p53 is required for etoposide-induced apoptosis of human embryonic stem cells.

The molecular mechanisms controlling DNA-damage-induced apoptosis of human embryonic stem cells (hESC) are poorly understood. Here we investigate the role of p53 in etoposide-induced apoptosis. We show that p53 is constitutively expressed at high levels in the cytoplasm of hESC. Etoposide treatment results in a rapid and extensive induction of apoptosis and leads to a further increase in p53 and PUMA expression as well as Bax processing. p53 both translocates to the nucleus and associates with the mitochondria, accompanied by colocalization of Bax with Mcl1. hESC stably transduced with p53 shRNA display 80% reduction of endogenous p53 and exhibit an 80% reduction in etoposide-induced apoptosis accompanied by constitutive downregulation of Bax and an attenuated upregulation of PUMA. Our data further show that undifferentiated hESC that express Oct4 are much more sensitive to etoposide-induced apoptosis than their more differentiated progeny. Our study demonstrates that p53 is required for etoposide-induced apoptosis of hESC and reveals, at least in part, the molecular mechanism of DNA-damage-induced apoptosis in hESC.

Neural progenitors from human embryonic stem cells.

The derivation of neural progenitor cells from human embryonic stem (ES) cells is of value both in the study of early human neurogenesis and in the creation of an unlimited source of donor cells for neural transplantation therapy. Here we report the generation of enriched and expandable preparations of proliferating neural progenitors from human ES cells. The neural progenitors could differentiate in vitro into the three neural lineages--astrocytes, oligodendrocytes, and mature neurons. When human neural progenitors were transplanted into the ventricles of newborn mouse brains, they incorporated in large numbers into the host brain parenchyma, demonstrated widespread distribution, and differentiated into progeny of the three neural lineages. The transplanted cells migrated along established brain migratory tracks in the host brain and differentiated in a region-specific manner, indicating that they could respond to local cues and participate in the processes of host brain development. Our observations set the stage for future developments that may allow the use of human ES cells for the treatment of neurological disorders.

Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method.

BACKGROUND: Human embryonic stem (ES) cells originate from the inner cell mass of the blastocyst, and retain in culture the properties of pluripotent cells of the early embryo. The study aim was to determine whether the open pulled straw (OPS) vitrification method, which is highly effective for the cryopreservation of embryos, might be also efficient for human ES cells. METHODS AND RESULTS: All human ES cell clumps that were vitrified by the OPS method could be recovered upon thawing, and gave rise to ES cell colonies after plating. Vitrified colonies were significantly smaller and showed an increased level of background differentiation compared with control colonies. However, these unwanted effects could be overcome by additional cultivation of the colonies for 1 and 2 days respectively. The vitrified human ES cells were cultivated for prolonged periods and retained the properties of pluripotent cells, including a normal karyotype, expression of the transcription factor Oct-4 and surface markers that are characteristic to human ES cells. When grafted into SCID mice, the vitrified cells gave rise to teratomas containing derivatives of all three embryonic germ layers. CONCLUSIONS: Vitrification by the OPS method is reliable and effective for the cryopreservation of human pluripotent embryonic stem cells.

Scientific considerations relating to the ethics of the use of human embryonic stem cells in research and medicine.

The recent development of embryonic stem (ES) cells from human blastocysts has the potential to revolutionize many of our approaches to human biology and medicine. Continued objection to the use of human ES cells on ethical grounds may inhibit progress or defer this opportunity indefinitely. It is essential that the ethical discussion proceed on a sound scientific basis. The ethical controversy surrounding human ES cells concerns their origin from human blastocysts and the perception of their developmental potential. It is likely that the worldwide requirement for human ES cells will be met by the development of a small number of cell lines, as has been the case in the mouse; current rates of success for human ES cell establishment suggest that only a modest number of embryos will be required to achieve this goal. It is in the public interest that human ES cell lines be derived under circumstances that will enable their widespread distribution with minimum encumbrances to academic researchers throughout the world. In considering the developmental potential of ES cells, an important distinction exists between pluripotentiality, or the ability to develop into a wide range of somatic and extraembryonic tissues, and totipotentiality, the ability of a cell or collection of cells to give rise to a new individual given adequate maternal support. There is no evidence that ES cells from any species can give rise to a new individual except when combined with cells which are the immediate progeny of a zygote. These developmental limitations of ES cells appear to relate to their inability to undergo axis formation and to generate the body plan. Alternatives to blastocyst-derived ES cells include embryonic germ cells, adult tissue stem cells, transdetermination of committed somatic cells, and therapeutic cloning. These research areas are complimentary and synergistic to ES cell research and it is premature and counterproductive to suggest that one avenue should be pursued in preference to another. The combination of cloning and ES cell technology has the potential to address many important issues in transplantation medicine and research, but a better understanding of the reprogramming of somatic cells is required before we can regard ES cells derived from normal and nuclear transfer blastocysts as equivalent.

Human pluripotent stem cells: a progress report.

The derivation of diploid human pluripotent stem cell lines from either human blastocysts or embryonic gonads in 1998 attracted a great deal of interest because of the widespread potential applications of these cells in research and in regenerative medicine. Since the initial reports, there has been some progress in the characterisation of blastocyst-derived stem cells, and some technical advances in their manipulation. Conditions for differentiation in vitro of pluripotent stem cells from either blastocysts or gonads have been defined. In some studies, committed progenitor cell populations have been isolated from mixed cultures of differentiating ES cells.

An orthotopic xenograft model of human nonseminomatous germ cell tumour.

We have established the first example of an orthotopic xenograft model of human nonseminomatous germ cell tumour (NSGCT). This reproducible model exhibits many clinically relevant features including metastases to the retroperitoneal lymph nodes and lungs, making it an ideal tool for research into the development and progression of testicular germ cell tumours.

Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei.

Pluripotent human stem cells isolated from early embryos represent a potentially unlimited source of many different cell types for cell-based gene and tissue therapies [1-3]. Nevertheless, if the full potential of cell lines derived from donor embryos is to be realised, the problem of donor-recipient tissue matching needs to be overcome. One approach, which avoids the problem of transplant rejection, would be to establish stem cell lines from the patient's own cells through therapeutic cloning [3,4]. Recent studies have shown that it is possible to transfer the nucleus from an adult somatic cell to an unfertilised oocyte that is devoid of maternal chromosomes, and achieve embryonic development under the control of the transferred nucleus [5-7]. Stem cells isolated from such a cloned embryo would be genetically identical to the patient and pose no risk of immune rejection. Here, we report the isolation of pluripotent murine stem cells from reprogrammed adult somatic cell nuclei. Embryos were generated by direct injection of mechanically isolated cumulus cell nuclei into mature oocytes. Embryonic stem (ES) cells isolated from cumulus-cell-derived blastocysts displayed the characteristic morphology and marker expression of conventional ES cells and underwent extensive differentiation into all three embryonic germ layers (endoderm, mesoderm and ectoderm) in tumours and in chimaeric foetuses and pups. The ES cells were also shown to differentiate readily into neurons and muscle in culture. This study shows that pluripotent stem cells can be derived from nuclei of terminally differentiated adult somatic cells and offers a model system for the development of therapies that rely on autologous, human pluripotent stem cells.

Localization, expression and genomic structure of the gene encoding the human serine protease testisin.

Testisin is a recently identified human serine protease expressed by premeiotic testicular germ cells and is a candidate tumor suppressor for testicular cancer. Here, we report the characterization of the gene encoding testisin, designated PRSS21, and its localization on the short arm of human chromosome 16 (16p13.3) between the microsatellite marker D16S246 and the radiation hybrid breakpoint CY23HA. We have further refined the localization to cosmid 406D6 in this interval and have established that the gene is approximately 4. 5 kb in length, and contains six exons and five intervening introns. The structure of PRSS21 is very similar to the human prostasin gene (PRSS8) which maps nearby on 16p11.2, suggesting that these genes may have evolved through gene duplication. Sequence analysis showed that the two known isoforms of testisin are generated by alternative pre-mRNA splicing. A major transcription initiation site was identified 97 nucleotides upstream of the testisin translation start and conforms to a consensus initiator element. The region surrounding the transcription initiation site lacks a TATA consensus sequence, but contains a CCAAT sequence and includes a CpG island. The 5'-flanking region contains several consensus response elements including Sp1, AP1 and several testis-specific elements. Analysis of testisin gene expression in tumor cell lines shows that testisin is not expressed in testicular tumor cells but is aberrantly expressed in some tumor cell lines of non-testis origin. These data provide the basis for identifying potential genetic alterations of PRSS21 that may underlie both testicular abnormalities and tumorigenesis.

Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.

We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines give rise to teratomas containing derivatives of all three embryonic germ layers. Both cell lines differentiate in vitro into extraembryonic and somatic cell lineages. Neural progenitor cells may be isolated from differentiating ES cell cultures and induced to form mature neurons. Embryonic stem cells provide a model to study early human embryology, an investigational tool for discovery of novel growth factors and medicines, and a potential source of cells for use in transplantation therapy.

Human embryonic stem cells.

Embryonic stem (ES) cells are cells derived from the early embryo that can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent; they share these properties with embryonic germ (EG) cells. Candidate ES and EG cell lines from the human blastocyst and embryonic gonad can differentiate into multiple types of somatic cell. The phenotype of the blastocyst-derived cell lines is very similar to that of monkey ES cells and pluripotent human embryonal carcinoma cells, but differs from that of mouse ES cells or the human germ-cell-derived stem cells. Although our understanding of the control of growth and differentiation of human ES cells is quite limited, it is clear that the development of these cell lines will have a widespread impact on biomedical research.

Human germ cell tumor cell lines express novel leukemia inhibitory factor transcripts encoding differentially localized proteins.

The polyfunctional cytokine leukemia inhibitory factor (LIF) has been implicated in the maintenance of many stem and progenitor cell populations and as an autocrine growth factor for many tumor cell populations, including germ cell tumors. Studies of LIF transcript expression in germ cell tumor cell lines identified two novel human LIF transcripts, hLIF-M and hLIF-T, containing noncoding alternate first exons that are conserved among all reported LIF genes. Embryonal carcinoma (EC) cell lines expressed these transcripts at consistent levels and hLIF-M was generally the predominant LIF transcript in these cells. This expression pattern was characteristic of EC cells since variable independently regulated expression of these transcripts was evident in other cell lines. Overexpression analysis demonstrated that each alternate hLIF transcript generated different levels of extracellular LIF activity as a consequence of the translation of distinct but partially overlapping sets of proteins. Secreted LIF proteins translated from alternate initiation codons were expressed from the hLIF-D and hLIF-M transcripts. Intracellular, potentially cell-autonomous, proteins were encoded by the hLIF-M and hLIF-T transcripts. Since EC cell lines also expressed LIF receptor transcripts, the novel LIF transcription profiles and proteins identified here suggest a role for autocrine and/or cell-autonomous LIF signaling during germ cell tumorigenesis.

CD30 and its ligand: possible role in regulation of teratoma stem cells.

Like the oocyte, the cells of the early embryo, and primordial germ cells, human teratocarcinoma stem cells are pluripotent, capable of giving rise to a wide range of somatic and extraembryonic tissues. Growth factors which regulate the growth of multipotent stem cells in the mouse have been identified, but none of these have been shown conclusively to have similar effects on human or primate multipotent stem cells. CD30 is a member of the tumour necrosis factor receptor superfamily with a restricted pattern of tissue distribution, limited to immune cells, decidual tissue, and human embryonal carcinoma: in common with other embryonal carcinoma markers, CD30 is found in foci of cells in a sub-population of seminomas. CD30 ligand is a transmembrane protein, structurally related to tumour necrosis superfamily members TNF alpha, TNF beta, and CD40. CD30 ligand is expressed by T and B lymphocytes, macrophages, and a variety of normal haematopoietic cells and tumours derived from them, and exerts pleiotropic effects on normal and malignant lymphoid cells, including death, differentiation, or cell division. Studies on cultured cell lines derived from human embryonal carcinomas and yolk sac carcinomas confirm CD30 expression in the former but not the latter, and show that CD30 expression is down-regulated during stem cell differentiation in vitro. Transcripts for CD30 ligand are found at highest levels in yolk sac carcinoma cell lines, but are also found in embryonal carcinoma. CD30 ligand protein is detected in yolk sac carcinoma and nullipotent embryonal carcinoma cell lines. Exogenous CD30 ligand has no effect on multipotent human stem cell growth in vitro. However, the receptor-ligand pair may function in autocrine regulation of embryonal carcinoma stem cells. CD30 and its ligand are candidate stem cell identity factors, juxtacrine regulators whose sole function is to identify a cell's position in a developmental hierarchy.

Human growth-differentiation factor 3 (hGDF3): developmental regulation in human teratocarcinoma cell lines and expression in primary testicular germ cell tumours.

We describe the cloning and initial characterization of a novel cDNA from human embryonal carcinoma (EC) cells. This cDNA, which we named human growth differentiation factor 3 (hGDF3), encodes the homologue of mouse GDF3, a TGFbeta superfamily member belonging to the Growth/Differentiation Factors. We have analysed the expression of hGDF3 in human embryonal carcinoma cell lines and in primary testicular germ cell tumours of adolescents and adults (TGCTs). Expression of hGDF3 in human EC cell lines is stem cell-specific, is down-regulated upon RA-mediated differentiation and is increased upon culture of the cells in the presence of activin A. In TGCTs, hGDF3 expression is low in seminomas, while expression in non-seminomas is readily detectable and appears to be associated with the EC and yolk sac components in the tumours. We have also mapped the hGDF3 locus to the short arm of human chromosome 12, a region consistently overrepresented in human testicular germ cell tumours. Thus, hGDF3 represents an embryonal carcinoma stem cell-associated marker both in vitro and in vivo.

Differentiation of human pluripotent teratocarcinoma stem cells induced by bone morphogenetic protein-2.

Pluripotent human teratocarcinoma stem cells cultured in vitro provide a resource for the study of early embryonic development in man, as well as a means for discovery of novel factors controlling cell differentiation and commitment. We previously reported that the human teratocarcinoma stem cell line GCT 27X-1 could be induced to differentiate into an endodermal progenitor cell by treatment with high doses of retinoic acid. A search for polypeptide inducers of differentiation in this system has identified bone morphogenetic protein-2 (BMP-2) as a potent inducer of differentiation. In cell line GCT 27X-1, treatment with BMP-2 reduces proliferation, induces morphological changes similar to obtained following treatment with retinoic acid, and causes a decrease in the expression of transcripts for the stem cell markers CD30 and Oct-4. Preliminary immunochemical studies indicate that the differentiated cells produced by BMP-2 are endodermal precursors with a pattern of marker expression similar to that found in retinoic acid treated cells. Models of endoderm differentiation in humans will be useful for identifying the molecules which mediate cell interactions in development, and in achieving directed differentiation of cells for use in transplantation.

Expression of CD30 and CD30 ligand in cultured cell lines from human germ-cell tumors.

Embryonal carcinoma (EC) cells are the multipotent stem cells of human teratocarcinomas. EC cell lines isolated from human testicular teratocarcinomas retain pluripotentiality and show cell-surface marker phenotype and growth characteristics similar to those of primate embryonic stem cells. CD30, a member of the tumor necrosis factor superfamily of cytokine receptors, is expressed on the surface of human EC cells, but little is known regarding its function or the expression of its ligand in this context. Northern blot analysis, reverse transcriptase-PCR, and immunoprecipitation were used to study the expression of CD30 ligand and CD30 in cell lines representing multipotent and nullipotent EC as well as yolk sac carcinoma. Antibodies, recombinant CD30-Fc, and recombinant CD30 ligand were used in bioassays to assess the ability of exogenous CD30 ligand to promote the growth of multipotent human EC stem cells. Nullipotent and multipotent human EC cells, but not yolk sac carcinoma, expressed surface CD30, as demonstrated by immunoblotting and immunofluorescence; in multipotent cells, expression was decreased during differentiation induced by retinoic acid. Transcripts for CD30L were found at high levels in a yolk sac carcinoma cell line by Northern analysis and reverse transcriptase-PCR and were present at lower levels in EC cells as well. Immunoprecipitation confirmed expression of CD30 ligand protein in yolk sac carcinoma and in nullipotent embryonal carcinoma. Antibodies with CD30 cross-linking activity failed to promote the growth of multipotent EC cell line GCT 27 X-1. Anti-CD30 ligand antibodies and CD30-Fc both failed to block the promotion of EC cell growth induced by yolk-sac cell culture supernatants or feeder-cell monolayers. CD30 ligand is expressed in cell lines representative of human yolk sac carcinoma and nullipotent embryonal carcinoma. The membrane-bound cytokine may have an autocrine role in human EC stem-cell renewal, but other exogenous factors are required to maintain the growth of multipotent EC cells in vitro.

Exceptional sensitivity of testicular germ cell tumour cell lines to the new anti-cancer agent, temozolomide.

Metastatic testicular germ cell tumours are cured in approximately 85% of patients using cisplatin-based combination chemotherapy. Patients who fail to respond have a poor prognosis, and there is a need for more effective treatments for cisplatin-resistant disease. In this study, it is shown that two of four cell lines derived from human non-seminomatous testicular germ cell tumours are exceptionally sensitive to temozolomide, a new imidazotetrazine which can cross the blood-brain barrier in mice. In addition, three pairs of cisplatin-resistant sublines show little cross-resistance to temozolomide. These data suggest that temozolomide might have activity against non-seminomatous testicular germ cell tumours which have relapsed following cisplatin-containing chemotherapy, and could have a role in the treatment of patients with metastatic lesions in the brain.

Hepatocytic transcription factor expression in human embryonal carcinoma and yolk sac carcinoma cell lines: expression of HNF-3 alpha in models of early endodermal cell differentiation.

Retinoic acid treatment of the multipotent human embryonal carcinoma cell line GCT 27 X-1 induced differentiation into an epithelial cell type which morphologically resembled rodent visceral endoderm in vitro. The differentiated cells expressed some markers characteristic of yolk sac, such as cytokeratin 19 and extracellular matrix proteins, but none of the secreted serum proteins produced by normal yolk sac or liver. HNF-3 alpha expression increased upon retinoic acid-induced GCT 27 X-1 differentiation. This increase in HNF-3 alpha expression may characterize an early stage in the differentiation of extraembryonic endoderm. In support of this hypothesis, we found that a yolk sac carcinoma cell line with a phenotype similar to rodent visceral endoderm expressed transcripts for HNF-1 alpha, -1 beta, -3 alpha, -3 beta, -3 gamma, and 4, whereas yolk sac carcinoma cell lines resembling rodent parietal endoderm expressed transcripts for HNF-3 alpha. The results support the conclusion that hepatocytic transcription factors, particularly HNF-3 alpha, may play an important role in early endodermal differentiation in man.

Glycolipids of germ cell tumors: extended globo-series glycolipids are a hallmark of human embryonal carcinoma cells.

Glycolipids of human germ cell tumor lines were analyzed to define the most common immunohistochemical profiles of embryonal carcinoma (EC), differentiated derivatives of EC, yolk sac carcinoma (YC) and choriocarcinoma (CC). Glycolipid composition was examined by high-performance thin-layer chromatography (HPTLC) combined with immunostaining with a panel of anti-carbohydrate monoclonal antibodies (MAbs). All EC cell lines were found to contain high levels of globo-series glycolipids, including globotriosylceramide (Gb3), globoside (Gb4), Gb5 (Gal beta 1-->3Gb4) and GL7 (sialyl Gal beta 1-->3Gb4). Somatic differentiated derivatives (e.g., EC cells treated with retinoic acid) contained decreased levels of globo-series glycolipids and increased levels of lacto- and ganglio-series glycolipids, including GD3, GT3 and GD2. CC cell lines contained relatively large amounts of Gb3 but did not contain extended globo-series glycolipids Gb5 and GL7. CC cell lines also contained a macroglycolipid reactive with the antibody to SSEA-1 (Lex). Glycolipids were not detected in two YC cell lines, while other YC cell lines contained globo-series core glycolipids (Gb3 and Gb4) and gangliosides. We conclude that EC, YC and CC have distinct patterns of membrane glycolipid expression that can be identified by HPTLC and immunostaining. Our results indicate that globo-series glycolipids Gb5 and GL7, which carry stage-specific embryonic antigens 3 and 4 (SSEA-3 and SSEA-4), are a hallmark of human EC cells. Cell lines derived from human germ cell tumors that do not express Gb5 and GL7 deserve to be re-evaluated, since they may represent different stem cells, most likely equivalent to somatic cells and their developmentally committed precursors (e.g., neuroblasts).

Cultured cell lines from human teratomas: windows into tumour growth and differentiation and early human development.

Cultured cell lines from human teratomas provide models to study molecules which might regulate tumour growth or normal embryonic development. Here, we describe efforts to delineate the role of transcription factors, extracellular matrix, and growth factors in controlling growth and differentiation of human embryonal carcinoma and yolk sac tumour.

A novel keratan sulphate proteoglycan from a human embryonal carcinoma cell line.

We describe here the purification and partial characterization of a 200 kDa keratan sulphate proteoglycan found in the pericellular matrix of human embryonal carcinoma cells. Previously we have shown that this molecule is recognized by a monoclonal antibody (GCTM-2). The antigen was isolated using ion-exchange chromatography and gel filtration, purification being monitored by e.l.i.s.a. using GCTM-2. Metabolic labelling of GCT 27 C-4 embryonal carcinoma cells with sodium [35S]sulphate resulted in the incorporation of [35S]sulphate into the purified molecule. Throughout the purification procedure, the peaks of 35S radioactivity were coincident with the peaks of immunoreactivity, and this label was released both by digestion with keratanase and chondroitinase, confirming the proteoglycan nature of the antigen. The intact molecule ran as a single broad band of 200 kDa, which has been identified by silver staining and immunoblotting following gel electrophoresis. Amino acid analysis of the purified antigen indicated a high content of serine, glycine and aspartic acid/asparagine residues. Visualization by rotary-shadowing electron microscopy suggests that the purified material forms large aggregates, even under denaturing conditions. Deglycosylation of this preparation with trifluoromethanesulphonic acid yielded a major band of 55 kDa and a minor band of 48 kDa. The biochemical nature of the molecule described here, along with tissue distribution studies using GCTM-2, indicates that the antigen is not related to previously described keratan sulphate proteoglycans.