Embryoid bodies cultured in in vivo diffusion chambers show reduced tumorigenicity while retaining expression of F9 antigens

Embryoid bodies of the mouse teratocarcinoma OTT6050 were dissociated into single cells and cultured in diffusion chambers implanted into the peritoneal cavities of mice. The syngeneic host mice, into which the cells of embryoid bodies cultured in the diffusion chambers had been injected, survived much longer than those which received the original cells of embryoid body. But in the case of the F9 cells, obtained in the same culture conditions, only a slight decrease in tumorigenicity was observed. By contrast, the F9 antigenic expression was observed on both F9 and embryoid body cells cultured in diffusion chambers. Judging from the determination of adult-type antigenic expressions, the differentiation of the cells in chamber was negligible. These results suggest that the tumorigenic activity of the embryoid body cells cultured in vivo in a diffusion chamber is almost suppressed, but that they continue in an undifferentiated state.     

Teratocarcinoma differentiation: plasminogen activator activity associated with embryoid body formation.

Changes in plasminogen activator activity have been examined as a clonal line of mouse embryonal carcinoma cells aggregate and differentiate to form cystic embryoid bodies in vitro. Within the first 10 days of study, the pluripotent embryonal carcinoma cells aggregate; a layer of endodermal cells appears on the outside of the aggregate forming an embryoid body; a basement membrane forms between the outer layer of endodermal cells and the internal cells; a cyst forms within the embryoid body; and the internal cells assume a columnar appearance along the inner portion of the basement membrane. After the formation of the endodermal layer, there is a rise in intracellular plasminogen activator activity. This rise continues for up to 25 days in culture, providing that the three-dimensional integrity of the embryoid bodies is maintained by culturing them on bacterial petri dishes. Selective removal of the outer endodermal layer of cells reduces the plasminogen activatory activity of the resulting embryoid body cores. Intracellular and secreted plasminogen activator activity of simple embryoid bodies composed of only two cell types can be increased by culturing the embryoid bodies in dbcAMP, theophylline, or cholera toxin. These results suggest that the embryoid body endodermal cells are the source of a cAMP-inducible plasminogen activator activity.     

IN VIVO STUDY ON THE PROCESS OF SOLID TUMOR FORMATION FROM EMBRYOID BODIES OF MOUSE TERATOCARCINOMA

Embryoid bodies of mouse teratocarcinoma OTT 6050 were studied with special reference to their growth and differentiation in vivo . They were grown either in the peritoneal cavity or in the lung. When injected intraperitoneally, embryoid bodies doubled their number every three days. Some of them attached to a small intra-peritoneal fat body and were soon surrounded by mesenchymal cells of host origin. They grew, fused with each other and became large solid tumors which contained many differentiated tissues. When injected intravenously, almost all the embryoid bodies lodged in the lungs and individually grew into discrete solid tumors which doubled in volume every 2.9 days. After about thirty days, some tumors were composed of only one type of tissue while others contained several types of tissues. The possible mechanisms for the uni- and multi-tissue types of differentiation are discussed.     

Generation of embryoid bodies from mouse embryonic stem cells cultured on STO feeder cells

Embryoid bodies, which are similar to post-implantation egg-cylinder stage embryos, provide a model for the study of embryo development and stem cell differentiation. We describe here a novel method for generating embryoid bodies from murine embryonic stem (ES) cells cultured on the STO feeder layer. The ES cells grew into compact aggregates in the first 3 days of coculture, then became simple embryoid bodies (EBs) possessing primitive endoderm on the outer layer. They finally turned into cystic embryoid bodies after being transferred to Petri dishes for 1-3 days. Evaluation of the EBs in terms of morphology and differentiating potential indicates that they were typical in structure and could generate cells derived from the three germ layers. The results show that embryoid bodies can form not only in suspension culture but also directly from ES cells cultured on the STO feeder layer.     

Osteogenic differentiation within intact human embryoid bodies result in a marked increase in osteocalcin secretion after 12 days of in vitro culture, and formation of morphologically distinct nodule-like structures

Osteogenic lineages derived from human embryonic stem cells hold much promise for clinical application in bone regeneration, in addition to providing a useful research model in developmental biology, and for pharmacological and cytotoxicity screening of bone-related biomaterials and drugs in vitro. Previously, osteogenic differentiation of human embryonic stem cells was achieved through dissociation of embryoid bodies by trypsinization, prior to culture with osteogenesis-promoting medium. This study therefore attempted a new approach: that is to achieve osteogenesis within intact human embryoid bodies. After 22 days of culture in osteogenesis-promoting medium comprising a cocktail of ascorbic acid, beta-glycerophosphate and dexamethasone, the attached embryoid bodies exhibited much cellular outgrowth and migration, and formed morphologically distinct nodule-like structures. These were somewhat similar to osteogenic nodules formed by mesenchymal stem cells, as reported by previous studies. Immunohistochemical staining and RT-PCR analysis confirmed the presence of osteogenic cells within these nodule-like structures. Additionally, the quantitative assay of osteocalcin secretion demonstrated a rapid sharp increase in osteocalcin expression on day 12 of in vitro culture, which could suggest the appearance of differentiated osteoblasts from day 12 onwards. Future work will attempt to investigate whether other cytokines, growth factors and chemical compounds could further enhance osteogenesis within intact human embryoid bodies.     

体外诱导鸡胚胎生殖细胞分化为神经干细胞

本研究探讨体外诱导鸡胚胎生殖细胞(EGCs)分化为神经干细胞(NSCs)的可能性.EGCs经类胚体(EB)阶段,以维生素A酸(RA)等进行诱导,在NSCs选择性培养基中筛培养扩增7 d,观察形态变化;采用RT-PCR法检测nestin基因表达及免疫细胞化学法检测nestin等NSCs特异性标志物,并对其扩增及分化能力进行观察.结果显示:EGCs经初级诱导,NSCs选择性培养基筛选培养7 d后,形成大量神经球样结构,可扩增传代;绝大部分神经球样结构呈nestin抗原阳性,表达nestin基因,且可分化为神经上皮样及少突胶质细胞.研究结果表明:RA等诱导的EGCs,经选择性培养基筛选培养可获得NSCs,有望为眼部神经变性疾病的治疗提供新的技术参考.     

THE PROCESS OF DIFFERENTIATION OF EMBRYOID BODIES IN THE LUNG OF SYNGENEIC MICE

The process of differentiation of embryoid bodies of mouse teratocarcinoma OTT6050 transplanted into the lung of syngeneic mice (129/Sv) is described. Embryoid bodies took more than 2 weeks to differentiate, and several kinds of differentiated tissues appeared often in the colonies derived from a single embryoid body. All the colonies with differentiated tissues were larger than 100μm in diameter.
Three steps on the differentiation of embryoid bodies can be distinguished by microscopic observations on histological preparations of tumors at different periods after injection. The first step is the deformation of the embryoid bodies and the disappearance of the outer endodermal cells, which occurs within a few days after injection. In the second step, which begins 5–7 days after injection, clusters of embryonal carcinoma cells in the colony are identified by the PAS reaction. The third step starts about 10 days after injection, and is characterized by the formation of tubular structures in some clusters.     

Culture of human haemopoietic cells in diffusion chambers. I. Growth of normal peripheral blood cells.

Growth of mononuclear cells from human peripheral blood from 10 normal individuals was tested in diffusion chamber culture over a period up to 17 or 21 days. After an initial decrease during the first few days an increase of the total cell number was observed with maximal values on day 13. In all individuals growth of undifferentiated blast cells, lymphocytes, plasma cells, immature and mature granulopoietic cells, macrophages, and megakaryocytes occurred. In all individuals the different cell types had similar growth patterns in diffusion chamber culture. The considerable numerical variations which were seen in the granulopoietic cells were probably due to different stem cell concentrations in the peripheral blood of the investigated individuals. The results indicate that the diffusion chamber technique is a valuable method for the detection of haemopoietic stem cells and the culture of lymphocytic cells in man.     

In Vitro Differentiation of Mouse Embryonic Stem Cells into Neurons of the Dorsal Forebrain

Pluripotent embryonic stem cells (ESCs) are able to differentiate into all cell types in the organism including cortical neurons. To follow the dynamic generation of progenitors of the dorsal forebrain in vitro, we generated ESCs from D6-GFP mice in which GFP marks neocortical progenitors and neurons after embryonic day (E) 10.5. We used several cell culture protocols for differentiation of ESCs into progenitors and neurons of the dorsal forebrain. In cell culture, GFP-positive cells were induced under differentiation conditions in quickly formed embryoid bodies (qEBs) after 10–12 day incubation. Activation of Wnt signaling during ESC differentiation further stimulated generation of D6-GFP-positive cortical cells. In contrast, differentiation protocols using normal embryoid bodies (nEBs) yielded only a few D6-GFP-positive cells. Gene expression analysis revealed that multiple components of the canonical Wnt signaling pathway were expressed during the development of embryoid bodies. As shown by immunohistochemistry and quantitative qRT-PCR, D6-GFP-positive cells from qEBs expressed genes that are characteristic for the dorsal forebrain such as Pax6, Dach1, Tbr1, Tbr2, or Sox5. qEBs culture allowed the formation of a D6-GFP positive pseudo-polarized neuroepithelium with the characteristic presence of N-cadherin at the apical pole resembling the structure of the developing neocortex.     

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.     

The insulin secretion of a minced neonatal rat pancreas cultured in a pancreatic chamber, in response to various insulin secretagogues

The minced pancreas of the neonatal rat was cultured for 35 days in a pancreatic chamber which was constructed of a plastic tube and an ultrafiltration membrane. Insulin and amylase secreted from this pancreatic chamber into the culture medium were measured. During the experiment, the concentration of glucose in the culture medium was changed between 5.5 and 16.5 mM at 2-3 day intervals in order to determine the insulin secretory response of the pancreatic tissue. Insulin secretion was markedly increased in response to 16.5 mM glucose. The ratio of insulin secretion to amylase secretion in the culture medium increased with the advance of culture days although secretions of both insulin and amylase decreased individually. On the 7th culture day, short term incubations were performed to test with various insulin secretagogues; obvious insulin release into the incubation medium was observed. These results show that the pancreatic chamber also in vitro secretes insulin rapidly and significantly in response to various stimuli; that by longer culture of a neonatal rat pancreas in this device, insulin secretory cells without exocrine tissue would be obtained without using digestive enzymes; that application of a pancreatic chamber for a pancreatic transplantation may be feasible.     

X-chromosome inactivation in differentiating mouse embryonic stem cells carrying X-linked GFP and lacZ transgenes

Three new female ES cell lines (GLM1, GLP1 and GLP2) were established from mouse embryos carrying GFP (green fluorescent protein) and HMG-lacZ transgenes on one of two X chromosomes in cis. Using these cell lines, we studied the temporal relationships among three events relevant to X-chromosome inactivation: replication asynchrony of the X chromosome, and quenching of GFP fluorescence and beta-galactosidase (beta-gal) activity, during cell differentiation induced by embryoid body (EB) formation and retinoic acid (RA) treatment. In embryoid bodies adhering to the bottom of culture dishes, GFP-negative cells appeared first in the peripheral outgrowths 4 days after the initiation of EB formation, followed about 24 hours later by the appearance of cells negative for beta-gal and those having a single allocyclic X chromosome. Although the frequency of cells with an allocyclic X chromosome could reach 80% in adherent embryoid bodies, it tended to remain low and variable in embryoid bodies maintained in suspension. In spite of apparently parallel extinction of GFP and lacZ in embryoid bodies, their concurrent occurrence did not always characterize RA-induced differentiation. Moreover, an allocyclic X chromosome was identified in not more than 20 percent of informative metaphase cells up to 10 days after initiation of RA treatment. These findings suggest that RA-induced differentiation of female ES cells does not always accompany X-inactivation.     

Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation.

Mouse embryonic stem cells are pluripotent cells that are derived from the inner cell mass of blastocysts. When induced to synchronously enter a program of differentiation in vitro, they form embryoid bodies that contain cells of the mesodermal, hematopoietic, endothelial, muscle, and neuronal lineages. Here, we used a panel of marker genes with early expression within the germ layers (oct-3, Brachyury T, Fgf-5, nodal, and GATA-4) or a variety of lineages (flk-1, Nkx-2.5, EKLF, and Msx3) to determine how progressive differentiation of embryoid bodies in culture correlated with early postimplantation development of mouse embryos. Using RNA in situ hybridization, we found that the temporal and spatial relationships existing between these marker genes in vivo were maintained also in vitro. Studying the onset of marker gene expression allowed us also to determine the time course of differentiation during the formation of embryoid bodies. Thus, stages equivalent to embryogenesis between implantation and the beginning of gastrulation (4.5-6.5 d.p.c.) occur within the first two days of embryoid body differentiation. Between days 3 and 5, embryoid bodies contain cell lineages found in embryos during gastrulation at 6.5 to 7.0 d.p.c., and after day 6 in culture, embryoid bodies are equivalent to early organogenesis-stage embryos (7.5 d.p.c.). In addition, we demonstrate that the panel of developmental markers can be applied in a screen for stage- or lineage-specific genes. Reporter gene expression from entrapment vector insertions can be co-localized with expression of specific markers within the same cell during embryoid body formation as well as during embryogenesis. Our results thus demonstrate the power of embryoid body formation as an in vitro model system to study early lineage determination and organogenesis in mammals, and indicate that they will prove to be useful tools for identifying developmental genes whose expression is restricted to particular lineages.     

Mouse embryonic stem cell-derived feeder cells support the growth of their own mouse embryonic stem cells

Feeder cells are usually used in culturing embryonic stem cells (ESCs) to maintain their undifferentiated and pluripotent status. To test whether mouse embryonic stem cells (mESCs) may be a source of feeder cells to support their own growth, 48 fibroblast-like cell lines were isolated from the same mouse embryoid bodies (mEBs) at three phases (10th day, 15th day, 20th day), and five of them, mostly derived from 15th day mEBs, were capable of maintaining mESCs in an undifferentiated and pluripotent state over 10 passages, even up to passage 20. mESCs cultured on the feeder system derived from these five cell lines expressed alkaline phosphatase and specific mESCs markers, including SSEA-1, Oct-4, Nanog, and formed mEBs in vitro and teratomas in vivo. These results suggest that mEB-derived fibroblasts (mEB-dFs) could serve as feeder cells that could sustain the undifferentiated growth and pluripotency of their own mESCs in culture. This study not only provides a novel feeder system for mESCs culture, avoiding a lot of disadvantages of commonly used mouse embryonic fibroblasts as feeder cells, but also indicates that fibroblast-like cells derived from mESCs take on different functions. Investigating the molecular mechanisms of these different functional fibroblast-like cells to act on mESCs will contribute to the understanding of the mechanisms of mESCs self-renewal.     

Differential Dialysis Culture for Separation and Concentration of a Macromolecular Product

A differential dialysis flask, constructed with three chambers and two membranes of different porosity, was used to effect the separation and concentration of enterotoxin B produced extracellularly by a culture of Staphylococcus aureus. Variables were examined that affected the diffusion of glucose, as measured by half-equilibration time and permeability coefficient; the relative chamber volume, type of membrane, membrane masking, and mixing all exerted a substantial influence on diffusion rates. A number of membrane filters were tested for usefulness; one type, made with vinylidene fluoride, had desirable physical and diffusional properties, but neither it nor others consistently withheld the bacteria for more than a marginally useful period of about 50 hr. In ordinary two-chambered dialysis culture, the amount of enterotoxin reached 10 times that in control culture; in differential, three-chambered dialysis culture the comparable factor of increase was about 7, with about two-thirds of this amount being separated from cells in the product chamber of the flask.     

Parallel development of cardiomyocytes and neurons in embryonic stem cell culture

Recent studies suggest that there are strong parallels between development and patterning of the vertebrate vascular system and the nervous system. While previous observations reported generation of vascular and neuronal progenitors from embryonic stem (ES) cells, the question of parallel development of vascular and neuronal cells in the same culture has not yet been investigated. Mouse D3 ES cells were cultured for 4 days in differentiation medium IMDM with 15% FBS in 100 mm non-adhesive Petri dishes to allow cells to aggregate and form embryoid bodies. At day 5, fibronectin or all-trans retinoic acid with fibronectin was added to the culture. On day 9, the embryoid bodies were seeded on poly-L-ornithine/fibronectin-coated plates. After plating, half of the plates were treated with laminin for 3 days and maintained for 1 week in Neurobasal media with B27. Here we show that ES cells differentiate into interconnected rhythmically contracting aggregates of functional cardiomyocytes and neurons. Double immunofluorescence with anti-phospholamban, anti-SERCA2 antibodies to detect cardiomyocytes and with anti-MAP2 antibodies to detect neurons revealed the cell aggregates consisting entirely of cardiomyocytes with neuronal cells located on the periphery or covering the aggregate's surface. The observed concurrent development of cardiomyocytes and neurons suggests bidirectional communication between both cell types. We propose that crosstalk between cardiovascular and neuronal progenitors is an important mechanism for the development of both systems.     

Morphological criteria for the in vitro differentiation of embryoid bodies produced by a transplantable teratoma of mice

Embryoid bodies are produced when a transplantable testicular teratoma from strain 129 mice is serially passaged in the peritoneal cavity of these mice. These bodies are roughly spherical containing two morphologically distinct cell types. Scanning and transmission electron microscopy have been employed to show that the endodermal cells of embryoid bodies, like those of the mouse embryo, are on the outer surface and have highly convoluted surfaces containing numerous microvilli-like projections. The inner, embryonal carcinoma cells, are the pluripotent stem cells of this tumor. Intracisternal A-type particles have been observed in electron micrographs and are almost exclusively located in the endodermal cells of the embryoid bodies. The A-type complement-fixing antigen has been identified in extracts prepared from this tumor. When embryoid bodies are placed in culture and allowed to attach to the surface of a petri dish, a large number of new morphologically distinct cell types appear. Attachment to the petri dish surface is required for the generation of these new cell types. Cells of similar morphology in culture, display a distinctly “clonal” distribution on the petri dish surface.     

定向诱导小鼠ES细胞向心肌细胞的分化

为了提高体外诱导ES细胞向心肌细胞分化的效率 ,对以往的诱导方法加以改进 ,采用直接悬浮培养和 0 8%DMSO诱导 ,建立了简便、高效的定向诱导ES细胞向心肌细胞分化的体系 .诱导第 9d起可见自发性、有节律跳动的类胚体出现 ,第 14d达到高峰 ,约有 70 %的拟胚体产生跳动 .用RT PCR的方法在跳动的拟胚体中检测到心肌细胞特异性标志物的表达 ,采用免疫荧光染色的方法在蛋白水平检测到心肌特异的α辅肌动蛋白 (α actinin)的表达 ,并可见清晰肌小节 ,表明在改进的体外诱导条件下ES细胞可分化为成熟的心肌细胞 .