The Transcription Factor-microRNA Regulatory Network during hESC-chondrogenesis

Griffiths, Rosie; Woods, Steven Paul; Cheng, Aixin; Wang, Ping; Griffiths-Jones, Sam; Ronshaugen, Matthew; Kimber, Susan J.

doi:10.1038/s41598-020-61734-4

Cited by 12 publications

(11 citation statements)

References 102 publications

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“…To understand the importance of SIRT1 in early chondrogenic development, we first sought to determine its expression during the generation of chondroprogenitors, using the 14‐day hESC‐developmental protocol (DPP) shown in Figure 1. In this protocol cells are induced, by serial growth factor applications, through a series of developmental steps to a primitive streak‐like stage, then to mesodermal progenitor followed by chondroprogenitors 3–5,18 …”

Section: Resultsmentioning

confidence: 99%

“…In this protocol cells are induced, by serial growth factor applications, through a series of developmental steps to a primitive streak-like stage, then to mesodermal progenitor followed by chondroprogenitors. [3][4][5]18 SIRT1 gene and protein expression significantly increased between days 2-5 of differentiation, then decreased steadily back to the original level by day 14 (Figure 1B,C). Alternate Sirtuins were also assessed; however, their transcript expression did not significantly change during the differentiation process (Figure 1B).…”

Section: Sirt1 Expression Is Dynamic and Is Elevated During Early Sta...mentioning

confidence: 93%

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SIRT1 activity orchestrates ECM expression during hESC‐chondrogenic differentiation

et al. 2022

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Sirt1 Expression Is Dynamic and Is Elevated During Early Sta...mentioning

confidence: 93%

SIRT1 activity orchestrates ECM expression during hESC‐chondrogenic differentiation

et al. 2022

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“…and shown in Fig 1. In this protocol cells are induced, by serial growth factor application, through a series of developmental steps to a primitive streak like stage, then to mesodermal progenitor followed by chondroprogenitors [3–5,18].…”

Section: Resultsmentioning

confidence: 99%

SIRT1 activity orchestrates ECM expression during hESC-chondrogenic differentiation

Smith

Humphreys

Bates

et al. 2020

Preprint

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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). HESCchondroprogenitors were treated with SIRT1 activator; SRT1720, or inhibitor; EX527, at different development stages. Activation of SIRT1 during 3D-pellet culture led to significant increases in 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 ACAN -10 enhancer. Unexpectedly, while ACAN was enhanced, GAG retention in the matrix was reduced when SIRT1 was activated. Significantly, ARID5B and COL2A1 were positively correlated, with Co-IP indicating binding of ARID5B to SIRT,1 suggesting that COL2A1 expression is promoted by an ARID5B and SIRT1 interaction. In conclusion, SIRT1 activation positively impacts on the expression of the main ECM components, whilst mitigating ECM 2 organization and GAG content during cartilage development. These results suggest that SIRT1 activity is beneficial to cartilage development and matrix synthesis, but that use in cartilage regeneration would require tailoring. SIRT1 modulation during the 2D early chondrogenic differentiation protocol, support the idea that transition from mesoderm to a chondroprogenitor, in stages 1 and 2 of the protocol, does not require SIRT1 activity, since its inhibition led to an increase in expression of the chondrogenic transcription factor SOX9. Activation of SIRT1 in a three-dimensional (3D) pellet culture increased chondrogenic gene expressionAs previous reports showed that SIRT1 modulation in a 3D setting resulted in augmented COL2 expression [23], we next assessed hESC-chondroprogenitors in a 3D culture system. Specifically, after 14 days of differentiation ( Figure 1A) hESC-chondroprogenitors were pelleted in day 14 medium, cultured for 3 days to establish the 3D pellets, then pellets were incubated in pellet culture medium containing SIRT1 activity modulators SRT1720 or EX527 (Figure 2A).Results indicated that SIRT1 activation using 5 µM SRT1720, increased chondrogenic gene expression compared to untreated controls during the first week of pellet culture ( Figure S2A). Prolonged SIRT1 activation (i.e. between 3 and 14 days after pelleting) resulted in enlarged pellets compared to controls, while pellets treated with EX527 did not display any change in size ( Figure 2B).Further qRT-PCR analysis revealed a significant increase in expression of chondrogenic genes (i.e. SOX5, ARID5B, COL2A1 and ACAN) in SRT1720 treated pellets compared to day 14 monolayer (2D) cultures, and untreated pellet controls. Conversely, there was a significant decrease in expression of the fibroblast/hypertrophy-associated genes COL1 and RUNX2 in these pellets compared to untreated pellet controls ( Figure 2C), while COLX was not detected i...

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“…It is now standard to assess gene expression using quantitative reverse transcription PCR (RT-qPCR) for transcription factor and matrix molecule genes expressed during chondrocyte development [43,55,113] or in mature chondrocytes or osteoblasts [114,115]. This can be extended to assessment of the whole transcriptome through RNAseq [116], e.g., comparison with developing limb tissue [88], osteogenesis compared to calvaria [115] and evaluation of chondrogenesis-associated microRNAs [116,117]. More recently it has been possible to delve into the nature of the different cells developing to form scaffold-free cartilage using singlecell RNAseq [80].…”

Section: Characterization Methodsmentioning

confidence: 99%

Pluripotent stem cells for skeletal tissue engineering

Ferreira

Humphreys

Ogene

et al. 2021

Critical Reviews in Biotechnology

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Here, we review the use of human pluripotent stem cells for skeletal tissue engineering. A number of approaches have been used for generating cartilage and bone from both human embryonic stem cells and induced pluripotent stem cells. These range from protocols relying on intrinsic cell interactions and signals from co-cultured cells to those attempting to recapitulate the series of steps occurring during mammalian skeletal development. The importance of generating authentic tissues rather than just differentiated cells is emphasized and enabling technologies for doing this are reported. We also review the different methods for characterization of skeletal cells and constructs at the tissue and single-cell level, and indicate newer resources not yet fully utilized in this field. There have been many challenges in this research area but the technologies to overcome these are beginning to appear, often adopted from related fields. This makes it more likely that cost-effective and efficacious human pluripotent stem cell-engineered constructs may become available for skeletal repair in the near future.

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The Transcription Factor-microRNA Regulatory Network during hESC-chondrogenesis

Cited by 12 publications

References 102 publications

SIRT1 activity orchestrates ECM expression during hESC‐chondrogenic differentiation

SIRT1 activity orchestrates ECM expression during hESC‐chondrogenic differentiation

SIRT1 activity orchestrates ECM expression during hESC-chondrogenic differentiation

Pluripotent stem cells for skeletal tissue engineering

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