Aim: To analyze neural tissue differentiation in a unique, chemically-defined in vitro culture model of gastrulating rat embryo proper by use of transmission electron microscopy (TEM), proliferating cell nuclear antigen (PCNA) expression, and in vivo transplantation after a 2-week culture in serum-free or serum-supplemented media. Influence of protein-free medium unfavorable for differentiation of neural tissue in vitro was compared with favorable serum-free media enriched with transferrin or albumin. Differentiation of mesodermal derivatives in transplants was also investigated.
Methods: We cultivated 9.5-day-old Fischer rat embryos on the gas-liquid interface in the protein-free Eagle's Minimum Essential Medium (MEM), in MEM with either iron-saturated holotransferrin (50 microg/mL) or iron-free apotransferrin (50 microg/mL), and in medium saturated with either bovine serum albumin (BSA) (4 mg/mL or 400 microg/mL) or rat serum (50%). After the two-week culture period, light microscopy, TEM, and immunohistochemical method for detection of PCNA were done. Some explants were transplanted under the kidney capsule of adult male rats to be cultured in vivo for additional two weeks. Chi-square test or Fisher exact test were used to compare the proportion of tissues developed.
Results: Proportion of differentiated neural tissue was similar in explants cultivated in apotransferrin- and holotransferrin-supplemented media (13/33 and 9/20, respectively), but higher than in explants cultivated in protein-free medium (1/13). Neurons and glia cells produced a neuropil structure. Myelinization occurred only in serum-supplemented medium. PCNA expression was detected in a small number of neural tissue cells, even in serum-free cultivated embryos. Differentiation of brain-like tissue, cerebrospinal, and vegetative ganglionic cells occurred in all groups of transplants. However, in the transplants derived from protein-free medium, the proportion of neural tissue, cartilage, bone, skeletal and smooth muscle was significantly lower than in transferrin-supplemented media (p<0.01). Albumin seemed to promote differentiation of all tissues except vegetative ganglionic cells.
Conclusion: Nerve tissue differentiated to a rather high degree in a two-week in vitro postimplantation embryo culture. Transferrin or albumin, as the only proteins used for serum-free precultivation, significantly improved subsequent differentiation of nerve tissue and mesodermal derivatives in transplants in vivo.