SIRT1 activity orchestrates ECM expression during hESC‐chondrogenic differentiation

Abstract

Epigenetic modification is a key driver of differentiation, and the deacetylase Sirtuin1 (SIRT1) is an established regulator of cell function, ageing, and articular cartilage homeostasis. Here we investigate the role of SIRT1 during development of chondrocytes by using human embryonic stem cells (hESCs). HESC‐chondroprogenitors were treated with SIRT1 activator; SRT1720, or inhibitor; EX527, during differentiation. Activation of SIRT1 early in 3D‐pellet culture led to significant increases in the expression of ECM genes for type‐II collagen (COL2A1) and aggrecan (ACAN), and chondrogenic transcription factors SOX5 and ARID5B, with SOX5 ChIP analysis demonstrating enrichment on the chondrocyte specific –10 (A1) enhancer of ACAN. Unexpectedly, when SIRT1 was activated, while ACAN was enhanced, glycosaminoglycans (GAGs) were reduced, paralleled by down regulation of gene expression for N‐acetylgalactosaminyltransferase type 1 (GALNT1) responsible for GAG chain initiation/elongation. A positive correlation between ARID5B and COL2A1 was observed, and co‐IP assays indicated association of ARID5B with SIRT1, further suggesting that COL2A1 expression is promoted by an ARID5B‐SIRT1 interaction. In conclusion, SIRT1 activation positively impacts on the expression of the main ECM proteins, while altering ECM composition and suppressing GAG content during human cartilage development. These results suggest that SIRT1 activity has a differential effect on GAGs and proteins in developing hESC‐chondrocytes and could only be beneficial to cartilage development and matrix protein synthesis if balanced by addition of positive GAG mediators.

Modulation of sirtuins during monolayer chondrocyte culture influences cartilage regeneration upon transfer to a 3D culture environment

“…This highlights the importance of specificity of selected activators to sirtuin family members. Smith et al (2022) recently used pellet culture to induce chondrogenic differentiation in human embryonic stem cells and found that activation of SIRT1 using 5 μM SRT1720 during pellet culture leads to increased collagen type II and aggrecan gene and protein expression, but reduced sGAG accumulation. This contrasts with our findings with mature freshly isolated bovine chondrocytes where we observed a decrease in total collagen with a 50 × lower dose of SIRT1720 ( Figure 7B ), indicating that the response to SIRT1 activation may depend on dose and the differentiation state of the chondroprogenitor cell or chondrocyte.…”

Directed differentiation of hPSCs through a simplified lateral plate mesoderm protocol for generation of articular cartilage progenitors

“…Day 11 prechondrocytes were removed from tissue culture plastic and centrifuged in 14ml polypropylene tubes at 300 xRCF in day 11 medium for 5 minutes to sediment cells. Caps were left loose to enable gaseous transfer and tubes incubated for 3 days at 37˚C to facilitate the production of a spherical pellet, as detailed previously [12]. At day 11+3, medium was changed for chondrogenic-basal medium (CM) containing GDF5 (10 ng/mL), TGFβ3 (10 ng/mL) (Peprotech) and BMP2 (0.5x EC66).…”

“…Samples were taken from both 2D and 3D culture as described previously [12]. Samples were transferred to RLT buffer directly and RNA extracted by use of a RNeasy QiAgen kit.…”

“…In the first step ActivinA, FGF2, BMP2 and the GSK3 inhibitor CHIR99021 (to replace Wnt3a) were used to generate mid-primitive streak like cells as employed previously by ourselves [12] and others [27]. Additionally, the PI3K inhibitor PIK90 was used to help inhibit endodermal differentiation [32].…”

“…Our group have developed directed differentiation protocols to produce prechondrocytes from hPSCs [9,10], with updated protocols replacing BMP4 with BMP2 to improve chondrogenesis [11], or substituting small molecules for growth factors to remove batch variability. However, though these differentiation pathways can produce prechondrocytes, they are not able to produce osteogenic cells, and only give moderate quality of cartilage pellets when cultured further [12]. One way of ensuring the production of cells present during the formation of the fetal tissue is to attempt to recapitulate the development process more closely [13].…”