Although DNA methylation epigenetically regulates development, data on global DNA methylation during development of limb buds (LBs) are scarce. We aimed to investigate the global DNA methylation developmental dynamics in rat LBs cultivated in a serum-supplemented (SS) and in chemically defined serum- and protein-free (SF) three-dimensional organ culture. Fischer rat front- and hind-LBs at 13th and 14th gestation days (GD) were cultivated at the air-liquid interface in Eagle's Minimal Essential Medium (MEM) or MEM with 50% rat serum for 14 days, as SF and SS conditions, respectively. The methylation of repetitive DNA sequences (SINE rat ID elements) was assessed by pyrosequencing. Development was evaluated by light microscopy and extracellular matrix glycosaminoglycans staining by Safranin O. Upon isolation, weak Safranin O staining was present only in more developed GD14 front-LBs. Chondrogenesis proceeded well in all cultures towards day 14, except in the SF-cultivated GD13 hind-LBs, where Safranin O staining was almost absent on day 3. That was associated with a higher percentage of DNA methylation than in SF-cultivated GD13 front-LBs on day three. In SF-cultivated front-LBs, a significant methylation increase between the 3rd and 14th day was detected. In SS-cultivated GD13 front-LBs, methylation increased significantly on day three and then decreased. In older GD14 SS-cultivated LBs, there was no increase of DNA methylation, but they were significantly hypomethylated relative to the SS-cultivated GD13 at days 3 and 14. We confirmed that the global DNA methylation increase is associated with less developed limb organ primordia that strive towards differentiation in vitro, which is of importance for regenerative medicine strategies.

E-selectin, ICAM-1 (intercellular adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1) play a role in atopic dermatitis (AD). This study aimed to evaluate their expression in skin biopsy specimens of patients diagnosed with AD using an optimized computer program. A descriptive analysis and comparison of digitally measured surface area and cell number were performed. The number of E-selectin-positive cells did not vary between the groups. In patients with AD, decreases of 1.2-fold for ICAM-1- and 1.3-fold for VCAM-1- positive cells were observed. The E-selectin-positive epidermal surface area increased (p < 0.001), while ICAM1 and VCAM1 decreased 2.5-fold and 2-fold, respectively, compared to controls. In the AD-affected skin, the E-selectin-positive endothelial area was 3.5-fold larger (p < 0.001), and the ICAM1-positive area was almost 4-fold larger (p < 0.001). E-selectin and ICAM-1 were expressed in the control dermis moderately and weakly, respectively. A strong E-selectin signal was detected in the AD-affected skin macrophages and a strong ICAM-1 signal in the dermal vessel endothelium. In the endothelial cells of AD-affected skin, no VCAM-1 signal could be found. E-selectin, ICAM-1, and VCAM-1 expression show significant disease-specific changes between AD-affected and control skin. The combination of digital analysis and a pathologist’s evaluation may present a valuable follow-up of AD activity parameters.

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Cartilage differentiates in rat limb buds cultivated in a chemically defined protein‐free medium in the same manner as in the richer serum‐supplemented medium. We aimed to investigate the remaining differentiation potential of pre‐cultivated limb buds by subsequent transplantation in vivo. Rat front (FLBs) and hind‐limb buds (HLBs) were isolated from Fischer rat dams at the 14th gestation day (GD 14) and cultivated at the air‐liquid interface in Eagle's Minimum Essential Medium (MEM) alone; with 5 μM of 5‐azacytidine (5azaC) or with rat serum (1:1). Overall growth was measured seven times during the culture by an ocular micrometre. After 14 days, explants were transplanted under the kidney capsule of adult males. Growth of limb buds was significantly lower in all limb buds cultivated in MEM than in those cultivated with serum. In MEM with 5azaC, growth of LBs was significantly lower only on day 3 of culture. Afterwards, it was higher throughout the culture period, although a statistically significant difference was assessed only for HLBs. In transplants, mixed structures developed with the differentiated transmembranous bone, cartilage with enchondral ossification, bone‐marrow, sebaceous gland, and hair that have never been found in vitro. Nerves differentiated only in transplants precultivated in the serum‐supplemented medium. We conclude that pre‐cultivation of LBs in a chemically defined protein‐free medium does not restrict osteogenesis and formation of epidermal appendages but is restrictive for neural tissue. These results are important for understanding limb development and regenerative medicine strategies.