Studies on polydisperse systems using an air-driven ultracentrifuge: application to phosphatidylcholine vesicles.

A high-speed air-driven ultracentrifuge (Airfuge) has been used to determine the molecular weight and effective specific volume of phosphatidylcholine vesicles. The method used to determine the effective specific volume involved varying the solution density until zero sedimentation of the vesicles occurred. The value obtained for the effective specific volume of 0.9885 ml/g agrees well with previously reported values. The determination of the molecular weight of the vesicles is based on a method in which the fraction of vesicles remaining in an upper fraction of the solution column is compared with the values obtained using standard proteins. The values obtained for the molecular weight of the vesicles range from 1.7 X 10(6) to 2.3 X 10(6) and are in good agreement with results obtained using the analytical ultracentrifuge and with previously reported results. Possible effects due to the polydispersity of the solute are assessed using theoretical calculations and the possibility of using the Airfuge for the study of other polydisperse systems is discussed.     

A simple and effective method for the isolation of inner cell mass samples from human blastocysts for gene expression analysis

The isolation of pure inner cell mass (ICM) and trophectoderm (TE) cells from a single human blastocyst is necessary to obtain accurate gene expression patterns of these cells, which will aid in the understanding of the primary steps of embryo differentiation. However, previously developed pure ICM isolation methods are either time-consuming or alter the normal gene expression patterns of these cells. Here, we demonstrate a simple and effective method of ICM samples isolation from human blastocysts. In total, 35 human blastocysts of all stages with expanded and good morphology were incubated in calcium/magnesium-free HEPES medium for 5 min before micromanipulation. With the aid of a laser, a biopsy pipette was inserted directly into the blastocoel for the suction-based removal of ICM samples. The ICM samples were obtained through simple mechanical pulling force or laser assistance, and each isolation process required 3–4 min. The isolated ICM and TE fractions were subjected to single-cell real-time quantitative RT-PCR to evaluate keratin 18 (KRT18) expression. Finally, 33 paired ICM and TE samples were verified using gene expression analysis. KRT18 was readily detectable in all TE cells but absent in 30 ICM counterparts, indicating a pure ICM isolation rate of 90.9% (30/33). The relative KRT18 expression of three TE samples compared with their three contaminated ICM counterparts was 19-fold (P?<?0.001), indicating that the contamination was very weak. These results demonstrate that our ICM isolation method is simple and effective.     

A method for replacing intravesicular contents of golgi vesicles using an air-driven ultracentrifuge

Golgi membrane vesicles can be easily and very rapidly (within 10 min.) loaded with solutions of desired composition by centrifugation of the vesicles at high g force in an air-driven ultracentrifuge and subsequent resuspension of the vesicle pellet. This centrifugal/mechanical loading procedure does not destroy the integrity of these vesicles, as demonstrated by the ability of loaded vesicles to (i) retain their contents, (ii) maintain a K+ gradient when loaded with K+ ions, and (iii) exchange internal UMP for external [3H]UMP when loaded with UMP. When radiolabeled solutes are loaded into vesicles, the displaced internal volume can be measured using a rapid filtration assay. This simple and rapid technique of replacing the intravesicular contents of Golgi membrane vesicles should prove useful in studying transport across this membrane and may have a variety of other applications, such as intravesicular volume measurements, macromolecule and drug delivery protocols, and the study of membrane fusion events.     

A convenient large-scale method for the isolation of membrane vesicles permeable to a specific inorganic ion: Isolation and characterization of functional acetylcholine receptor-containing vesicles from the electric organ of Electrophorus electricus

A convenient, large-scale method for the isolation of membrane vesicles permeable to specific inorganic ions has been developed. The general principle of this method involves the exchange of Na⁺ within the vesicles for external Cs⁺. Vesicles in which this exchange rapidly occurs can be separated on the basis of their density from vesicles in which the exchange occurs slowly (G. P. Hess and J. P. Andrews (1977) Proc. Nat. Acad. Sci. USA74, 482–486). This approach has been adapted to develop a method suitable for the large-scale isolation of vesicles that contain functional acetylcholine receptors from the Electrophorus electricus electroplax. The new procedure involves a discontinuous sucrose gradient for an initial purification of the vescles. This allows the use of a low-speed centrifuge, which has a capacity up to 30 times greater than the Beckman ultracentrifuge previously used. A self-forming CsCl-Percoll gradient and low-speed centrifugation are then used for the isolation of the functional acetylcholine receptor-containing vesicles. The isolation step leads close to the theoretically possible fourfold purification of the vesicles that contain functional receptors. The yield, up to 12 mg membrane protein/centrifugal run, is about 100-fold higher than the yield from the sucrose-CsCl density gradient previously (Hess and Andrews, see above) used. The gradients are self-forming and an equilibrium is reached after centrifugation for only 30 min. In 12 experiments with membrane preparations from 12 different ceis, the functional vesicles had an internal volume of 2.0 ± 0.3 μl/mg vesicle protein and a receptor concentration of 1.2 ± 0.02 μm (1.2 μmol/liter of internal volume). Electron micrographs of these vesicles show an average vesicle radius of 1600 ± 300 Å. From these results, an average of 12 receptor molecules/membrane vesicle is calculated.     

On the isolation of embryonic stem cells: Comparative behavior of murine, porcine and ovine embryos

The efficiency of isolation and the characteristics of embryo-derived cell lines from murine, porcine, and ovine embryos cultured on STO feeders or homologous embryonic fibroblasts (HEF) feeders were compared. While murine isolated ICM or intact embryos plated on STO or HEF feeders gave rise to cell lines with embryonic stem cell-like (ES-like) morphology, ovine embryos did not. Cell lines with ES-like morphology were isolated from porcine intact embryos and isolated ICM when plated on STO feeders but not when plated on HEF. Neither murine nor porcine ES-like cell lines expressed cytokeratin 18 or vimentin. Unlike murine ES-like cell lines, porcine ES-like cells did not undergo observable differentiation in vitro or in vivo. Cell lines with epithelial-like morphology were isolated from porcine and ovine embryos. Both porcine and ovine epithelial-like cell kines expressed cytokeratin 18. When induced to differentiate in vitro, porcine and ovine epithelial-like cell lines formed vesicular structures. Electron microscopy revealed that the porcine vesicles were composed of polarized epithelial cells, each with a basally-located nucleus and an apical border containing numerous microvilli with a well organized microfilament core. The results of this study show that conditions which allow isolation of ES cells from murine embryos allow the isolation of porcine embryo-derived cell lines sharing some, but not all, the characteristics of murine ES cells.     

Survival of porcine inner cell masses in culture and after injection into blastocysts

Isolation of embryonic stem cells has been documented only in the mouse and perhaps the hamster and cow. We report results of experiments designed to determine the effect of age of porcine embryos (6 through 10 d after the first day of estrus) on isolation of cell lines with embryonic stem cell-like morphology. The capacity of fresh and short-term cultured inner cell mass (ICM) cells to differentiate into normal tissues after injection into blastocysts was also measured. Few Day-6 ICM survived in culture to the first passage onto fresh feeder cells, but cell lines with embryonic stem cell-like morphology developed from Day-7 through Day-10 ICM. Isolation of embryonic stem cell-like colonies was achieved at a higher frequency from ICM isolated from older embryos, but embryonic stem cell-like colonies from older embryos also tended to differentiate spontaneously in culture. Viable porcine chimeras were born after injection of fresh ICM into blastocysts that were transferred to recipients for development to term; no chimeras were born from blastocysts injected with ICM subjected to short-term (1 to 6 d) culture. Germ-cell chimerism was confirmed in one of the chimeras. These results document that undifferentiated cells can be removed from porcine blastocysts, transplanted to other embryos, and contribute to development of normal differentiated tissues, including germ cells. Cells with embryonic stem-like morphology can be isolated in culture from ICM at various embryonic ages, but ICM from young blastocysts (e.g., Day-7 embryos) yield embryonic stem cell-like colonies at lower frequency than do ICM from older blastocysts (e.g., Day-10 embryos).     

Trophoblast regeneration by inner cell masses isolated from cultured mouse embryos

The developmental potential of the inner cell mass (ICM) of the cultured mouse embryo was determined by testing the ability of the ICM to regenerate trophoblast in vitro. ICM's isolated by immunosurgery from either single or chimeric embryos were able to regenerate trophoblast when they were isolated at 69 hours of culture from the 2-cell stage, but they had lost this capacity by 93 hours of culture. Trophoblast regeneration by isolated ICM's did not appear to require either a critical cell mass at the time of isolation or cell proliferation during regeneration.     

Derivation and characterization of human embryonic stem cell lines from poor quality embryos

Poor quality embryos discarded from in vitro fertilization （IVF） laboratories are good sources for deriving human embryonic stem cell （hESC） lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses （ICMs） could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods （P〉0.05）. As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.     

Potential of isolated mouse inner cell masses to form trophectoderm derivatives in vivo

Recent in vitro experiments on immunosurgically isolated mouse inner cell masses (ICMs) have suggested that some ICM cells may retain the potential to form trophectoderm after initial blastocyst formation. These experiments relied almost solely on in vitro morphology for identification of trophectoderm derivatives and provided no proof that the putative trophectoderm cells were capable of functioning in utero. We present clear in vivo evidence that at least some cells in ICMs isolated from early blastocysts do retain the potential to form postimplantation trophectoderm derivatives. Early ICMs occasionally contributed to trophoblast fractions in ICM/morula aggregation chimeras. More strikingly, when aggregated with each other, these ICMs were capable of implanting in the uterus, a property of trophectoderm cells alone. Indeed, some aggregates reconstituted complete egg cylinders. However, ICMs isolated from later blastocysts rarely produced in vivo trophoblast, and it appears that the ability to form trophectoderm is lost around the 16–19 cell ICM stage. These results are discussed in relation to changing patterns of gene activity in early development.     

The interaction of calmodulin with erythrocyte membrane proteins

The method of sedimentation equilibrium in an air-driven ultracentrifuge (Airfuge) has been employed to investigate the interaction of 125I-calmodulin with the cytoskeletal components of the human red cell membrane. The results indicate significant calcium-dependent calmodulin binding activity in the low and high ionic strength extracts of the human erythrocyte membrane. The interaction of 125I-calmodulin with the low ionic strength extract proteins is analysed quantitatively. Further purification of the high ionic strength extract comprising mainly band 2.1 and band 4.1 results in the elution of calmodulin binding activity in a purified fraction of band 4.1.     

Influence of feeder layer type on the efficiency of isolation of porcine embryo-derived cell lines

Experiments were conducted to determine the effects of feeder layers composed of different cell types on the efficiency of isolation and the behavior of porcine embryo-derived cell lines. Inner cell masses (ICM) isolated from 7- to 8-d-old embryos were plated on feeder layers composed of Buffalo rat liver cells (BRL), a continuous cell line of murine embryonic fibroblasts (STO), STO combined with BRL at a 9:1 and 1:1 ratio, STO with BRL-conditioned medium (STO + CM), porcine embryonic fibroblasts (PEF), PEF combined with BRL at a 9:1 and 1:1 ratio, porcine uterine epithelial cells (PUE), murine embryonic fibroblasts (MEF), or an epithelial-like porcine embryo-derived cell line (PH3A). It was found that embryo-derived cell lines could be isolated only from the STO and the STO with BRL-conditioned medium treatments. The isolated cell lines were of epithelial-like and embryonic stem cell-like (ES-like) morphology. The feeders tested had an effect on the behavior of plated ICM. Some feeders, represented by PUE, BRL, STO:BRL (1:1), PEF:BRL (1:1), and PH3A, did not promote attachment of the ICM to the feeder layer; others, represented by STO and MEF, allowed attachment, differentiation and proliferation. On PEF feeders the ICM spread onto the feeder layer after attachment without apparent signs of proliferation or differentiation. None of the feeders tested increased the efficiency of isolation or the growth characteristics of embryo-derived (both ES-like and epithelial-like) cell lines over that of STO feeders.     

Cytoplasmic and nuclear protein synthesis during in vitro differentiation of murine ICM and embryonal carcinoma cells

Protein syntheses during in vitro differentiation of inner cell masses (ICM) isolated from mouse blastocysts and of pluripotent embryonal carcinoma cells (ECC) were compared by two-dimensional electrophoretic analysis of [³⁵S]methionine-labeled cells. While most of the polypeptides found in ICM, ECC, and embryoid bodies (EB) derived from them were common to all four preparations, some distinct differences were noted. More polypeptides changed in intensity during the differentiation of ICM than during the differentiation of ECC. Analysis of ECC prior to differentiation revealed that only some of the polypeptides abundant in ICM were present, while at the same time, some of the polypeptides abundant in ICM-EB were being synthesized. These data indicate that ECC represent cells further advanced in development than the cells of ICM isolated from 4-day-old blastocysts. The EB derived from ECC also differ from those from ICM. Comparison of EB derived from ICM and ECC with cells of the parietal yolk sac line, PYS, indicates that all three synthesize two polypeptides abundant in EB. These two polypeptides can, therefore, be used as biochemical markers of parietal entoderm differentiation. Pluripotent ECC synthesize small amounts of characteristic EB proteins and the 10-nm filament protein (also found in PYS cells but not in EB). This indicates that small numbers of differentiated or differentiating cells are present in pluripotent ECC cultures.     

The possible dual origin of the ectoderm of the chorion in the mouse embryo.

The origin of the extraembryonic ectoderm of the chorion in the mouse embryo has long been the source of some controversy. Various manipulative studies suggested that it arose from the trophectoderm and not the inner cell mass (ICM) of the blastocyst. However, recent studies on the development of isolated ICMs in vitro have reported the formation of tissues morphologically resembling extraembryonic ectoderm. One explanation not excluded by previous studies is that the chorionic ectoderm is of dual origin, from both ICM and trophectoderm. The present study provides a more detailed analysis than previously possible of the in vivo fate of ICMs in chimeras, using a sensitive assay for glucose phosphate isomerase (GPI) isozymes which permits study of the chorionic ectoderm alone. In a large series of blastocyst injection chimeras, no donor ICM contribution to the mature chorionic ectoderm could be detected, donor activity appearing only in the embryonic fraction. Thus, the in vitro results cannot be readily explained by dual origin of the chorionic ectoderm and remain in conflict with existing in vivo data. Analysis of most ICM/morula chimeras revealed the same pattern, but a few showed ICM contributions to the trophoblast fractions, suggesting that some ICM cells retain the potential to form trophectoderm derivatives in vivo.     

Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation

Isolation of a microsomal membrane fraction is a common procedure in studies involving membrane proteins. By conventional definition, microsomal membranes are collected by centrifugation of a postmitochondrial fraction at 100,000g in an ultracentrifuge, a method originally developed for large amounts of mammalian tissue. We present a method for isolating microsomal-type membranes from small amounts of Arabidopsis thaliana plant material that does not rely on ultracentrifugation but instead uses the lower relative centrifugal force (21,000g) of a microcentrifuge. We show that the 21,000g pellet is equivalent to that obtained at 100,000g and that it contains all of the membrane fractions expected in a conventional microsomal fraction. Our method incorporates specific manipulation of sample density throughout the procedure, with minimal preclearance, minimal volumes of extraction buffer, and minimal sedimentation pathlength. These features allow maximal membrane yields, enabling membrane isolation from limited amounts of material. We further demonstrate that conventional ultracentrifuge-based protocols give submaximal yields due to losses during early stages of the procedure; that is, extensive amounts of microsomal-type membranes can sediment prematurely during the typical preclearance steps. Our protocol avoids such losses, thereby ensuring maximal yield and a representative total membrane fraction. The principles of our method can be adapted for nonplant material.     

PHAGOCYTOSIS OF LATEX BEADS BY ACANTHAMOEBA CASTELLANII (NEFF) : III. Isolation of the Phagocytic Vesicles and Their Membranes

A method is described for the rapid and efficient isolation of phagocytic vesicles from large scale cultures of Acanthamoeba castellanii (Neff) that have been incubated with polystyrene latex beads. Cells were allowed to phagocytose latex beads for 30 min and then were homogenized, and the phagocytic vesicles were isolated by one centrifugation through several layers of sucrose. Identity and purity of the phagocytic vesicles were determined by electron microscopy, chemical analyses, and assays of acid phosphatase, α- and β-glucosidase, and reduced nicotinamide adenine dinucleotide dehydrogenase. When phagocytosis was allowed to occur for longer periods the phagocytic vesicles appeared to fuse with each other and perhaps with digestive vacuoles. The resultant vesicles which contained many beads were heavier than those which consisted of only one bead or a few beads with a closely applied membrane. Ultrasonication ruptured the isolated vesicles, and the membranes could then be isolated in 30–50% yield based on phospholipid analysis. These membranes were essentially free of acid hydrolases and, presumably, other soluble proteins, as was also indicated by their low ratio of protein to phospholipid. The membranes have been prepared both as closed vesicles and as open sheets.     

THE STRUCTURE AND FORMATION OF PROTEIN GRANULES IN THE FAT BODY OF AN INSECT

In the larva of the butterfly Calpodes ethlius, the fat body begins to store protein in the form of granules at about 30 to 35 hours before pupation, at a time when the endocuticle is being resorbed. At least two sorts of granule can be distinguished. The first granules to arise are those within vesicles of the Golgi complex. These may increase in size by incorporating material from microvesicles at their surface and by coalescence with one another. Later, at about 10 hours before pupation, another sort of granule arises by the isolation of regions of the endoplasmic reticulum (ER) within paired membranes derived from Golgi vesicles. Several of these ER isolation bodies coalesce, with fusion of their outer isolating membranes. The ribosomes and membranes may then disappear and the granules become indistinguishable from the protein granules formed from Golgi vesicles, or the ribosomes may remain and be embedded in dense crystalline protein, forming a storage body for both protein and RNA. Mitochondria are isolated within paired membranes in the same way as regions of the ER. The isolated mitochondria also coalesce in a similar manner. When the inner membranes are lost, the structure of a group of isolation bodies is indistinguishable from that of a cytolysome. Isolation within paired membranes, as described here, may be of general importance in segregating regions of massive lysis or massive sequestration.     

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.