Abstract
Background Given regenerative therapies, the utilization of primary human cells is desired and requested in the development of in vitro systems and disease models. After a few passages in vitro, all cells from the connective tissue end up in a similar fibroblastoid cell type marked by loss of the specific expression pattern. It is still under discussion whether different de-differentiated mesenchymal cells have similar or identical differentiation capacities in vitro.
Methods Chondrocytes isolated from patients with late-stage osteoarthritis were cultured for several passages until de-differentiation was completed. The mRNA level of cartilage markers was investigated, and the adipogenic, osteogenic and chondrogenic differentiation capacity was examined. By adding 5-aza-2’-deoxycytidine (5-aza-dC) to the media, the influence of DNA methylation on the differentiation capacity was analyzed.
Results The chondrocytes used in this work were not affected by the loss of specific gene expression upon cell culture. The mRNA levels of SOX5, SOX6, SOX9, aggrecan, and proteoglycan-4 remained unchanged. The underlying mechanisms of cartilage marker maintenance in osteoarthritic (OA) chondrocytes were investigated with a focus on the epigenetic modification by DNA methylation. The treatment of de-differentiated chondrocytes with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) displayed no appreciable impact on the observed maintenance of marker gene expression, while the chondrogenic differentiation capacity was compromised. On the other hand, the pre-cultivation with 5-aza-dC improved the osteogenesis and adipogenesis of OA chondrocytes. Contradictory to these effects, the DNA methylation levels were not reduced after treatment with 1 μM 5-aza-dC for four weeks.
Conclusion Chondrocytes isolated from late-stage osteoarthritic patients represents a reliable cell source for in vitro studies as wells as disease models since the chondrogenic differentiation potential remains. 5-aza-2’-deoxycytidine could not further improve their chondrogenic potential.
Footnotes
shirin.kadler{at}tu-berlin.de, oezlem.vural{at}tu-berlin.de, luzia.reiners-schramm{at}tu-berlin.de, roland.lauster{at}tu-berlin.de, mark.rosowski{at}tu-berlin.de
List of abbreviations
- 5-aza-C
- 5-aza-cytidine
- 5-aza-dC
- 5-aza-2’-deoxycytidine
- 5mC
- 5-methyl-cytosine
- ACAN
- aggrecan
- COL2A1
- collagen type II chain alpha 1
- DNMT
- DNA methyltransferase
- FABP
- fatty acid binding protein
- GAG
- glycosaminoglycan
- NGS
- Next generation sequencing
- OA
- osteoarthritis
- OPN
- osteopontin
- PRG4
- proteoglycan-4
- UBE2D2
- ubiquitin-conjugating enzyme E2 D2