sheep blastocysts. Embryos were produced and vitrified at the expanded blastocyst stage. Ten inner cell masses arising from day 6-7 blastocysts were isolated by immunosurgery, disaggregated, and cultured onto mitomocin-C-inactivated mouse STO fibroblasts (MIF). After 5 or 6 days of culture the primary cell colonies were disaggregated, seeded in a new MIF, and cultured for 3 or 4 days to form new colonies called Passage 1. These cells were then disaggregated and cultured for other two passages. The primary cell colonies and Passage 2 colonies expressed stage specific embryonic markers SSEA-1, SSEA-3, and SSEA-4, and were alkaline phosphatase positive. In the absence of feeder layer and human leukemia inhibitory factor (LIF), these cells differentiated into variety of cell types and formed embryoid bodies. When cultured for an extended period of time, embryoid bodies differentiated into derivatives of three embryonic germ (EG) layers. These were characterized by detection of specific markers for differentiation such early mesoderm (FE-C6), embryonic myosin (F1-652), neural precursor (FORSE-1), and endoderm (anti-cytokeratin 18). To our knowledge, this is the first time that embryonic cell lines from in vitro produced and vitrified ovine blastocysts have been isolated and examined for detection of SSEA markers, and embryoid bodies have been cultured and examined for specific cell surface markers for differentiation.