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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. For further resources related to this article, please visit the WIREs website.

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Developmental processes in gastrulating rat embryos were investigated by using an original, serum-free, chemically defined model system. 9.5-day-old rat embryos, without extraembryonic membranes, were cultivated at the air–liquid interface in a serum-free medium, with and without a protein supplement, for 2 weeks. A teratogenic, demethylating agent, 5-azacytidine, was added to serum-free and protein-free culture medium and to serum-free medium supplemented with human transferrin. A single dose of 5-azacytidine impaired the survival, growth and differentiation of embryos in protein-free medium and serum-free medium with transferrin. In contrast, repeated exposure to 5-azacytidine was required to impair growth in serum-supplemented medium. It was concluded that the activity of 5-azacytidine was easier to detect in a simple, chemically defined medium than in a serum-supplemented medium. This serum-free in vitro method could be useful in screening for teratogenic or embryotoxic substances during gastrulation, the most critical stage of mammalian development.

The spin-trap free radical scavenger N -tert-butyl-α-phenylnitron (PBN) ameliorated effects of several teratogens involving reactive oxygen species (ROS). We investigated for the first time whether PBN could ameliorate teratogenesis induced by a DNA hypomethylating hematological therapeutic 5-azacytidine (5azaC). At days 12 and 13 of gestation, Fisher rat dams were pretreated by an i.v. injection of PBN (40 mg/kg) and 1 h later by an i.p. injection of 5azaC (5mg/kg). Development was analyzed at gestation day 15 in embryos and day 20 in fetuses. PBN alone did not significantly affect development. PBN pretreatment restored survival of 5azaC-treated dams' embryos to the control level, restored weight of embryos and partially of fetuses, and partially restored crown-rump lengths. PBN pretreatment converted limb adactyly to less severe oligodactyly. PBN pretreatment restored global DNA methylation level in the limb buds to the control level. Cell proliferation in limb buds of all 5azaC-treated dams remained significantly lower than in controls. In the embryonic liver, PBN pretreatment normalized proliferation diminished significantly by 5azaC; whereas in embryonic vertebral cartilage, proliferation of all 5azaC-treated dams was significantly higher than in PBN-treated dams or controls. Apoptotic indices significantly enhanced by 5azaC in liver and cartilage were not influenced by PBN pretreatment. However, PBN significantly diminished ROS or reactive nitrogen species markers nitrotyrosine and 8-hydroxy-2′deoxyguanosine elevated by 5azaC in embryonic tissues, and, therefore, activity of this DNA hypomethylating agent was associated to the activation of free radicals. That pretreatment with PBN enhanced proliferation in the liver and not in immature tissue is interesting for the treatment of 5azaC-induced hepatotoxicity and liver regeneration.

AimTo 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.MethodsFischer 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 Eagle's 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.ResultsAt 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.ConclusionOur 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.

SummaryWe 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.

Mesenchymal stromal cells (MSC) hold significant potential for tissue engineering applications. Modular tissue engineering involves the use of cellularized “building blocks” that can be assembled via a bottom-up approach into larger tissue-like constructs. This approach emulates more closely the complexity associated hierarchical tissues compared with conventional top-down tissue engineering strategies. The current study describes the combination of biodegradable porous poly(DL-lactide-co-glycolide) (PLGA) TIPS microcarriers with canine adipose-derived MSC (cAdMSC) for use as implantable conformable building blocks in modular tissue engineering applications. Optimal conditions were identified for the attachment and proliferation of cAdMSC on the surface of the microcarriers. Culture of the cellularized microcarriers for 21 days in transwell insert plates under conditions used to induce either chondrogenic or osteogenic differentiation resulted in self-assembly of solid 3D tissue constructs. The tissue constructs exhibited phenotypic characteristics indicative of successful osteogenic or chondrogenic differentiation, as well as viscoelastic mechanical properties. This strategy paves the way to create in situ tissue engineered constructs via modular tissue engineering for therapeutic applications.