A comparison of surface proteins in embryonal carcinoma cells and their differentiated derivatives

Surface proteins from five cell lines (three embryonal carcinoma cell lines (F9, PCC4 and PCC3), teratocarcinoma-derived endodermal cells (PYS) and fibroblasts (line 3/A/1-D-3 differentiated from PCC3)) were compared by two dimensional polyacrylamide gel electrophoresis after selective iodination with ¹²⁵I in the presence of lactoperoxidase. The labeled proteins were solubilized either in Nonidet P40/urea/ampholyte/mercaptoethanol solution or in Nonidet P40 only. In total, about thirty major ¹²⁵I-labeled surface proteins were identified by their isoelectric point and molecular weight. 14 proteins are present in all five cell types, although their quantity or accessibility for labeling differs between differentiated and undifferentiated cells. Three proteins (200, 160 and 150 kilodaltons) are present in undifferentiated cells only. Two of them (160 and 150 kilodaltons) were solubilized by Nonidet P40/urea/ampholyte/mercaptoethanol, but not by Nonidet P40. One protein (50 kilodaltons) was found in nullipotent F9 cells only. About 14–15 proteins (including fibronectin) were released by Nonidet P40/urea/ampholyte/mercaptoethanol but not by Nonidet P40. They are presumably bound to submembrane or cytoskeleton structures by non-covalent bonds.     

Minor histocompatibility antigens are developmentally regulated on murine embryonal carcinoma cells and their early differentiated derivatives

Differences in the expression of minor histocompatibility (Hm) alloantigens on two mouse embryonal carcinoma (EC) cell lines and the PYS-2 and T.D.M.-1 differentiated derivatives have been demonstrated by their ability to elicit a cytolytic T lymphocyte response. Experiments involving the use of various responder-target strain combinations and recombinant inbred mice strains have shown that: (1) there are major differences in Hm expression on EC cells compared with differentiated derivatives whose Hm expression appears more like that of adult splenocytes; (2) although both EC cell lines show reduced Hm immunogenicity compared with adult splenocytes, major differences in the expression and possible presentation of Hm between the F9 and PCC3 EC cell lines can be detected by in vivo priming and by in vitro cold competition target experiments. These observations are discussed in relation to the differences in allograft rejection patterns observed with PCC3 and F9 and to possible differences in developmental staging of these cell lines.     

Plasma membrane proteomics of human embryonic stem cells and human embryonal carcinoma cells

Human embryonic stem cells (hESCs) are of immense interest in regenerative medicine as they can self-renew indefinitely and can give rise to any adult cell type. Human embryonal carcinoma cells (hECCs) are the malignant counterparts of hESCs found in testis tumors. hESCs that have acquired chromosomal abnormalities in culture are essentially indistinguishable from hECC. Direct comparison of karyotypically normal hESCs with hECCs could lead to understanding differences between their mechanisms of growth control and contribute to implementing safe therapeutic use of stem cells without the development of germ cell cancer. While several comparisons of hECCs and hESCs have been reported, their cell surface proteomes are largely unknown, partly because plasma membrane proteomics is still a major challenge. Here, we present a strategy for the identification of plasma membrane proteins that has been optimized for application to the relatively small numbers of stem cells normally available, and that does not require tedious cell fractionation. The method led to the identification of 237 and 219 specific plasma membrane proteins in the hESC line HUES-7 and the hECC line NT2/D1, respectively. In addition to known stemness-associated cell surface markers like ALP, CD9, and CTNNB, a large number of receptors, transporters, signal transducers, and cell-cell adhesion proteins were identified. Our study revealed that several Hedgehog and Wnt pathway members are differentially expressed in hESCs and hECCs including NPC1, FZD2, FZD6, FZD7, LRP6, and SEMA4D, which play a pivotal role in stem cell self-renewal and cancer growth. Various proteins encoded on chromosome 12p, duplicated in testicular cancer, were uniquely identified in hECCs. These included GAPDH, LDHB, YARS2, CLSTN3, CSDA, LRP6, NDUFA9, and NOL1, which are known to be upregulated in testicular cancer. Distinct HLA molecules were revealed on the surface of hESCs and hECCs, despite their low abundance. Results were compared with genomic and proteomic data sets reported previously for mouse ESCs, hECCs, and germ cell tumors. Our data provides a surface signature for HUES-7 and NT2/D1 cells and distinguishes normal hESCs from hECCs, helping explain their 'benign' versus 'malignant' nature.     

Expression of the Duchenne muscular dystrophy gene products in embryonic stem cells and their differentiated derivatives.

Three protein products of the Duchenne muscular dystrophy (DMD) gene were identified so far. These include the two very similar muscle and brain type dystrophins, which are encoded by 14-kilobase (kb) mRNAs, and Dp71, which is much smaller. Dp71 is encoded by a 6.5-kb mRNA, which is transcribed from approximately 6% of the giant dystrophin gene. The present investigation shows that Dp71 is the first product of the DMD gene detectable during development. It is already expressed in the pluripotent embryonic stem cells. The two 14-kb mRNAs encoding the dystrophins are detectable only after differentiation of specialized cell types. The possible implication of these findings with regard to the ontogenetic activation and the evolution of the DMD gene are discussed.     

Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells.

In a previous paper, we have shown that in the absence of stress, mouse embryonal carcinoma cells, like mouse early embryo multipotent cells, synthesize high levels of 89- and 70-kilodalton heat shock proteins (HSP)(O. Bensaude and M. Morange, EMBO J. 2:173-177, 1983). We report here the pattern of proteins synthesized after a short period of hyperthermia in various mouse embryonal carcinoma cell lines and early mouse embryo cells. Among the various cell lines tested, two of them, PCC4-Aza R1 and PCC7-S-1009, showed an unusual response in that stimulation of HSP synthesis was not observed in these cells after hyperthermia. However, inducibility of 68- and 105-kilodalton HSP can be restored in PCC7-S-1009 cells after in vitro differentiation triggered by retinoic acid. Similarly, in the early mouse embryo, hyperthermia does not induce the synthesis of nonconstitutive HSP at the eight-cell stage, but induction of the 68-kilodalton HSP does occur at the blastocyst stage. Such a transition in the expression of HSP has already been described for Drosophila melanogaster and sea urchin embryos and recently for mouse embryos. It may be a general property of early embryonic cells.     

Expression of c-myb in embryonal carcinoma cells and embryonal stem cells

Mouse c-myb has been implicated in the regulation of differentiation and proliferation of haematopoietic cells. Analysis of the chromatin structure of the promoter region of c-myb in embryonal carcinoma (EC) cells and embryonal stem (ES) cells reveals a DNAse I-hypersensitive site coincident with a site found in c-myb-expressing haematopoietic cells, but absent in murine fibroblasts (which do not express c-myb). EC and ES cells were found to express c-myb mRNA, albeit at a level lower than found in haematopoietic cells. Differentiation of ES cells into embryoid bodies resulted in an elevated level of c-myb expression.     

Octamer-dependent regulation of the kFGF gene in embryonal carcinoma and embryonic stem cells.

Expression of kFGF, which belongs to the family of fibroblast growth factor genes, is restricted to undifferentiated embryonal carcinoma and embryonic stem cells. Stem cell specific expression of kFGF is controlled by a distally localized enhancer, conferring both positive and negative regulation to the kFGF and tk promoters. This enhancer contains a consensus octamer binding sequence that controls positive regulation in EC and ES cells. The octamer sequence binds Oct1 and Oct4 in nuclear extracts from undifferentiated EC cells, while only Oct1 is bound in nuclear extracts from RA differentiated cells. These results suggest that the kFGF gene is a target for positive regulation by Oct4 and implicate Oct4 as target for regulation by the retinoic acid receptors.     

Interactions between diploid embryonal carcinoma cells and early embryonic cells

Two diploid embryonal carcinoma (EC) cell lines, P10 and P19, differ in their response to the embryonic environment. P10 produces mostly normal chimeras following injection into blastocysts, whereas P19 produces mostly abnormal chimeras. In this study, P10 cells were aggregated with morulae, and all resulting fetuses were chimeric with very large contributions from the EC cells. However, all embryos were abnormal. Following aggregation of P19 cells with morulae, very few embryos were recovered and they were all non-chimeric. Both P10 and P19 were capable of forming functional gap junctions with morula cells and with the ICM of the blastocyst but not with trophoblast, showing that differences in the ability to make junctional contact with the embryo cannot explain the differences between the two cell lines.     

DNA repair in murine embryonic stem cells and differentiated cells

Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.     

Two specific markers for neural differentiation of embryonal carcinoma cells

Two multipotential embryonal carcinoma (EC) cell lines, 1003 and 1009, can be induced to form preferentially neural derivatives in vitro. Synthesis of specific proteins during neural differentiation was followed by two-dimensional gel electrophoresis. The comparison of protein patterns obtained with neural and non-neural derivatives of these EC cell lines indicates that two changes are specific for the neural pathway: (i) the appearance of a new beta-tubulin isoform and (ii) the accumulation of the brain isozyme of creatine phosphokinase already present in small amounts in EC stem cells. These changes were found to take place early in the course of differentiation and to occur even when neurite outgrowth was prevented.     

Production of PDGF-like growth factors by embryonal carcinoma cells and binding of PDGF to their endoderm-like differentiated cells

In this report, we demonstrate that F9 and PC-13 embryonal carcinoma (EC) cells do not bind significant amounts of platelet-derived growth factor (PDGF), whereas the endoderm-like differentiated cells derived from EC cells do. The F9-differentiated cells exhibit approximately 8300 receptors per cell, with an apparent dissociation constant of 30 pM. Two endoderm-like cell lines, PSA-5E and PYS-2, also bind PDGF and exhibit approximately 4800 and 23,500 receptors per cell, respectively. The lack of PDGF binding by the parental EC cells is consistent with their release of a factor(s) that is closely related to PDGF. This factor(s) competes with PDGF for binding to membrane receptors and is recognized by antibodies raised against PDGF. However, this factor(s) does not appear to be antigenically identical to PDGF. We also show that production of this PDGF-like factor(s) is reduced more than 90% when F9 EC cells differentiate into cells that bind PDGF. Thus, our findings indicate that EC cells release a factor(s) that should be capable of binding to their differentiated cells. This raises the possibility that PDGF, or a closely related factor, can influence cell proliferation and/or cell behavior of early embryonic cells.     

Production and utilization of growth factors related to fibroblast growth factor by embryonal carcinoma cells and their differentiated cells

Previous studies have established that embryonal carcinoma (EC) cells produce several different growth factors, but express few, if any, receptors for epidermal growth factor, platelet-derived growth factor, or transforming growth factor type-beta. In this study, the production and utilization of fibroblast growth factor (FGF) by EC cells and their differentiated cells were investigated. We have determined that EC cells produce a heat-labile, heparin-binding factor that competes with FGF for binding to membrane receptors and appears to be immunologically related to FGF. The same or a similar factor is produced by three different EC cell lines, including a multipotent human EC cell line. However, production of this factor is apparently reduced when each EC cell line differentiates. Unlike the parental EC cells, the differentiated cells respond to FGF by growth stimulation and the growth responses to FGF correlate with increased binding of FGF. Although the binding data indicate that both the EC cells and their differentiated cells exhibit high affinity receptors for FGF, the differentiated cells express these receptors at levels approximately 10-fold higher. These findings suggest that the FGF-related growth factor could influence the growth of EC cells or their differentiated cells.     

Use of lectins for probing differentiated human embryonic stem cells for carbohydrates

The carbohydrates present on the surface of differentiated human embryonic stem cells (hESCs) are not yet well established. Here, we have employed a panel of lectins and several anti-carbohydrate antibodies to determine the carbohydrates that are present at day 12 of hESC differentiation as embryoid bodies (EBs). On the basis of staining with fluorescein-labeled lectins, we have determined the presence of both terminal and internally linked alpha-d-mannopyranosyl groups, poly-N-acetyllactosaminyl chains, both alpha2,3- and alpha2,6-linked N-acetylneuraminic acid (Neu5Ac), alpha1,6-linked l-fucosyl, and beta-D-galactosyl groups, and more specifically, the T, Tn, and sialyl-Tn antigens. However, no alpha1,2-linked l-fucosyl, terminal nonreducing alpha-D-galactosyl, N-acetyl-beta-D-glucosaminyl, nor N-acetyl-alpha-D-galactosaminyl groups were found by this approach. We also established the presence of Neu5Acalpha2,3/2,6-Galbeta1,4 GlcNAc-terminated chains on the surfaces of 12-day-old EBs, as indicated by the great enhancement of staining by Erythrina cristagalli agglutinin (ECA) after treatment with neuraminidase. In each case, inhibition of binding by a haptenic sugar or treatment with neuraminidase was used to eliminate the possibility of nonspecific binding of the lectins. A comparison with undifferentiated cell staining revealed an increase in alpha2,3-linked Neu5Ac as well as a change to exclusively alpha1,6-linked l-fucose upon differentiation.     

Presynaptic dopaminergic properties of differentiated mouse embryonic stem cells

This study characterized the presynaptic dopaminergic properties of neuronally differentiated mouse embryonic stem (ES) cells. Approximately 30% of the ES cells expressed tyrosine hydroxylase (TH) immunoreactivity when co-cultured with PA6 cells. These cultures expressed high affinity, sodium-dependent dopamine uptake as well as depolarization-induced and calcium-dependent dopamine release of this transmitter. These and other important dopaminergic genes found expressed in these cultures by RT-PCR included Nurr1, vesicular monoamine transporter 2 (VMAT2), TH, dopamine transporter (DAT), and glial cell line-derived neurotrophic factor (GDNF) receptors c-Ret and GFRalpha1. These results demonstrate that differentiated ES cells have the presynaptic functions for maintaining dopaminergic homeostasis, which may be essential for their long-term use in restoring CNS levels of this transmitter.     

High expression of stathmin in multipotential teratocarcinoma and normal embryonic cells versus their early differentiated derivatives

Stathmin is a ubiquitous cytoplasmic protein, phosphorylated in response to agents regulating the proliferation, the differentiation and the specialized functions of cells, in a way possibly integrating the actions of diverse concomitant regulatory signals. Its expression is also regulated in relation with cell proliferation and differentiation and reaches a peak at the neonatal stage. To assess the possible role of stathmin at earlier stages of development, we examined its expression and regulation in embryonal carcinoma (EC) and derived cell lines as well as in the early mouse embryo. Interestingly, stathmin is highly abundant in the undifferentiated, multipotential cells of the F9, 1003 and 1009 EC cell lines. Its high expression markedly decreased, both at the protein and mRNA levels, when F9 cells were induced to differentiate into endodermal-like cells with retinoic acid and dibutyryl-cAMP. Stathmin was also much less abundant in differentiated cell lines such as the trophectodermal line TDM-1, as well as in several F9- and 1003-derived cell lines committed to differentiate towards the mesodermal and neuroectodermal lineages but still proliferating. Therefore, the observed decrease of stathmin expression is not related to the reduced proliferation rate but rather to the differentiation of the multipotential EC cells. The immunocytochemical pattern of stathmin expression during early mouse development indicated that stathmin is also highly abundant in the multipotential cells of the inner cell mass of the blastula, whereas it is much lower in the differentiated trophectodermal cells. These results confirm the physiological relevance of the observations with EC cells, and suggest that stathmin, in addition to its high expression at later stages of development and in the adult nervous system, may be considered as a new marker of the multipotential cells of the early mouse embryo.