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

Abstract

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).

Transmembranous and enchondral osteogenesis in transplants of rat limb buds cultivated in serum‐ and protein‐free culture medium

“…Our results obtained previously regarding transplantation under the kidney capsule of in vitro cultivated gastrulating embryos-proper showed that this site is optimal for the continuation of chondrogenesis, osteogenesis, neurogenesis, and development of skin appendages. In vivo results also reflected results obtained by previous cultivation in vitro (Belovari et al, 2001;Bulic-Jakus et al, 2001).…”

5-Azacytidine enhances proliferation in transplanted rat fetal epiglottis

“…The classical ectopic in vivo transplantation model used here (9), confirmed its value for this particular developmental study dealing with embryonic epiglottis, because 17-days-old epiglottis survived well and its differentiation proceeded towards the mature elastic cartilage. Subcapsular kidney space has been exploited extensively before for investigation of developmental potential of a number of embryonic tissues such as the embryonic shield (19), single germ layers (20), retina (21), lacrimal gland (22), mandible (23) and lensectomized eyes (24,25). Results obtained with epiglottis are in concordance with those studies because development always progressed and tissues could exert at least a part of their full developmental potential even if pre-cultivated in poor metabolic conditions such as chemically defined media without any proteins.…”

“…Results obtained with epiglottis are in concordance with those studies because development always progressed and tissues could exert at least a part of their full developmental potential even if pre-cultivated in poor metabolic conditions such as chemically defined media without any proteins. The reason for this lies probably in the optimal vascularization of the kidney capsule that provides an optimal microenvironment (19).…”

Embryo‐derived teratoma in vitro biological system reveals antitumor and embryotoxic activity of valproate

“…As shown previously, by subsequent isotransplantation of the precultivated embryo-derived teratoma to the metabolically richer environment in vivo for another 14 days, the residual potential for further growth or differentiation (e.g., of skin appendages, bone, neural tissue, and even organotypic structures) or the restriction of such potential was assessed [32,33]. A similar teratoma assay in rodents in vivo is used to confirm pluripotency [34] or discover a residual potential for malignant transformation of various cells precultivated in vitro for regenerative medicine purposes [12,35].…”