Neo-cartilage engineered from primary chondrocytes is epigenetically similar to autologous cartilage, in contrast to using mesenchymal stem cells

Hollander, Wouter den; Suchiman, H. Eka D.; Houtman, E.; Slieker, Roderick C.; Heijmans, Bastiaan T.; Slagboom, P. Eline; Nelissen, Rob G H H; Ramos, Yolande F M; Meulenbelt, Ingrid

doi:10.1016/j.joca.2016.03.009

Cited by 33 publications

(33 citation statements)

References 49 publications

(44 reference statements)

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“…By RBBS only limited changes in promoter methylation were identified during MSC chondrogenesis in a 3D alginate model (Herlofsen et al , 2013), but more recently by Infinium 450K methylation array 1116 differentially methylated CpGs were identified across a timecourse of chondrogenic differentiation in a pellet model, albeit starting at Dayl4 (to Day49) in contrast to our comparison of undifferentiated vs. differentiated cells (Day0 vs. Dayl4) (Bomer et al , 2016). The vast majority of differentially methylated CpGs during chondrogenesis herein become hypomethylated indicative of a loss of repression at these locations.…”

Section: Discussionmentioning

confidence: 56%

“…In contrast DNA methylation at gene promoters, assessed by reduced representation bisulfite sequencing (RBBS), did not correlate with gene expression (Herlofsen et al , 2013). Utilising the Infinium 450K methylation array the effect of ageing on cartilage DNA methylation and the similarity between MSC-derived cartilage methylation in comparison with cartilage engineered from articular chondrocytes have been studied (Bomer et al , 2016; Peffers et al , 2016).…”

Section: Introductionmentioning

confidence: 99%

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DNA hypomethylation during MSC chondrogenesis occurs predominantly at enhancer regions

Barter

Bui

Cheung

et al. 2019

Preprint

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SummaryRegulation of transcription occurs in a cell type specific manner by epigenetic mechanisms including DNA methylation and histone modifications. Methylation changes during stem cell differentiation may play a key role in lineage specification. We sought to characterise DNA methylation changes during chondrogenesis of mesenchymal stem cells (MSCs) in order to further our understanding of epigenetic regulation in chondrocytes. The consequences of which has potential to improve cartilage generation for tissue engineering purposes and also to provide context for observed methylation changes in cartilage diseases such as osteoarthritis. We identified significant DNA hypomethylation during chondrogenesis including changes at many key cartilage gene loci. Importantly characterisation of significant CpG loci indicated their predominant localisation to enhancer regions. Comparison with adult cartilage and other tissue methylation profiles identified chondrocyte-specific regulatory regions. Taken together we have associated methylation at many CpGs with the chondrocyte phenotype.AbstractRegulation of transcription is determined in a cell type specific manner by epigenetic mechanisms including DNA methylation and histone modifications. Methylation changes during stem cell differentiation may play a role in lineage specification. Multipotent mesenchymal stem cell (MSC) differentiation into chondrocytes not only serves as a model for chondrocyte development but also provides an important source of cartilage for tissue engineering purposes. We sought to characterise DNA methylation changes during chondrogenesis to further understanding of epigenetic regulation but to also provide context for the changes identified during disease.DNA cytosine methylation changes during human MSC differentiation into chondrocytes were measured by Infinium 450K methylation array. Methylation changes at gene loci were contrasted with gene expression changes. Chromatin states of significant methylation loci were interpreted by intersection with chondrogenesis histone modification ChlP-seq data. Chondrogenic and cartilage specific hypomethylation was utilised in order to identify a chondrocyte methylome. Articular cartilage and tissue panel DNA methylation was compared and alterations during osteoarthritis cartilage disease classified.Significant DNA hypomethylation was identified following chondrogenic differentiation of MSCs including changes at many key cartilage gene loci. Highly upregulated genes during chondrogenesis were more likely to exhibit a reduction in DNA methylation. Characterisation of significant CpG loci indicated their predominant localisation in CpG poor regions which importantly are most likely to correspond to enhancer regions. Methylation level at certain CpGs following chondrogenesis corresponds to the level found in adult cartilage.Taken together, considerable demethylation changes to the epigenetic landscape occur during MSC chondrogenesis especially at sites marked by enhancer modifications. Comparison with other tissues, including healthy and OA cartilage, associates CpGs to the chondrocyte phenotype and provides context for changes in disease.

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Section: Discussionmentioning

confidence: 56%

Section: Introductionmentioning

confidence: 99%

DNA hypomethylation during MSC chondrogenesis occurs predominantly at enhancer regions

Barter

Bui

Cheung

et al. 2019

Preprint

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“…Cartilage tissue engineering is no exception, and hence large scale expansion of chondrocytes is required either for novel scaffold testing, determination of potential cytotoxic effects of medical devices and implants for orthopaedic use (Bomer et al, 2016; Camarero-Espinosa et al, 2016; Makris et al, 2015). Cultivation of such high cell counts is a demanding task, especially considering the low number of obtained cells in the primary culture, and an often limited amount of available tissue.…”

Section: Discussionmentioning

confidence: 99%

“…Over the past decade a number of viable options of cartilage regeneration have been introduced into clinical practice (Camarero-Espinosa et al, 2016; Hettrich, Crawford & Rodeo, 2008; Schrobback et al, 2011). Among these, autologous chondrocyte implantation (ACI) seems the most promising since it relies on the use of biodegradable materials that serve as temporary cell-carriers, enabling in vitro cell growth and subsequent implantation into the defective cartilage (Bomer et al, 2016; Niemeyer et al, 2016; Robb et al, 2012). …”

Section: Introductionmentioning

confidence: 99%

Isolation and characterization of human articular chondrocytes from surgical waste after total knee arthroplasty (TKA)

Naranda

Gradišnik

Gorenjak

et al. 2017

PeerJ

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BackgroundCartilage tissue engineering is a fast-evolving field of biomedical engineering, in which the chondrocytes represent the most commonly used cell type. Since research in tissue engineering always consumes a lot of cells, simple and cheap isolation methods could form a powerful basis to boost such studies and enable their faster progress to the clinics. Isolated chondrocytes can be used for autologous chondrocyte implantation in cartilage repair, and are the base for valuable models to investigate cartilage phenotype preservation, as well as enable studies of molecular features, nature and scales of cellular responses to alterations in the cartilage tissue.MethodsIsolation and consequent cultivation of primary human adult articular chondrocytes from the surgical waste obtained during total knee arthroplasty (TKA) was performed. To evaluate the chondrogenic potential of the isolated cells, gene expression of collagen type 2 (COL2), collagen 1 (COL1) and aggrecan (ACAN) was evaluated. Immunocytochemical staining of all mentioned proteins was performed to evaluate chondrocyte specific production.ResultsCartilage specific gene expression of COL2 and ACAN has been shown that the proposed protocol leads to isolation of cells with a high chondrogenic potential, possibly even specific phenotype preservation up to the second passage. COL1 expression has confirmed the tendency of the isolated cells dedifferentiation into a fibroblast-like phenotype already in the second passage, which confirms previous findings that higher passages should be used with care in cartilage tissue engineering. To evaluate the effectiveness of our approach, immunocytochemical staining of the evaluated chondrocyte specific products was performed as well.DiscussionIn this study, we developed a protocol for isolation and consequent cultivation of primary human adult articular chondrocytes with the desired phenotype from the surgical waste obtained during TKA. TKA is a common and very frequently performed orthopaedic surgery during which both femoral condyles are removed. The latter present the ideal source for a simple and relatively cheap isolation of chondrocytes as was confirmed in our study.

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“…Primary chondrocytes were isolated from three independent donors and passaged twice or thrice, as described in [23]. Chondrocytes were transfected in duplo with antisense locked nucleic acid (LNA) GapmeR (Qiagen) targeting P3H2-AS1 (TGAGCAACTAGGTGTA) or GapmeR negative control (AACACGTCTATACGC) at 10 nM final concentration using Lipofectamine RNAiMax Transfection Reagent (Invitrogen) according to manufacturer's protocol.…”

Section: In Vitro Downregulation Lncrna Using Locked Nucleic Acid Gapmentioning

confidence: 99%

Elucidating another level of epigenetic regulation in osteoarthritis by identifying functionalcis-acting long non-coding RNAs and their targets in articular cartilage

Hoolwerff

Metselaar

Tuerlings

et al. 2020

Preprint

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Objective To identify robustly differentially expressed long non-coding RNAs (lncRNAs) with osteoarthritis (OA) pathophysiology in cartilage. Moreover to explore potential target mRNAs by establishing co-expression networks, followed by functional validation.Methods RNA sequencing was performed on macroscopically lesioned and preserved OA cartilage of patients who underwent a joint replacement surgery due to OA (N=98). Differential expression (DE) analysis was performed on lncRNAs that were annotated in GENCODE and Ensembl. To identify potential interactions, correlations were calculated between the identified DE lncRNAs and previously reported DE protein-coding genes in the same samples.Modulation of chondrocyte lncRNA expression was achieved using LNA GapmeRs.Results By applying our in-house pipeline we identified 5,053 lncRNAs to be robustly expressed, of which 191 were FDR significant differentially expressed between lesioned and preserved OA cartilage. Upon integrating mRNA sequencing data, we showed that intergenic and antisense DE lncRNAs show high, positive correlations with their flanking, respectively, sense genes. To functionally validate this observation we selected P3H2-AS1, which was downregulated in primary chondrocytes, resulting in downregulation of P3H2 gene expression levels. As such, we can confirm that P3H2-AS1 regulates its sense gene P3H2.Conclusion By applying an improved detection strategy, robustly differentially expressed lncRNAs in OA cartilage were detected. Integration of these lncRNAs with differential mRNA expression levels in the same samples showed insight into their regulatory networks. Our data signifies that intergenic, as well as antisense lncRNAs play an important role in regulating the pathophysiology of OA.

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Neo-cartilage engineered from primary chondrocytes is epigenetically similar to autologous cartilage, in contrast to using mesenchymal stem cells

Cited by 33 publications

References 49 publications

DNA hypomethylation during MSC chondrogenesis occurs predominantly at enhancer regions

DNA hypomethylation during MSC chondrogenesis occurs predominantly at enhancer regions

Isolation and characterization of human articular chondrocytes from surgical waste after total knee arthroplasty (TKA)

Elucidating another level of epigenetic regulation in osteoarthritis by identifying functionalcis-acting long non-coding RNAs and their targets in articular cartilage

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