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.     

In vitro pluripotency of epiblasts derived from bovine blastocysts

Two experiments were conducted to compare the utility of in vitro- and in vivo-derived bovine blastocysts for the isolation of pluripotent epiblasts. In experiment 1, the inner cell masses (ICMs) of in vivo-collected blastocysts yielded a higher proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts (P = .0157). In experiment 2, ICMs of in vivo-collected blastocysts that hatched on day 8 yielded a greater proportion of epiblasts after culture on STO feeder cells than ICMs from in vitro-produced blastocysts that hatched on day 8. The difference was reversed but smaller for blastocysts that hatched on day 9 (Interaction, P = .0125). Epiblasts from blastocysts that hatched on day 8 regardless of their source generated more differentiated cell lines in extended culture than did blastocysts that hatched on day 9. Extended epiblast culture yielded cells identifiable as products of the three embryonic germ layers that included epithelial cells, fibroblasts, neuronal cells, hepatocyte-like cells, and macrophage-like cells. Alkaline phosphatase activity combined with cell morphology identified the bovine epiblast cells and distinguished them from trophectoderm and endoderm that frequently contaminated epiblast cell cultures. In vivo-derived blastocysts, especially from early-hatching blastocysts, were a superior source of pluripotent epiblasts. Epiblast cells in this study all differentiated or senesced indicating that standard conditions for mouse embryonic stem cell culture do not maintain bovine epiblast cells in an undifferentiated state. © 1995 wiley-Liss, Inc.

1 This artilce is a US Government work and, as such, is in the public domain in the United States of America.

     

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.     

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.     

Culture of blastomeres from in vitro-matured,fertilized, and cultured bovine embryos

A culture system was devised to study the differentiation of bovine blastomeres. Blastomeres (2–13 per well) from embryos produced by in vitro maturation, fertilization, and culture of oocytes obtained from slaughterhouse ovaries were cultured for 10 days in 24-well culture plates on feeder layers in blastomere culture medium (BCM: equal parts tissue culture medium 199 and low-glucose Dulbecco's modified Eagle's medium with 10% fetal bovine serum). Ovine embryonic fibroblasts and STO cells were superior to bovine and mouse embryonic fibroblasts as mitotically inactivated feeder cells. Over five studies in which four blastomeres from an embryo were added to each culture well, an average of one colony per well formed from the blastomeres. The colonies continued to grow throughout the culture period, and most colonies resembled trophectoderm in their cellular characteristics, although some cultures contained a mixture of trophectoderm and endoderm. When the number of blastomeres cultured in each well was varied from 2–8, the number of colonies formed was proportional to the number of blastomeres added. Blastomeres from day 5 and day 6 embryos produced fewer colonies than did those from day 4 embryos, perhaps as a result of differentiation and tighter blastomere adhesion resulting in damage during their separation. The absence of serum did not alter the number of colonies formed. A number of growth factors, including LIF, OM, PDGFα, and FGF4, had no effect on the number of colonies, the size of colonies, or their alkaline phosphatase staining score beyond that provided by the feeder layer or serum when present. Blastomeres did not form colonies in the absence of feeder layers. Mol. Reprod. Dev. 48:238–245, 1997. © 1997 Wiley-Liss, Inc.     

Culturing the epiblast cells of the pig blastocyst

Summary 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 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.     

Isolation and characterization of embryonic stem cell-like cells from in vitro-produced buffalo (Bubalus bubalis) embryos

This study was carried out to isolate and characterize buffalo embryonic stem (ES) cell-like cells from in vitro-produced embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 120 blastocysts whereas 28 morulae were used for the isolation of blastomeres mechanically. The ICM cells/ blastomeres were cultured on mitomycin-C-treated feeder layer. Primary cell colony formation was higher (P < 0.05) for hatched blastocysts (73.1%, 30/41) than that for early/expanded blastocysts (25.3%, 20/79). However, no primary cell colonies were formed when blastomeres obtained from morulae were cultured. Primary colonies were formed in 14.1% (12/85) of intact blastocyst culture, which was significantly lower (P < 0.05) than that of 41.6% for ICM culture. These colonies were separated by enzymatic or mechanical disaggregation. Using mechanical disaggregation method, the cells remained undifferentiated and two buffalo ES cell-like cell lines (bES1, bES2) continued to grow in culture up to eight passages. However, disassociation through enzymatic method resulted in differentiation. Undifferentiated cells exhibited stem cell morphological features, normal chromosomal morphology, and expressed specific markers such as alkaline phosphatase (AP) and Oct-4. Cells formed embryoid bodies (EBs) in suspension culture; extended culture of EBs resulted in formation of cystic EBs. Following prolonged in vitro culture, these cells differentiated into several types of cells including neuron-like and epithelium-like cells. Furthermore, the vitrified-thawed ES cell-like cells also exhibited typical stem cell characteristics. In conclusion, buffalo ES cell-like cells could be isolated from in vitro-produced blastocysts and maintained in vitro for prolonged periods of time.     

Isolation and initial culture of porcine inner cell masses derived from in vitro-produced blastocysts

The present study was conducted to isolate and culture inner cell mass (ICM) primarily derived from in vitro-produced blastocysts and to develop the culture conditions for the ICM cells. In Experiment 1, immunosurgically isolated ICMs of blastocysts derived from in vitro fertilization (IVF), somatic cell nuclear transfer (SCNT) or parthenogenetic activation (PA) were seeded onto STO cells. Primary colonies from each isolated ICM were formed with a ratio of 28.9, 30.0 and 4.9%, respectively. In Experiment 2, blastocysts collected from IVF were directly seeded onto a feeder layer with or without zona pellucida (ZP), or were subjected to ICM isolation by immunosurgery. Primary colonies were formed in 36.8% of isolated ICMs and 19.4% in intact blastocysts without ZP. In Experiment 3, ICMs from IVF blastocysts were seeded onto STO cells, mouse embryonic fibroblast (MEF) or porcine uterine epithelial cells (PUEC). On STO and MEF cells, 34.5 and 22.2% of primary colonies were formed, respectively. However, no primary colony was formed on the PUEC or in feeder-free condition. In Experiment 4, ICMs from IVF blastocysts were cultured in DMEM + Ham's F10 (D/H medium), DMEM + NCSU-23 (D/N medium) or DMEM alone. When D/H medium or D/N medium was used, 21.7 or 44.4% of primary colony were formed, respectively, while no primary colony was formed in DMEM alone. These cells showed alkaline phosphatase activity and could be maintained for up to five passages. In suspension culture, cells formed embryoid bodies. These results demonstrate that porcine ICM could be isolated and cultured primarily from in vitro-produced blastocysts with a suitable culture system.     

Optimization of a lipitoid-based plasmid DNA transfection protocol for bovine trophectoderm CT-1 cells

Embryo-derived cell lines are important in vitro models for investigating the molecular mechanisms directing embryonic tissue lineage segregation and maintenance. The bovine trophectoderm-derived CT-1 cell line has been widely used to identify regulatory mechanisms of interferon tau gene expression, and it possesses potential as a model for characterizing the gene regulatory network controlling trophoblast lineage differentiation and development. This functional potential, however, is severely limited as CT-1 cells are very recalcitrant to standard transfection methods. The focus of this study was to test the cationic lipitoid reagent as an effective transfection reagent for DNA plasmid delivery. Optimization of liptoid-based transfection of plasmid DNA resulted in 9% transfection efficiency averaged across entire CT-1 colonies, with many subregions of CT-1 colonies achieving transfection rates of 15%. These rates are a substantial improvement over near-zero efficiencies achieved using other standard transfection techniques. CT-1 cells were also successfully adapted to substrate-free culture for over 20 passages, eliminating the need to culture CT-1 colonies on feeder cells or matrix-coated cultureware. Together, these results increase the utility of the CT-1 cell line as an in vitro bovine trophoblast model and provide insight into overcoming DNA delivery difficulties in other cell lines not amenable to genetic manipulation.     

Ultrastructural and karyotypic examination of in vitro produced bovine embryos developed in the sheep uterus

This study examined whether development of bovine in vitro produced (IVP) blastocysts in the sheep uterus resulted in morphologically and karyotypically normal elongation stage bovine blastocysts. Seven day IVP bovine blastocysts, resulting from either in vitro maturation and fertilization, nuclear transfer (NT), or parthenogenic activation, were surgically transferred at the blastocyst stage into sheep uteri. Sheep were sacrificed after 7-9 days, and blastocysts were flushed from their uteri. One of each kind of IVP bovine blastocyst was recovered from sheep uteri for analysis by transmission electron microscopy, and nine NT blastocysts were used to establish cell cultures that were analysed for chromosome complement. TEM analysis of in vivo-derived elongation stage bovine and ovine blastocysts was done for comparative purposes. Most ultrastructural features of the 13-19 day blastocysts were similar to earlier stage blastocysts except that distinct alternative mitochondrial morphologies were found between epiblast and trophectoderm cells. Monociliated cells, presumably nodal cells, were observed in the bovine epiblast and hypoblast, and retrovirus-like particles were elaborated by cells in these same areas. Development in the sheep uterus of IVP bovine blastocysts resulted in the presence of crystalloid bodies in the trophectoderm cells, and apoptotic and necrotic cells were observed in the epiblast tissue. Thus, in vivo incubation in the sheep uterus allowed nearly normal development to the elongated blastocyst stage and may be useful for assessment of NT bovine blastocyst developmental competence. Cell cultures derived from the NT blastocysts had normal chromosome complements suggesting that activation by ionomycin and 6-dimethyl-aminopurine did not cause detrimental changes in ploidy in those blastocysts that developed.     

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.     

Conventional pluripotency markers are unspecific for bovine embryonic-derived cell-lines

Bovine embryonic stem cells are of potentially big value in transgenic research and studies of lineage commitment and development. Nevertheless, key aspects of the establishment of bovine embryonic stem cells such as the identification of specific pluripotency markers need to be clarified to achieve successful results. Bovine blastocysts were produced in vitro and cultured for 8 days up to the expanded or hatched stage. The trophectoderm, the inner cell mass and its embryonic stem cell-derived lines, all showed a common positive immunocytochemical staining for stage-specific embryonic antigen-4, tumour-rejection antigen gp96 and NANOG proteins. The antigenic profile obtained partially agrees with previous data from bovine and other species. Until a validated pluripotent bovine stem cell marker can be identified, it might be advisable to combine the use of epiblast and trophoblast-specific markers to rule out the presence of early committed trophectoderm cells in bovine embryonic stem cell cultures.     

Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos

Inner cell mass (ICM) and trophectoderm cell lineages in preimplantation mouse embryos were studied by means of iontophoretic injection of horseradish peroxidase (HRP) as a marker. HRP was injected into single blastomeres at the 2- and 8-cell stages and into single outer blastomeres at the 16-cell and late morula (about 22- to 32-cell) stages. After injection, embryos were either examined immediately for localization of HRP (controls) or they were allowed to develop until the blastocyst stage (1 to 3.5 days of culture) and examined for the distribution of labeled cells. In control embryos, HRP was confined to one or two outer blastomeres. In embryos allowed to develop into blastocysts, HRP-labeled progeny were distributed into patches of cells, showing that there is limited intermingling of cells during preimplantation development. A substantial fraction of injected blastomeres contributed descendants to both ICM and trophectoderm (95, 58, 44, and 35% for injected 2-cell, 8-cell, 16-cell, and late morula stages, respectively). Although more than half of the outer cells injected at 16-cell and late morula stages contributed descendants only to trophectoderm (53 and 63%, respectively), some outer cells contributed also to the ICM lineage even at the late morula stage. Although the mechanism for allocation of outer cells to the inner cell lineage is unknown, our observation of adjacent labeled mural trophectoderm and presumptive endoderm cells implicated polarized cell division. This observation also suggests that mural trophectoderm and presumptive endoderm are derived from common immediate progenitors. These cells appear to separate into inner and outer layers during the fifth cleavage division. Our results demonstrate the usefulness of HRP as a cell lineage marker in mouse embryos and show that the allocation of cells to ICM or trophectoderm begins after the 2-cell stage and continues into late cleavage.     

A method for cultivating morphologically undifferentiated embryonic stem cells from porcine blastocysts

Variable conditions were tested to determine an in-vitro cultivation method for the formation of morphologically undifferentiated embryonic stem cells from the inner cell mass (ICM) derived outgrowth of porcine blastocysts. Although all 16 Day-9 embryos failed to form colonies, 14 such colonies were obtained from a total of 69 Day-10 embryos when they were co-cultivated with porcine uterine fibroblast (PUF) cells over a 6-day period. The best results were obtained in Dulbecco's modified Eagle medium (DMEM) with 10% fetal calf serum and 10% porcine serum supplemented with bovine insulin and beta-mercaptoethanol, in which six out of seven embryos formed adequate ICM-derived colonies. Since murine fibroblasts were not found to be suitable feeder cells in this procedure, an endocrine synergistic interaction, which promotes embryonic attachment and colony formation, between porcine blastocysts and PUF cells is hypothesized. Continued propagation of the ICM-derived cells was not dependent on these factors; a total of seven cell lines were obtained after three to five subsequent passages on murine feeder-layers that resembled morphologically undifferentiated embryonic cells.     

Role of the extracellular matrix protein thrombospondin in the early development of the mouse embryo

The distribution of the extracellular matrix protein thrombospondin (TSP) in cleavage to egg cylinder staged mouse embryos and its role in trophoblast outgrowth from cultured blastocysts were examined. TSP was present within the cytoplasm of unfertilized eggs; in fertilized one- to four-cell embryos; by the eight-cell stage, TSP was also densely deposited at cell-cell borders. In the blastocyst, although TSP was present in all three cell types; trophectoderm, endoderm, and inner cell mass (ICM), it was enriched in the ICM and at the surface of trophectoderm cells. Hatched blastocysts grown on matrix-coated coverslips formed extensive trophoblast outgrowths on TSP, grew slightly less avidly on laminin, or on a 140-kD fragment of TSP containing its COOH terminus and putative cell binding domains. There was little outgrowth on the NH2 terminus heparin-binding domain. Addition of anti-TSP antibodies (but not GRGDS) to blastocysts growing on TSP strikingly inhibited outgrowth. Consistent with its early appearance and presence in trophoblast cells during implantation, TSP may play an important role in the early events involved in mammalian embryogenesis.     

用饲养层分离胚胎干细胞集落的实验初探

目的 用饲养层分离胚胎干细胞集落。方法 用胚龄为13~14 d的小鼠胚胎分离原代成纤维细胞,制成饲养层,用于囊胚的培养。结果 小鼠原代胚胎成纤维细胞（PMEF）贴壁能力较好,增殖快,易铺层。囊胚和内细胞团（ICM）在饲养层上贴壁生长良好,当培养4~5 d时,其增殖率为16/28（57%）。在ICM离散48 h后,各种胚胎干细胞（ES）集落开始出现。此种集落经碱性磷酸酶染色成阳性。结论 用饲养层分离胚胎干细胞获得初步成功。     

Differential expression of ezrin/radixin/moesin (ERM) and ERM-associated adhesion molecules in the blastocyst and uterus suggests their functions during implantation

Development of the blastocyst to implantation competency, differentiation of the uterus to the receptive state, and a cross talk between the implantation-competent blastocyst and the uterine luminal epithelium are all essential to the process of implantation. In the present investigation, we examined the possibility for a potential cross talk between the blastocyst and uterus involving the ezrin/radixin/moesin (ERM) proteins and ERM-associated cytoskeletal cross-linker proteins CD43, CD44, ICAM-1, and ICAM-2. In normal Day 4 blastocysts and after rendering dormant blastocysts to implantation-competent by estrogen in vivo (activated), the outer surface of mural trophectoderm cells showed much higher levels of radixin as compared to those in the polar trophectoderm cells, inner cell mass (ICM), and primitive endoderm. In contrast, ezrin was present on both the mural and the polar trophectoderm cell surfaces of normal Day 4 and activated blastocysts at higher intensity than dormant blastocysts. A distinct localization was noted in the primitive endoderm of dormant blastocysts that was not apparent in activated or normal Day 4 blastocysts. The expression of moesin was modestly higher at the mural trophectoderm of implantation-competent blastocysts, while the localization appeared to be present primarily on the polar trophectoderm cell surface of Day 4 blastocysts. The localization of ERM-associated adhesion molecules CD43, CD44, and ICAM-2 was more intense in the implantation-competent blastocysts compared with the dormant blastocysts. However, while CD44 was present both in the trophectoderm and in ICM, CD43 and ICAM-2 were localized primarily to the trophectoderm. The signal for ICAM-1 was very intense in the ICM but was modest in the trophectoderm. No significant changes in fluorescence intensity were noted between activated and dormant blastocysts. In the receptive uterus on Day 4 of pregnancy, ERM proteins were localized to the uterine epithelium, while on Day 5 the localization, especially of radixin and moesin, extended to the stroma surrounding the implantation chamber. With respect to ERM-associated adhesion molecules, while CD44 and ICAM-1 were exclusively localized in the stroma on Day 4, CD43 and ICAM-2 were localized to the epithelium. On Day 5, the localization of CD44 and ICAM-1 became highly concentrated in the antimesometrial stroma of the implantation chamber. The localization of CD43 and ICAM-2 remained mostly epithelial, although some stromal localization of CD43 was noted on Day 5. These results suggest that differential expression and distribution of ERM proteins and ERM-associated adhesion molecules are involved in the construction of the cellular architecture necessary for blastocyst activation and uterine receptivity leading to successful implantation.     

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.