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 Ë‚ .0001) and enhanced differentiation of both myotubes (P Ë‚ .01) and cylindrical epithelium (P Ë‚ .05). Hyperthermia of 43°C applied each day for 30 minutes during the first week (CEM43°C 210 minutes) impaired overall growth (P Ë‚ .01) and diminished cell proliferation (P Ë‚ .0001). Long-term hyperthermia of 40.5°C applied for two weeks (CEM43°C 630 minutes) significantly impaired survival (P Ë‚ .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 Ë‚ .0001), overall growth (P Ë‚ .01) and cartilage differentiation (P Ë‚ .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.

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.

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 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. 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.

Modular Orthopaedic Tissue Engineering With Implantable Microcarriers and Canine Adipose-Derived Mesenchymal Stromal Cells.

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.

A Free Radical Scavenger Ameliorates Teratogenic Activity of a DNA Hypomethylating Hematological Therapeutic.

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.

Epigenetic deregulation through DNA demethylation seems not to interfere with the differentiation of epithelia from pre-gastrulating rat embryos in vitro.

One of the epigenetic mechanisms controlling differentiation during mammalian development is the process of DNA methylation. The differentiation of tissues in pre-gastrulating rat embryos cultivated in vitro under the influence of the demethylating agent 5-azacytidine (5azaC) was investigated. Eight-day-old Fisher rat embryos consisting of epiblast and hypoblast (primitive ectoderm and primitive endoderm) were isolated and cultivated in serum-supplemented medium by air-lifting method in vitro. A single dose of 5azaC (30 microM) was added to the culture medium on day 5 of cultivation. After 14 days, teratoma-like structures developed and were processed by routine histology. When compared to controls, the explants treated with 5azaC showed a statistically significant higher incidence of neuroblasts, myotubes, cartilage, and blood islands. On the other hand, the incidence of stratified squamous, columnar and glandular epithelium was not statistically different from controls. It seems that differentiation of epithelia was not sensitive to DNA demethylation caused by 5azaC like differentiation of other tissues, especially mesodermal derivatives.

Development of epithelia in experimental teratomas derived from rodent embryos.

Investigation of developmental potential of various embryonal tissues is important for design of new approaches to regenerative medicine aimed at supplementing tissues damaged by trauma or disease. Rodent embryos have been extensively used in experiments designed for investigation of developmental potential to give rise to various types of epithelia such as superficial epithelia, neuroepithelium and sometimes even malignantly transformed epithelium in teratoma-like structures. These experiments have been done in vitro, in transplants in vivo and by combined in vitro-in vivo methods.

Development of epithelia in the ectopic transplant of the fetal rat epiglottis.

Embryonic in situ development is strictly regulated within the specific microenvironment of developing tissues. However, for regenerative medicine purposes (supplementation of damaged tissues/organs), transplantation to ectopic sites has been considered. To investigate developmental potential of fetal epiglottic epithelia at an ectopic site, fetal epiglottis was transplanted under the kidney capsule and its development compared to fetal and adult epiglottis. Seventeen-day-old Fischer rat epiglottides were microsurgically isolated under a dissecting microscope and transplanted under the kidney capsule of adult males. After 14 days, classic histology and immunohistochemical detection of the Proliferating Cell Nuclear Antigen (PCNA) were done in isolated and accordingly fixed transplants. The 17-day-old fetal epiglottis and adult epiglottis were processed in the same way. The 17-day-old fetal epiglottides were covered with immature epithelium expressing PCNA in almost all cells. Adult epiglottis was covered with two types of epithelia (stratified squamous epithelium and ciliated pseudostratified epithelium). In the stratified squamous epithelium PCNA was abundantly expressed in the basal cell layer and absent from more superficial and more differentiated cells. Transplants survived well during the experimental period. On their surface ciliated pseudostratified epithelium could be easily recognized, but squamous epithelium was almost absent. PCNA was expressed in basal cells of the ciliated pseudostratified epithelium and was absent from the more differentiated superficial cells. It seems that at this ectopic site further differentiation of the epiglottic epithelia can proceed but differentiation of squamous epithelium seems not to be favored. It seems that this ectopic site is optimal for further differentiation of the epiglottic epithelium towards ciliated pseudostratified epithelium.

Differentiation of epiglottal epithelia during prenatal and postnatal human development.

Differentiation of epiglottal epithelia during human development was for the first time investigated by the light microscopy and documented in celoidine collection of human embryos from the Archive of the Department of Histology and Embryology, School of Medicine University of Zagreb, Croatia. At 6 weeks epiglottal swelling was found to be covered by a simple squamous epithelium consisting of a single layer of cells. At 8 weeks epithelium changed to a two-layered cuboidal epithelium which at the end of the 8th week transformed to multilayered columnar epithelium without cilia and goblet cells. In the one-day-old newborn, the majority of epiglottis was found to be covered by the mature ciliated columnar pseudostratified epithelium with goblet cells while only a minor part of the oral surface next to the tongue by the stratified squamous epithelium. This unexpected finding is in contrast to the domination of the stratified squamous epithelium found at the age of 13 years and in 35-years-old adult. Reversal of proportion covered by different types of epithelia between birth and puberty /adulthood is probably connected to the establishment of the air-flow which could be stimulating for differentiation of stratified squamous epithelium.

Development of the fetal neural retina in vitro and in ectopic transplants in vivo.

Investigation of the developmental potential of immature tissues is important for novel approaches to human regenerative medicine. Development of the fetal neural retina has therefore been investigated in two experimental systems. Retinas were microsurgically isolated from 20-days-old rat fetuses and cultivated in vitro for 12 days or transplanted in vivo under the kidney capsule of adult males for as long as 6 months. Shedding of the photoreceptor outer segment which is a process occurring at the terminal stage of photoreceptor differentiation was observed in culture by transmission electron microscopy (TEM). In transplants, no photoreceptors were found although markers of terminal neural and glial differentiation (e,g. synaptophysin, chromogranin and glial fibrilary acidic protein--GFAP) along with the molecules involved in the process of differentiation (guidance molecule semaphorin IIIA and chondroitin sulfate proteoglycan) were expressed. Semaphorin was differentially expressed being absent from older transplants. Proliferating cell nuclear antigen and nestin (marker of undifferentiated neural cells) were still weakly expressed even in six-months-old transplants. We could conclude that in both our experimental systems fetal neural retina proceeded to differentiate further on. However, even in long-term ectopic transplants a small population of cells still retained the potential for proliferation and has not yet reached the stage of terminal differentiation.

DNA methylation as a regulatory mechanism for gene expression in mammals.

Epigenetics refers to the study of heritable changes in gene expression that occur without a change in DNA sequence. In the last decade, it has been shown that epigenetic mechanisms provide an "extra" layer of transcriptional control that regulates genes expression. Three distinct mechanisms appear intricately related in initiating and sustaining epigenetic modifications: RNA-associated silencing, DNA methylation and histone modification. These mechanisms are critical components in the normal development and cell growth. DNA methylation is involved in transcriptional silencing of genes, regulation of expression of imprinted genes, a number of tumour suppressor genes in cancer and silencing of genes located on the inactive X chromosome. In this review, we are focused on the basic principles of DNA methylation as the main epigenetic mechanism for normal embryonic development and epigenetic alterations that contribute to carcinogenesis.

Of mice and men: teratomas and teratocarcinomas.

Teratomas and teratocarcinomas are tumors containing tissue derivatives of all three germ-layers. They can be induced by transplantation of animal embryos to ectopic microenvironment. Development of malignant teratocarcinomas depends on embryonic stage, species-specificity and immunological competence of the host. In the man, teratomas and teratocarcinomas usually represent a subtype of germ-cell tumors but sacrococcygeal teratomas arise from the remnants of the pluripotent primitive streak. Undifferentiated embryonal carcinoma (EC) cells are responsible for the malignancy of experimental mouse teratocarcinomas. Mouse EC cells injected to the adult give rise to tumors and upon injection to early embryos to differentiated tissues--thus resembling normal mouse embryonic stem cells (mESC). Epigenetic changes rather than mutations are associated with transformation of mESC to EC cells. Human EC and ES cell-lines (hESC) contain chromosomal abnormalities and can form teratocarcinoma after transplantation. ES cells are among those proposed for cell replacement therapy in the man. Suicide gene introduction should be recommended prior to their use in vivo to ablate them in case of malignant transformation.

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

5-azacytidine enhances proliferation in transplanted rat fetal epiglottis.

Fetal rat epiglottis and its developmental potential in ectopic transplants under the influence of the epigenetic drug was investigated. Epiglottises from 17-days-old rat embryos were transplanted under kidney capsules of adult rats for 14 days. 5-azacytidine (5 mg/kg) was injected intraperitoneally during first three days and controls were sham treated. TEM, immunohistochemical detection and quantitative stereological analysis of the Proliferating Cell Nuclear Antigen (PCNA) expression (numerical density N(v)) were performed. Typical chondroblasts with long surface processes and sparse lipid droplets were found in fetal epiglottis and chondrocytes with shorter processes, numerous lipid droplets and elastic fibers in adult. PCNA was expressed in almost all cells of the fetal epiglottis while in the adult it was expressed in minority of cells. In transplants, differentiation progressed towards the differentiation found in the adult. Application of 5-azacytidine increased the capacity for proliferation (N(v PCNA)) in comparison to controls but no difference in differentiation was observed. Data about the developmental potential and induction of proliferation in mammalian epiglottis by epigenetic modulation is of importance for regenerative medicine.