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.     

Differentiation-promoting effects of mammary-derived growth inhibitor (MDGI) on pluripotent mouse embryonic stem cells

Pluripotent embryonic stem cells (line BLC6), when cultivated in vitro as embryoid bodies and injected subcutaneously into syngeneic mice, form teratocarcinomas consisting of embryonal carcinoma cells and differentiated tissues of all three primary germ layers. In order to study the possible effects of the mammary-derived growth inhibitor (MDGI) on the differentiation pattern of the tumors developing in the mice, BLC6 cell-derived embryoid bodies were treated in vitro for 4 days with either MDGI or a synthetic peptide composed of the C-terminal 11 amino acids of MDGI. In those tumors, significantly more differentiated neural tissue and lesser proportions of undifferentiated embryonic carcinoma cells (ECC) were found in the MDGI-and peptide-treated groups, compared with controls. The results are discussed with respect to a possible differentiation-promoting capacity of MDGI.     

DIFFUSION CHAMBER CULTURE OF A SINGLE EMBRYOID BODY FROM THE TESTICULAR TERATOMA OF STRAIN 129 MOUSE

When single embryoid bodies of teratocarcinoma OTT 6050 were cultured by the diffusion chamber technique in the peritoneal cavity of a mouse, they lost their characteristic three-dimensional structure early in the culture period and proliferated logarithmically up to the 60th day of culture with a doubling time of 3.7 days, forming cell layers that adhered to the surface of the membrane filters of the diffusion chamber. They continued further to proliferate at a lower rate up to the 80th day of culture. At the 60th day, many round cells, classified by diameter into about three classes, were observed on the membrane filters. The tumorigenicity of these cells derived from the chamber cultures was much less than that of embryoid bodies injected directly into the abdominal cavity, judging from the number of days the mice survived.     

Differentiation-promoting effects of mammary-derived growth inhibitor (MDGI) on pluripotent mouse embryonic stem cells

Pluripotent embryonic stem cells (line BLC6), when cultivated in vitro as embryoid bodies and injected subcutaneously into syngeneic mice, form teratocarcinomas consisting of embryonal carcinoma cells and differentiated tissues of all three primary germ layers. In order to study the possible effects of the mammary-derived growth inhibitor (MDGI) on the differentiation pattern of the tumors developing in the mice, BLC6 cell-derived embryoid bodies were treated in vitro for 4 days with either MDGI or a synthetic peptide composed of the C-terminal 11 amino acids of MDGI. In those tumors, significantly more differentiated neural tissue and lesser proportions of undifferentiated embryonic carcinoma cells (ECC) were found in the MDGI- and peptide-treated groups, compared with controls. The results are discussed with respect to a possible differentiation-promoting capacity of MDGI.     

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.     

Avian pluripotent stem cells

Pluripotent embryonic stem cells are undifferentiated cells capable of proliferation and self-renewal and have the capacity to differentiate into all somatic cell types and the germ line. They provide an in vitro model of early embryonic differentiation and are a useful means for targeted manipulation of the genome. Pluripotent stem cells in the chick have been derived from stage X blastoderms and 5.5 day gonadal primordial germ cells (PGCs). Blastoderm-derived embryonic stem cells (ESCs) have the capacity for in vitro differentiation into embryoid bodies and derivatives of the three primary germ layers. When grafted onto the chorioallantoic membrane, the ESCs formed a variety of differentiated cell types and attempted to organize into complex structures. In addition, when injected into the unincubated stage X blastoderm, the ESCs can be found in numerous somatic tissues and the germ line. The potential give rise to somatic and germ line chimeras is highly dependent upon the culture conditions and decreases with passage. Likewise, PGC-derived embryonic germ cells (EGCs) can give rise to simple embryoid bodies and can undergo some differentiation in vitro. Interestingly, chicken EG cells contribute to somatic lineages when injected into the stage X blastoderm, but only germ line chimeras have resulted from EGCs injected into the vasculature of the stage 16 embryo. To date, no lines of transgenic chickens have been generated using ESCs or EGCs. Nevertheless, progress towards the culture of avian pluripotent stem cells has been significant. In the future, the answers to fundamental questions regarding segregation of the avian germ line and the molecular basis of pluripotency should foster the full use of avian pluripotent stem cells.     

Conditions Permitting Solid Tumor Formation of Teratocarcinoma Cells in Allogeneic Mice

In this report, the growth capacity of murine ascitic teratocarcinomas (embryoid bodies, EBs) was studied in certain strains of allogeneic mice. When EB was intravenously injected into Balb/c mice, various types of tumor colonies were formed with various types of differentiated tissues in the lung, as in the case of syngeneic 129/Sv mice. Tumor colonies in the abdomen of Balb/c and other allogeneic mice formed by the intraperitoneal injection of EB, however, contained merely undifferentiated embryonal carcinoma cells (EC cells) and sometimes some primitive tissues, too. Analysis of lung colonies (LCs) from EBs in Balb/c mice indicated that EC cells are a potent target for being rejected but they can escape this rejection by differentiating into various types of tissues cells.     

The characterization of SV40-transformed cell lines derived from mouse teratocarcinoma: growth properties and differentiated characteristics.

Mouse teratocarcinoma cells derived from embryoid bodies of 129SVsl mice were cultured in vitro to permit their differentiation. These cells were then infected with simiam virus 40 (SV40) and 31 cloned cell lines (SVTER) were derived from these cultures. All 31 SVTER cell lines contained the SV40 tumor (T) antigen and grew as permanent lines in culture. Mock-infected embryoid body cultures did not give rise to permanent cell lines. The morphology of each SVTER cell line was distinct and did not change during successive subclonings. The growth properties and tumorigenic potential of all 31 SVTER cell lines were investigated. None of these lines produced tumors in 129SVsl mice. Each cell line was tested for its ability to (1) grow in medium containing 1% serum, (2) plate on cell monolayer, and (3) form clones in methocel suspension. Only three of the SVTER cell lines were transformed with respect to all three of these criteria. Most of these cell lines were minimal transformants. The SVTER cell lines were tested for creatine phospholinase (CPK), an enzyme activity chracteristic of mouse brain and muscle tissue, and the protease, plasminogen activator (PA) which is found in embryoid bodies and several differentiated cell types. Some of the SVTER cell lines contained high levels of CPK, while others had high levels of PA and a third group of cells contained neither enzyme activity. No SVTER cell line was found with high levels of both these enzyme activities. This result suggests that mutually exclusive sets of genes are expressed in these cells as might be expected from the distinct tissue distribution of the two enzyme activities studied. These SVTER cell lines may be useful in reconstructing developmental pathways of differentiating teratomas in vitro.     

Differentiation of human testicular embryonal carcinoma and teratocarcinoma grown in nude mice and soft-agar cultures

The differentiation pattern of two human germ cell tumors, grown in nude mice and in vitro is described. Tumor A was an embryonal carcinoma (EC) of borderline histology with characteristics of yolk sac tumor and of seminoma; tumor B was a teratocarcinoma with yolk sac elements and syncytiotrophoblastic giant cells. The morphology of an EC as well as cytogenetic characteristics were maintained during 20 passages in nude mice from tumor A and over 11 passages from tumor B. Tumor A did not grow in vitro. Cell suspensions prepared from xenografted tumor B grew into cystic embryoid bodies in semi-solid tissue culture medium. These embryoid bodies showed cuboidal and flattened cells with microvilli, junctional complexes, peripheral microfilaments, and annulated lamellae, reminiscent of the 'inner cell mass' of a blastula and of endoderm, respectively. When such colonies were transplanted into nude mice, however, only tumors with the morphology found in the transplants appeared.     

胚胎干细胞治疗心肌梗死的研究进展

胚胎干细胞 (ES细胞 )是一种多能细胞 ,来源于囊胚期胚胎 ,具有很强的自我更新能力 ,并能分化成很多细胞类型。体外 ,ES细胞能自发聚集形成胚胎体 (EB) ,分化成许多种细胞类型 ;ES细胞注射到免疫缺陷的小鼠体内 ,产生畸胎瘤 ,其中包含有三个胚层的细胞。添加生长因子或与其它细胞共培养等方法可以促进ES细胞体外分化为心肌细胞 ,筛选后移植到梗死的心肌 ,可以提高心脏功能 ,是治疗心肌梗死的一种很有潜力的方法     

Alpha-fetoprotein in tumours derived from cystic and simple embryoid bodies.

Cellular aggregates called embryoid bodies (EB) have been obtained from the experimental teratocarcinoma (TC) 0TT6050. Two morphological types of EB can be differentiated, which are injected subcutaneously into isogenic 129/Sv mice. The tumors are collected 20 and 30 days after EB injection and processed histologically, and immunohistochemically with anti-alpha-fetoprotein (alpha-FP) antibodies. Our results indicate that the histological pattern of the tumors is related to the degree of morphological organization of the EB used.     

Forced expression of the homeobox-containing gene Pem blocks differentiation of embryonic stem cells.

Similarities in the differentiation of mouse embryos and ES cell embryoid bodies suggest that aspects of early mammalian embryogenesis can be studied in ES cell embryoid bodies. In an effort to understand the regulation of cellular differentiation during early mouse embryogenesis, we altered the expression of the Pem homeobox-containing gene in ES cells. Pem is normally expressed in the preimplantation embryo and expressed in a lineage-restricted fashion following implantation, suggesting a role for Pem in regulating cellular differentiation in the early embryo. Here, we show that the forced expression of Pem from the mouse Pgk-1 promoter in ES cells blocks the in vitro and in vivo differentiation of the cells. In particular, embryoid bodies produced from these Pgk-Pem ES cells do not differentiate into primitive endoderm or embryonic ectoderm, which are prominent features of early embryoid bodies from normal ES cells. This Pgk-Pem phenotype is also different from the null phenotype, as embryoid bodies derived from ES cells in which endogenous Pem gene expression has been blocked show a pattern of differentiation similar to that of normal ES cells. When the Pgk-Pem ES cells were introduced into subcutaneous sites of nude mice, only undifferentiated EC-like cells were found in the teratomas derived from the injected cells. The Pem-dependent block of ES cell differentiation appears to be cell autonomous; Pgk-Pem ES cells did not differentiate when mixed with normal, differentiating ES cells. A block to ES cell differentiation, resulting from the forced expression of Pem, can also be produced by the forced expression of the nonhomeodomain region of Pem. These studies are consistent with a role for Pem in regulating the transition between undifferentiated and differentiated cells of the early mouse embryo.     

一种利用STO饲养层细胞制备拟胚体的新方法

建立了一种利用STO饲养层细胞制备拟胚体的新方法。该方法选用生长至80%饱和密度的STO细胞,经丝裂霉素C(10 mg/ml)处理4 h后以8×104 cm-2的密度接种培养12 h,制备饲养层,再将ES-D3细胞以1×104 cm-2的密度接种其上,首先用含mLIF的DMEM培养液培养24 h,再更换拟胚体诱导培养液,5～9天后获得了各成熟阶段的拟胚体。形态结构和分化潜能等研究表明,该方法制备的拟胚体结构典型,具有产生3个胚层谱系来源的功能细胞的潜能。与传统拟胚体制作方法如悬滴培养法相比,具有操作简便,拟胚体形成率高,重复性好等优点,是开展哺乳动物早期胚胎发育和干细胞分化研究的理想工具。     

Wnt11 facilitates embryonic stem cell differentiation to Nkx2.5-positive cardiomyocytes

Wnt signaling plays a crucial role in the control of morphogenesis in several tissues. Herein, we describe the role of Wnt11 during cardiac differentiation of embryonic stem cells. First, we examined the expression profile of Wnt11 during the course of differentiation in embryoid bodies, and then compared its expression in retinoic acid-treated embryoid bodies with that in untreated. In differentiating embryoid bodies, Wnt11 expression rose along with that of Nkx2.5 expression and continued to increase. When the embryoid bodies were treated with retinoic acid, Wnt11 expression decreased in parallel with the decreased expression of cardiac genes. Further, treatment of embryoid bodies with medium containing Wnt11 increased the expression of cardiac marker genes. Based on these results, we propose that Wnt11 plays an important role for cardiac development by embryoid bodies, and may be a key regulator of cardiac muscle cell proliferation and differentiation during heart development.     

Biochemical criteria for the in vitro differentiation of embryoid bodies produced by a transplantable teratoma of mice. The production of acetylcholine esterase and creatine phosphokinase by teratoma cells

When embryoid bodies are grown in suspension culture in vitro, they undergo only a limited amount of morphological development. When these same embryoid bodies are permitted to attach to the surface of a culture dish, a wide variety of new morphological cell types appear. Suspension cultures of embryoid bodies do not contain significant detectable levels of acetylcholine esterase or creatine phosphokinase. These same enzymes however are produced in cell cultures derived from embryoid bodies attached to the culture dish surface. Polyacrylamide gel electrophoresis has been employed to demonstrate that the electrophoretic form of creatine phosphokinase produced by teratoma cells in culture is the brain form of the enzyme. Solid transplantable tumors containing only embryonal carcinoma cells (stem cells) do not contain either of these enzymatic activities. Well differentiated transplantable teratomas contain both enzymes.     

Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds

Embryonic stem (ES) cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy. The main catalyst for ES cells differentiation is the growth into embryoid bodies (EBs), which are utilized widely as the trigger of in vitro differentiation. In this study, a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established. When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds, they grew into aggregates gradually and formed simple EBs with circular structures. After 7 days' culture,they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers. Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types; they were also able to form into tissue-like structures. Moreover, with introduction of ascorbic acid, ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19. The results demonstrated that collagen/Matrigel scaffolds supported EBs formarion and their subsequent differentiation in a single three dimensional environment.     

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.     

Differentiation of Pluripotent Embryonic Stem Cells in the Peritoneal Cavity of Irradiated Mice

We studied the behavior and differentiation of pluripotent embryonic stem cells of R1 mice in vivo. Undifferentiated embryonic stem cells and differentiating embryoid bodies implanted in the abdominal cavity of irradiated mice were shown to form tumors containing the derivatives of all germ layers. Cells of the embryoid bodies form tumors two weeks after implantation, while undifferentiated embryonic stem cells form tumors only by week three.     

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.