Maja Vlahović

Teratoma: from spontaneous tumors to the pluripotency/malignancy assay

A teratoma is a benign tumor containing a mixture of differentiated tissues and organotypic derivatives of the three germ layers, while a teratocarcinoma also contains embryonal carcinoma cells (EC cells). Experimental teratomas and teratocarcinomas have been derived from early mammalian embryos transplanted into the adult animal (ectopic sites). In the rat, the pluripotency of the transplanted epiblast was demonstrated and a quantifiable restriction of developmental potential persisted after subsequent transplantation of chemically defined cultivated postimplantation embryos. The rat is nonpermissive for teratocarcinoma development and rat pluripotent cell lines have been established only recently. Transplantation of mouse embryos, epiblast, or embryonic stem cells (mESCs) gave rise to teratocarcinomas. The pluripotency of reprogrammed human cells has been tested by a ‘gold standard’ trilaminar teratoma assay in immunocompromised mice in vivo. Human pluripotent stem cells proposed for use in regenerative medicine such as human embryonic stem cell (hESC), human nuclear‐transfer/therapeutic cloning embryonic stem cell (NT‐ESC), or human induced pluripotent stem cell (hiPSC) lines, once differentiated in vitro to the desired cell type, should be again tested in a long‐term animal teratoma assay to exclude their malignancy. Such an approach led to a recently implemented human therapy with retinal pigmented epithelium. For greater biosafety, the teratoma assay should be standardized and complemented by assessments of mutations/epimutations, RNA/protein expression, and possible immunogenicity of autologous pluripotent cells. Furthermore, the standardized teratoma assay should be directed more to the assessment of EC/malignant cell features than of differentiated tissues, especially after a unique case of human therapy with neural stem cells was found to lead to malignancy. WIREs Dev Biol 2016, 5:186–209. doi: 10.1002/wdev.219

Chemically Defined Protein-Free in vitro Culture of Mammalian Embryo Does Not Restrict Its Developmental Potential for Differentiation of Skin Appendages

In a unique serum- and protein-free chemically defined in vitro culture model of postimplantation mammalian development the epidermis differentiates regularly, although the differentiation of other tissues is impaired due to the lack of the serum. The present study in that model was done to estimate more carefully the degree of epidermal differentiation in defined media supplemented with some growth- or differentiation-stimulating substances. The main objective was to discover by grafting in vivo to the richer environment whether simple protein-free culture conditions restrict an inherent embryonic potential for differentiation of skin appendages. Embryonic parts of E9.5 gastrulating Fischer rat embryos were cultivated for 2 weeks in the protein-free Eagle’s minimum essential medium supplemented with holotransferrin, apotransferrin, insulin and/or Na2SeO3 and in controls cultivated in protein-free medium or in serum-supplemented medium. In all experiments there was a high incidence of differentiation of the epidermis. A high level of epidermal differentiation was confirmed for the first time at the ultrastructural level. A well-differentiated cornified layer and cells connected with desmosomes containing keratohyaline masses and cytokeratin filaments were found. A strong immunohistochemical signal for the proliferating cell nuclear antigen was always detected in the basal layer of the epidermis showing that those cells were still able to proliferate. Finally, embryos precultivated for 1 or 2 weeks in the protein-free medium and media supplemented with apotransferrin or serum were grafted under the kidney capsule for an additional 2 weeks. It was discovered that even after spending 2 weeks in the simple protein-free medium in vitro, embryos retained their developmental potential for differentiation of skin appendages (hair and sebaceous glands).

Epigenetic drug 5-azacytidine impairs proliferation of rat limb buds in an organotypic model-system in vitro.

Aim To establish an organotypic in vitro model of limb bud development to verify whether epigenetic drug and teratogen 5-azacytidine (5azaC) has an effect on limb buds independent of its effects on the placenta. Methods Fischer strain rat fore- and hindlimb buds were microsurgically isolated from 13 days old embryos and cultivated in vitro for two weeks at the air-liquid interface in Eagles minimum essential medium (MEM) with 50% rat serum. 30 µmol of 5azaC was added to the fresh medium. Overall growth was measured by an ocular micrometer. Routine histology, immunohistochemical detection of the proliferating cell nuclear antigen (PCNA), and stereological quantification of PCNA expression were performed. Results At four time points, significantly lower overall growth was detected for fore- and hindlimb bud explants cultivated with 5azaC in comparison to controls. After the culture period, numerical density of the PCNA signal for both types of limb buds was lower than for controls (P < 0.001). Limb buds were initially covered by immature epithelium and contained mesenchyme, myotubes, single hemangioblasts, hemangioblast aggregates, blood islands, and capillaries. Regardless of the treatment, cartilage and epidermis differentiated, but cells and structures typical for vasculogenesis disappeared. Conclusion Our findings, obtained outside of the maternal organism, stress the importance of compromised cell proliferation for 5azaC impact on limb buds. This investigation points to the necessity to establish alternatives to in vivo research on animals using teratogenic agents.

Influence of hyperthermal regimes on experimental teratoma development in vitro

We screened for the impact of hyperthermal regimes varying in the cumulative equivalent minutes at 43°C (CEM43°C) and media composition on tumour development using an original teratoma in vitro model. Rat embryos (three germ layers) were microsurgically isolated and cultivated at the air‐liquid interface. During a two week period, ectodermal, mesodermal and endodermal derivatives developed within trilaminar teratomas. Controls were grown at 37°C. Overall growth was measured, and teratoma survival and differentiation were histologically assessed. Cell proliferation was stereologically quantified by the volume density of Proliferating Cell Nuclear Antigen. Hyperthermia of 42°C, applied for 15 minutes after plating (CEM43°C 3.75 minutes), diminished cell proliferation (P ˂ .0001) and enhanced differentiation of both myotubes (P ˂ .01) and cylindrical epithelium (P ˂ .05). Hyperthermia of 43°C applied each day for 30 minutes during the first week (CEM43°C 210 minutes) impaired overall growth (P ˂ .01) and diminished cell proliferation (P ˂ .0001). Long‐term hyperthermia of 40.5°C applied for two weeks (CEM43°C 630 minutes) significantly impaired survival (P ˂ .005). Long‐term hyperthermia of 40.5°C applied from the second day when differentiation of tissues begins (CEM43°C 585 minutes) impaired survival (P ˂ .0001), overall growth (P ˂ .01) and cartilage differentiation (P ˂ .05). No teratomas survived extreme regimes: 43°C for 24 hours (CEM43°C 1440 minutes), hyperthermia in the scant serum‐free medium (CEM43°C 630 minutes) or treatment with an anti‐HSP70 antibody before long‐term hyperthermia 40.5°C from the second day (CEM43°C 585 minutes). This in vitro research provided novel insights into the impact of hyperthermia on the development of experimental teratomas from their undifferentiated sources and are thus of potential interest for future therapeutic strategies in corresponding in vivo models.

Epigenetics and testicular germ cell tumors

Abstract Malignant testicular germ cell tumors (TGCTs) are the most frequent testicular cancers in Caucasian males, developing at the most productive age of man. We are briefly reviewing TGCT-tumorigenesis with an emphasis on epigenetics. Epigenetic mechanisms such as DNA methylation and histone modifications together with RNA interference that all change gene expression are driving early spermatogenesis. Deregulation of normal development might lead to a testicular germ cell neoplasia in situ (GCNIS), from which TGCTs originate. The breakthrough epigenetic research, both in normal development and TGCT tumorigenesis, has been going on to find better biomarkers and therapy for this type of tumors.

PO-362 Influence of epigenetic agents on mouse teratoma development in vitro

Introduction Testicular Germ Cell Tumours (TGCT) are the most frequent malignancies in young male population and believed to be initiated by epimutations, i.e. aberrant epigenetics. Among various, teratoma is the most differentiated TGCT type encompassing all three germ layer derived tissues. Mouse teratoma is a well-established in vitro model obtained by cultivating mouse embryo. It represents an ideal system to investigate the effect of most prominent epigenetic drugs and agents. Material and methods After isolation, embryos were treated for two hours with 5-azacytidine, Trichostatin A, Valproate, esiNanog, esiOct3/4 and esiTrrap, respectively. The embryos/teratomas were measured at day 0 and for the consequent 7 days of culturing in MEM enriched with rat serum. For analysis of proliferative and apoptotic activity, immunohistochemistry on paraffin embedded embryos/teratomas was performed using anti-Ki-67 and anti-Caspase-3, respectively. Signal intensity was measured by morphometric analysis. For gene expression analyses, embryos/teratomas were pooled into experimental groups from which specific gene related RNA quantity was analysed by both qPCR and ddPCR. Results and discussions Epigenetic modulators reduced significantly embryo/teratoma growth. Most prominent decrease was detected in 5-azacytidine and esiOct3/4 treated embryos/teratomas. Furthermore, 5-azacytidine almost completely disrupted tissue architecture and cellularity. Proliferative activity was not decreased by any epigenetic modulator. EsiNanog, esiTrrap and surprisingly 5-azacytidine, in fact, showed a slight increase in proliferation. Still, 5-azacytidine induced an increase in apoptotic activity as well. Even stronger incitement of apoptosis was found in Valproate treated embryos/teratomas, while other modulators had no effect on apoptotic activity at all. Expression of analysed stemness and differentiation genes panel was significantly disrupted by 5-azacytidine, Valproate and esiOct ¾. Other modulators induced slighter decrease or no change in gene expression. Conclusion This research presents a strong adverse influence of epigenetic modulators on experimental germ cell tumour development. It seems that this effect is consequent to induced change in stemness and differentiation genes expression.

Impact of 5-azacytidine on rat decidual cell proliferation

The DNA demethylating agent 5‐azacytidine (5‐azaC) has a teratogenic influence during rat development influencing both the embryo and the placenta. Our aim was to investigate its impact on early decidual cell proliferation before the formation of placenta. Thus, female Fischer rats received 5‐azaC (5 mg/kg, i.p.) on the 2nd, 5th or 8th day of gestation and the decidual tissues were harvested on gestation day 9. They were then analysed immunohistochemically for expression of cell proliferation marker proliferating cell nuclear antigen (PCNA) in decidual cells and for global DNA methylation using the coupled restriction enzyme digestion, random amplification and pyrosequencing assays. We found that 5‐azaC administered on the 5th and 8th (but not on 2nd) day of gestation led to increased PCNA expression in decidual cells compared with untreated controls. No significant changes in DNA methylation were detected, with either method, in any of the treated rat groups compared with untreated controls. Thus, we conclude that 5‐azaC can stimulate decidual cell proliferation without simultaneously changing global DNA methylation level in treated cells.