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

Mužić, Vedrana; Katušić Bojanac, Ana; Jurić-Lekić, Gordana; Himelreich, Marta; Tupek, Katarina; Šerman, Ljiljana; Marn, Nina; Sinčić, Nino; Vlahović, Maja; Bulić-Jakuš, Floriana (2013) Epigenetic drug 5-azacytidine impairs proliferation of rat limb buds in an organotypic model-system in vitro. Croatian Medical Journal, 54 (5). pp. 489-495. ISSN 0353-9504

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Abstract

Aim was 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. 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.

Item Type:	Article
Uncontrolled Keywords:	limb bud; in vitro culture; rat; 5-azacytidine
Subjects:	BIOMEDICINE AND HEALTHCARE > Clinical Medical Sciences
Divisions:	Division for Marine and Enviromental Research
Projects:	Project title Project leader Project code Project type Male and female reproductive tract: development, histophysiology and infertility (Muški i ženski spolni sustav: razvoj, normalna histofiziologija i neplodnost)- Davor Ježek 108-1080399-0383 MZOS Experimental embryonic tumors and mammalian development in vitro and in vivo (Eksperimentalni embrionalni tumori i razvoj zametaka sisavaca in vitro i in vivo)- Florijana Jakuš 108-1080399-0335 MZOS
Depositing User:	Nina Marn
Date Deposited:	28 Oct 2015 14:43
URI:	http://fulir.irb.hr/id/eprint/2187
DOI:	10.3325/cmj.2013.54.489

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