Global DNA methylation and chondrogenesis of rat limb buds in a three-dimensional organ culture system

Abstract

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.

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

“…The presented results show that cartilage originating from the early rat limb buds pre-cultivated in a serum-and protein-free medium (Mužić Radović et al, 2022) preserves the full potential for osteogenesis in vivo. This finding is in accordance with the results of other authors showing that the self-organizing and self-patterning capacity of the mouse embryonic cartilage is retained even when subjected to cellular dissociation (Fernando et al, 2018).…”

“…After deparaffinization, routine haematoxylin-eosin or Safranin O staining was done, and sections were then rehydrated and covered (Mužić Radović et al, 2022).…”

“…DNA methylation data were obtained and analysed by the PyroMark Q24 Advanced Software. Quality control of the data was determined during the pyrosequencing run as described before (Mužić Radović et al, 2022).…”

“…A specific global DNA methylation dynamics of repetitive sequences (SINE) associated with chondrogenesis was shown in the limb bud developmental system we use. A rise in global DNA methylation was associated with less differentiated organ primordia in cartilage development (Mužić Radović et al, 2022). 5-azacytidine (5azaC), a DNA demethylating epigenetic therapeutic (Voso et al, 2015) that is known to activate gene expression, significantly impaired the growth of limb buds cultivated in the model used previously (Muzic et al, 2013).…”

“…We have previously shown that terminal differentiation of the cartilage in rat limb buds cultivated in vitro can proceed in the simple chemically defined protein-free medium (Mužić Radović et al, 2022).…”