Directed differentiation of human embryonic stem cells toward chondrocytes

Oldershaw, Rachel A.; Baxter, M.; Lowe, Emma T.; Bates, Nicola; Grady, Lisa M; Soncin, Francesca; Brison, Daniel R.; Hardingham, Timothy E.; Kimber, Susan J.

doi:10.1038/nbt.1683

Cited by 276 publications

(298 citation statements)

References 57 publications

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“…To further assess whether the SIRT1 repressive effects on LEF1 are conserved throughout chondrogenesis, we used a previously described 3-step protocol in which human embryonic stem cells are efficiently directed toward chondrogenesis (32,33). At d 13 of chondrogenesis, SOX9, COL2A1, and ACAN gene expression was significantly increased compared to d 0 (Fig.…”

Section: The Transcription Factor Lef1 Is a Transactivator Of Mmp13 Amentioning

confidence: 99%

“…MAN7 human embryonic stem cells (hESCs) were cultured as previously described by Oldershaw et al (32) and Cheng et al (33) with slight modifications. hESCs were cultured in a feeder-free system on vitronectin-coated (Thermo Fisher Scientific) tissue culture plates with mTESR1 medium (StemCell Technology, Vancouver, BC, Canada) and subcultured using EDTA passage.…”

Section: Direct Chondrogenesis Differentiation Protocolmentioning

confidence: 99%

“…hESCs were cultured in a feeder-free system on vitronectin-coated (Thermo Fisher Scientific) tissue culture plates with mTESR1 medium (StemCell Technology, Vancouver, BC, Canada) and subcultured using EDTA passage. For differentiation toward chondroprogenitors, a defined 3-stage protocol was used (32). In brief, hESCs were transferred to fibronectin (EMD-Millipore)-coated tissue culture wells (d 0) and initially differentiated toward primitive streak mesentoderm by addition of Wnt3a, activin A, and fibroblast growth factor (FGF)-2 (d 1-3), followed by FGF2, bone morphogenic protein (BMP)-2 (substitution for BMP4; unpublished data), and follistatin, to promote mesoderm-like cells (d 4-7).…”

Section: Direct Chondrogenesis Differentiation Protocolmentioning

confidence: 99%

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LEF1‐mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes

et al. 2017

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Reduced SIRT1 activity and levels during osteoarthritis (OA) promote gradual loss of cartilage. Loss of cartilage matrix is accompanied by an increase in matrix metalloproteinase (MMP) 13, partially because of enhanced LEF1 transcriptional activity. In this study, we assessed the role of SIRT1 in LEF1-mediated MMP13 gene expression in human OA chondrocytes. Results showed that MMP13 protein levels and enzymatic activity decreased significantly during SIRT1 overexpression or activation by resveratrol. Conversely, MMP13 gene expression was reduced in chondrocytes transfected with SIRT1 siRNA or treated with nicotinamide (NAM), a sirtuin inhibitor. Chondrocytes challenged with IL-1b, a cytokine involved in OA pathogenesis, enhanced LEF1 protein levels and gene expression, resulting in increased MMP13 gene expression; however, overexpression of SIRT1 during IL-1b challenge impeded LEF1 levels and MMP13 gene expression. Previous reports showed that LEF1 binds to the MMP13 promoter and transactivates its expression, but we observed that SIRT1 repressed LEF1 protein and mRNA expression, ultimately reducing LEF1 transcriptional activity, as judged by luciferase assay. Finally, mouse articular cartilage from Sirt1 2/2 presented increased LEF1 and MMP13 protein levels, similar to human OA cartilage. Thus, demonstrating for the first time that SIRT1 represses MMP13 in human OA chondrocytes, which appears to be mediated, at least in part, through repression of the transcription factor LEF1, a known modulator of MMP13 gene expression.-Elayyan, J., Lee, E.-J., Gabay, O., Smith, C. A., Qiq, O., Reich, E., Mobasheri, A., Henrotin, Y., Kimber, S. J., Dvir-Ginzberg, M. LEF1-mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes. FASEB J. 31, 3116-3125 (2017). www.fasebj.orgArticular cartilage undergoes age-related degenerative changes leading to the joint disease osteoarthritis (OA). Gradual loss of hyaline joint cartilage during OA is evoked through chronic synovitis, which involves the accumulation of proinflammatory cytokines, as IL1b, TNF-a, and IL6 within the joint (1-5), subsequently leading to a steady increase in matrix metalloproteinases (MMPs) and a disintegrin and MMP with thrombospondin motifs (ADAMTS) proteins, which further promote cartilage destruction (6-12). Recent experimental attempts ABBREVIATIONS: ACAN, aggrecan gene; ADAMTS, a disintegrin and metalloproteinase with thrombospondin-like motifs; BMP, bone morphogenic protein; ChIP, chromatin immunoprecipitation; COL2A1, collagen-2 a-1 gene; FBS, fetal bovine serum; FGF, fibroblast growth factor; GDF, growth differentiation factor; GSK3b, glycogen synthase kinase-3b; hESC, human embryonic stem cell; KO, knockout; LEF, lymphoid enhancer factor; MMP, matrix metalloproteinase; NAM, nicotinamide adenine mononucleotide; OA, osteoarthritis; Res, resveratrol; qPCR, quantitative PCR; siRNA, small interfering RNA; SIRT1, silent mating type information regulation 2 homolog 1 (sirtuin-1); SOX9, sex-determining-region-on-the-Y-chromosome-relate...

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Section: The Transcription Factor Lef1 Is a Transactivator Of Mmp13 Amentioning

confidence: 99%

Section: Direct Chondrogenesis Differentiation Protocolmentioning

confidence: 99%

Section: Direct Chondrogenesis Differentiation Protocolmentioning

confidence: 99%

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LEF1‐mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes

et al. 2017

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“…To further explore this possibility, we compared the global gene expression profiles of TRA-1-60 ( þ ) cells purified on various days, original HDFs, established iPSCs and differentiated progenies (endoderm, EN; mesoderm, ME; neuroectoderm, NE; and primitive streak-like mesendoderm [11][12][13][14] , PSMN from ESCs/ iPSCs using DNA microarrays ( Supplementary Fig. 4).…”

Section: Resultsmentioning

confidence: 99%

Induction of pluripotency in human somatic cells via a transient state resembling primitive streak-like mesendoderm

et al. 2014

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During mammalian embryonic development, the primitive streak initiates the differentiation of pluripotent epiblast cells into germ layers. Pluripotency can be reacquired in committed somatic cells using a combination of a handful of transcription factors, such as OCT3/4, SOX2, KLF4 and c-MYC (hereafter referred to as OSKM), albeit with low efficiency. Here we show that during OSKM-induced reprogramming towards pluripotency in human cells, intermediate cells transiently show gene expression profiles resembling mesendoderm, which is a major component of the primitive streak. Based on these findings, we discover that forkhead box H1 (FOXH1), a transcription factor required for anterior primitive streak specification during early development, significantly enhances the reprogramming efficiency of human fibroblasts by promoting their maturation, including mesenchymal to epithelial transition and the activation of late pluripotency markers. These results demonstrate that during the reprogramming process, human somatic cells go through a transient state that resembles mesendoderm.

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“…We further demonstrate that the same technique has potential use for repairing defects in native articular cartilage, as evidenced by the formation of cartilage-cartilage interface with integration strengths of 400 kPa. Conceivably, the same technique could be extended to bioengineer other tissues (39), such as tendon or meniscus, and to the use of other cell sources, such as embryonic and induced pluripotent stem cells (40,41).…”

Section: Discussionmentioning

confidence: 99%

Large, stratified, and mechanically functional human cartilage grown in vitro by mesenchymal condensation

Bhumiratana

Eton

Oungoulian

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

171

189

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The efforts to grow mechanically functional cartilage from human mesenchymal stem cells have not been successful. We report that clinically sized pieces of human cartilage with physiologic stratification and biomechanics can be grown in vitro by recapitulating some aspects of the developmental process of mesenchymal condensation. By exposure to transforming growth factor-β, mesenchymal stem cells were induced to condense into cellular bodies, undergo chondrogenic differentiation, and form cartilagenous tissue, in a process designed to mimic mesenchymal condensation leading into chondrogenesis. We discovered that the condensed mesenchymal cell bodies (CMBs) formed in vitro set an outer boundary after 5 d of culture, as indicated by the expression of mesenchymal condensation genes and deposition of tenascin. Before setting of boundaries, the CMBs could be fused into homogenous cellular aggregates giving rise to well-differentiated and mechanically functional cartilage. We used the mesenchymal condensation and fusion of CMBs to grow centimeter-sized, anatomically shaped pieces of human articular cartilage over 5 wk of culture. For the first time to our knowledge biomechanical properties of cartilage derived from human mesenchymal cells were comparable to native cartilage, with the Young's modulus of >800 kPa and equilibrium friction coeffcient of <0.3. We also demonstrate that CMBs have capability to form mechanically strong cartilage-cartilage interface in an in vitro cartilage defect model. The CMBs, which acted as "lego-like" blocks of neocartilage, were capable of assembling into human cartilage with physiologic-like structure and mechanical properties.

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Directed differentiation of human embryonic stem cells toward chondrocytes

Cited by 276 publications

References 57 publications

LEF1‐mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes

LEF1‐mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes

Induction of pluripotency in human somatic cells via a transient state resembling primitive streak-like mesendoderm

Large, stratified, and mechanically functional human cartilage grown in vitro by mesenchymal condensation

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