This work was supported by the following grants: Natural Science Foundation of China (No. 81572198; No. 81260161; No. 81000460); Natural Science Foundation of Guangdong Province, China (No. 2015A030313772); China Postdoctoral Science Foundation Funded Project (No. 2013M530385); The Medical Research Foundation of Guangdong Province, China (No. A2016314); Shenzhen Science and Technology Projects (No. JCYJ20160301111338144; No. JSGG20151030140325149; No. JSGG20140519105550503; No.GJHZ20130412153906739; No. JCYJ20140414170821160; No.JCYJ20140414170821200).

This study was approved by the Human Ethics Committee of Shenzhen Second People&#39;s Hospital.
1. Human Articular Cartilage Tissue Collection and Chondrocyte Culture
<ol>
	<li>Preparation of materials
	<ol>
		<li>Prepare Dulbecco&#39;s modified eagle medium (DMEM) medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Prepare 1 mg/mL collagenase II, 0.25% trypsin-EDTA, phosphate-buffered saline (PBS), and a cell strainer (40 &#181;m nylon).</li>
	</ol>
	</li>
	<li>Human articular cartilage tissue collection
	<ol>
		<li>Isolate articular cartilage from the knee joints of donor patients after trauma. Obtain informed consent from all participants.</li>
		<li>Dice the cartilage into 1-2 mm3 pieces using a sterile scalpel and digest chondrocytes from the minced cartilage with 1 mg/mL collagenase II in DMEM at 37 &#176;C for 12-16 h.</li>
		<li>Filter the resulting cell suspension through a cell strainer (40 &#181;m) and wash twice with PBS.</li>
		<li>Count cells with a hemocytometer and seed at a density of 20,000-30,000 cells/cm2 in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.</li>
		<li>Culture in an incubator at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Monolayer chondrocyte expansion
	<ol>
		<li>Harvest sub-confluent cells using 0.25% trypsin-EDTA and re-plate at a density of 6,600 cells/cm2. Change the medium twice a week.</li>
		<li>Culture chondrocytes in monolayers for up to six passages and assess at passages 1, 2, 3, 4 and 5.</li>
	</ol>
	</li>
</ol>
2. Genomic DNA (gDNA) Extraction
<ol>
	<li>Collect cells and resuspend in 500 &#181;L lysis buffer (15 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 0.5% SDS, 200 &#181;g/mL RNase A) per 10 million cells. Resuspend cells by pipetting and rapid inversion, and then incubate for 1 h at 37 &#176;C.</li>
	<li>Add proteinase K at a concentration of 160 &#181;g/mL of cell lysate and invert the mixture vigorously. Incubate 6 h at 55 &#176;C.</li>
	<li>Add one volume of Tris pH 7.9 saturated phenol:chloroform:isoamyl alcohol (25:24:1) to the sample. Vortex or shake the sample by hand thoroughly for approximately 20 s.</li>
	<li>Centrifuge the sample at room temperature for 5 min at 13,000 &#215; g. Remove the upper aqueous phase and transfer the layer to a fresh tube.</li>
	<li>Extract with an equal volume of chloroform to remove phenol and transfer the top aqueous phase to a fresh tube.</li>
	<li>Precipitate DNA by adding 0.1 sample volume of 3 M sodium acetate pH 8.0 and 2 volumes of 100% ethanol.</li>
	<li>Store the tube at -20 &#176;C overnight to precipitate the gDNA.</li>
	<li>Centrifuge the sample at 4 &#176;C for 10 min at 16,000 x g to pellet gDNA.</li>
	<li>Wash the sample three times with 70% ethanol, and centrifuge at 4 &#176;C for 2 min at 13,000 x g.</li>
	<li>Remove the supernatant carefully and then air-dry. Resuspend the DNA in 10 mM Tris pH 8.0, 0.1 mM EDTA.</li>
</ol>
3. DNA Methylation Profiling
<ol>
	<li>Use a DNA methylation kit to perform the bisulfite conversion reaction using a total of 500 ng of the genomic DNA, according to manufacturer&#39;s protocol. Elute in 10 &#181;L of elution buffer (50 ng/&#181;L).</li>
	<li>Perform the DNA methylation profiling using a commercial kit according to manufacturer&#39;s protocol.</li>
</ol>
4. Dot Blot Analysis
<ol>
	<li>Denature the isolated DNA (1 mg per sample) in 0.1 M NaOH for 10 min at 95 &#176;C. Neutralize the DNA with 1 M NH4OAc on ice, and then dilute two-fold. Spot 2 &#181;L of the serial diluted genomic DNA on an N+ membrane.</li>
	<li>Blot the membrane at 80 &#176;C for 30 min.</li>
	<li>Block non-specific antibody binding sites by soaking the N+ membrane in 5% BSA in TBS-T for 1 h. Use a 10 cm Petri dish as a reaction chamber at room temperature.</li>
	<li>After washing 5 min three times in TBST, incubate the membrane with a mouse anti-5-methylcytosine (5-mC) monoclonal antibody (1:1,000) in TBS-T at 4 &#176;C overnight.</li>
	<li>Wash the membrane for 5 min three times in TBS-T, and then incubate with a secondary antibody, HRP-conjugated sheep anti-mouse immunoglobulin-G (IgG) (1:5,000) in TBS-T for 1 h at room temperature.</li>
	<li>Wash the membrane for 5 min three times in TBS-T.</li>
	<li>Add the enzyme substrate to the membrane and incubate for 5-10 min. Visualize the secondary antibody signal using a chemiluminescence kit according to the manufacturer&#39;s instructions.</li>
</ol>

<ol>
	<li>Brittberg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+deep+cartilage+defects+in+the+knee+with+autologous+chondrocyte+transplantation.">Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation.</a> N Engl J Med. 331, 889-895 (1994).</li><li>Pareek, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Term+Outcomes+After+Autologous+Chondrocyte+Implantation:+A+Systematic+Review+at+Mean+Follow-Up+of+11.4+Years.">Long-Term Outcomes After Autologous Chondrocyte Implantation: A Systematic Review at Mean Follow-Up of 11.4 Years.</a> Cartilage. 7 (4), 298-308 (2016).</li><li>Duan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+networking+of+chondrocyte+dedifferentiation+in+vitro+and+its+implications+for+cell-based+cartilage+therapy.">Cytokine networking of chondrocyte dedifferentiation in vitro and its implications for cell-based cartilage therapy.</a> Am J Transl Res. 7 (2), 194-208 (2015).</li><li>Ma, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+dedifferentiated+human+articular+chondrocytes+in+monolayer+culture.">Gene expression profiling of dedifferentiated human articular chondrocytes in monolayer culture.</a> Osteoarthritis Cartilage. 21 (4), 599-603 (2013).</li><li>Duan, L., Liang, Y., Ma, B., Zhu, W., Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+in+chondrocyte+phenotype+maintenance+for+cell-based+cartilage+repair.">Epigenetic regulation in chondrocyte phenotype maintenance for cell-based cartilage repair.</a> Am J Transl Res. 7 (11), 2127-2140 (2015).</li><li>Duan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+profiling+in+chondrocyte+dedifferentiation+in+vitro.">DNA methylation profiling in chondrocyte dedifferentiation in vitro.</a> J Cell Physiol. , (2016).</li><li>Bhat, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+detection+at+single+base+resolution+using+targeted+next+generation+bisulfite+sequencing+and+cross+validation+using+capillary+sequencing.">DNA methylation detection at single base resolution using targeted next generation bisulfite sequencing and cross validation using capillary sequencing.</a> Gene. , (2016).</li><li>Shi, X. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+Genome-wide+DNA+Methylation+Profiles+Altered+in+Kashin-Beck+Disease+Using+Infinium+Human+Methylation+450+Bead+Chips.">Exploring Genome-wide DNA Methylation Profiles Altered in Kashin-Beck Disease Using Infinium Human Methylation 450 Bead Chips.</a> Biomed Environ Sci. 29 (7), 539-543 (2016).</li><li>Li, X. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+an+HPLC+Method+for+Determining+the+Genomic+Methylation+Levels+of+Taxus+Cells.">Optimization of an HPLC Method for Determining the Genomic Methylation Levels of Taxus Cells.</a> J Chromatogr Sci. 54 (2), 200-205 (2016).</li><li>Maghbooli, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+DNA+methylation+as+a+possible+biomarker+fordiabetic+retinopathy.">Global DNA methylation as a possible biomarker fordiabetic retinopathy.</a> Diabetes Metab Res Rev. 31 (2), 183-189 (2015).</li><li>Barlic, A., Drobnic, M., Malicev, E., Kregar-Velikonja, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+gene+expression+in+human+articular+chondrocytes+assigned+for+autologous+implantation.">Quantitative analysis of gene expression in human articular chondrocytes assigned for autologous implantation.</a> J Orthop Res. 26 (6), 847-853 (2008).</li><li>Legendre, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+hyaline+cartilage+matrix+synthesis+in+collagen+sponge+scaffolds+by+using+siRNA+to+stabilizechondrocytes+phenotype+cultured+with+bone+morphogenetic+protein-2+under+hypoxia.">Enhanced hyaline cartilage matrix synthesis in collagen sponge scaffolds by using siRNA to stabilizechondrocytes phenotype cultured with bone morphogenetic protein-2 under hypoxia.</a> Tissue Eng Part C Methods. 19 (7), 550-567 (2013).</li><li>Niethammer, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+autologous+chondrocyte+quality+of+matrix-based+autologous+chondrocyte+implantation+in+the+knee+joint.">Analysis of the autologous chondrocyte quality of matrix-based autologous chondrocyte implantation in the knee joint.</a> Int Orthop. 40 (1), 205-212 (2016).</li><li>Hayatsu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bisulfite+genomic+sequencing+used+in+the+analysis+of+epigenetic+states,+a+technique+in+the+emerging+environmental+genotoxicology+research.">The bisulfite genomic sequencing used in the analysis of epigenetic states, a technique in the emerging environmental genotoxicology research.</a> Mutat Res. 659 (1-2), 77-82 (2008).</li><li>Haque, N., Nishiguchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisulfite+sequencing+for+cytosine-methylation+analysis+in+plants.">Bisulfite sequencing for cytosine-methylation analysis in plants.</a> Methods Mol Biol. 744, 187-197 (2011).</li><li>Ananiev, G. 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The authors declare that they have no competing financial interests.

Chondrocyte dedifferentiation in vitro severely compromises the outcome of ACI in the treatment of cartilage defect repair11,12. To optimize ACI outcome, it is crucial to avoid the use of dedifferentiated chondrocytes13. Studies have suggested that general DNA methylation level is associated with the extent of the chondrocyte dedifferentiation4,6. Thus, it is imperative to...

Autologous chondrocyte implantation (ACI) is a relatively new, state-of-the-art procedure to treat articular cartilage defects1,2. One of the crucial steps in ACI is amplification of chondrocytes via monolayer culture in vitro. During amplification, the hyaline chondrocytes easily lose their phenotype and become dedifferentiated, which is undesirable for ACI treatment3,4. To optimize the outcome of ACI treatment, the extent of chondrocyte dedifferentiation should be determined before replantation. It is imperative to establish an economic and rapid way to determine the chondrocytes&#39; status. Recently, the association between DNA methylation and chondrocyte dedifferentiation has attracted much attention4,5,6. DNA methylation is a process by which methyl groups are added to DNA, resulting in the conversion of cytosine residues to 5-methylcytosine (5-mC).
To elucidate the biology of DNA methylation in chondrocyte dedifferentiation, the first step is to evaluate the DNA methylation level of chondrocytes, which so far has proven challenging. Bisulfite genomic sequencing is the most widely used technique to analyze DNA methylation7,8. In this assay, bisulfite conversion causes DNA degradation, and thus a substantial amount of sample must be provided for the assay. Also, high-performance liquid chromatography (HPLC) has been used to quantify global DNA methylation levels9,10. However, HPLC analysis requires genomic DNA digestion. Furthermore, advanced and expensive experimental instruments are required. Therefore, in addition to the high cost, these experimental procedures are time consuming. Anti-5-mC antibodies have now become commercially available, which has created the possibility for immune blotting of 5-mC-containing genomic DNA from complex genomes.
In this report, we extracted genomic DNA from chondrocytes grown in a series of monolayer cultures. We used a dot blot assay to evaluate the 5-mC content in human chondrocytes with different numbers of passages. We found that 5-mC content was increased in highly dedifferentiated chondrocytes compared to chondrocytes with low-grade dedifferentiation. Additionally, we identified a relationship between dedifferentiation status and 5-mC levels. Finally, we reported that the changes in 5-mC content were associated with the chondrocyte phenotype. Therefore, the 5-mC dot blot assay is a reliable, simple, and rapid method to detect the DNA methylation level in chondrocytes.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM</td><td>Gibco Inc.</td><td>11965&amp;ndash;092</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>HyClone Inc.</td><td>SH30256.01B</td><td>D-PBS, free of Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>FBS</td><td>Gibco Inc.</td><td>10099-141</td><td></td></tr><tr><td>0.25% Trypsin/EDTA</td><td>Gibco Inc.</td><td>25200-056</td><td></td></tr><tr><td>1% Penicillin-Streptomycin</td><td>Gibco Inc.</td><td>15140-122</td><td></td></tr><tr><td>Chloroform</td><td>Mallinckrodt</td><td>4440</td><td></td></tr><tr><td>Isoamyl Alcohol</td><td>Sigma</td><td>I-3643</td><td></td></tr><tr><td>Phenol</td><td>Gibco BRL</td><td>15513-039</td><td></td></tr><tr><td>Proteinase K</td><td>Gibco BRL</td><td>24568-2</td><td></td></tr><tr><td>TAE buffer&amp;nbsp;</td><td>Bio Whittaker</td><td>16-011V</td><td></td></tr><tr><td>Distilled Water</td><td>Gibco BRL</td><td>15230-170</td><td></td></tr><tr><td>1 M Tris-HCl</td><td>Biosharp Inc.</td><td>BL514A</td><td></td></tr><tr><td>Tween20</td><td>Biotopped Inc.</td><td>C58H114O26</td><td></td></tr><tr><td>BSA</td><td>Proliant Inc.</td><td>68700</td><td></td></tr><tr><td>Collagenase, Type II</td><td>Sigma-Aldrich</td><td>C6885</td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td>ISOLAB Inc.</td><td>075.03.001</td><td></td></tr><tr><td>Falcon 100 mm dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>Centrifuge Tubes</td><td>TPP AG</td><td>91050</td><td>Gamma-sterilized</td></tr><tr><td>High-speed centrifuge</td><td>Eppendorf</td><td>5804R</td><td></td></tr><tr><td>ThermoMixer</td><td>MIULAB</td><td>MTH-100</td><td></td></tr><tr><td>Carbon dioxide cell incubator</td><td>Thermo scientific</td><td>3111</td><td></td></tr><tr><td>Chemi-imaging Analyse System</td><td>UVITEC Cambridge</td><td>ALLIANCE</td><td></td></tr></tbody></table>

a 5-mc dot blot assay quantifying the dna methylation level of chondrocyte dedifferentiation in vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

Chondrocytes were cultured in a monolayer up to passage 6 (P6). Chondrocytes showed progressive phenotypic changes with successive passages of the monolayer culture. The P0 chondrocyte morphology was round, whereas the cells became highly pinched and flattened with successive passages up to P6 (Figure 1). This morphology change is typical of the chondrocyte dedifferentiation process. Meanwhile, results of the heat map indicated that the general methylation level of CpG si...

Watch this Scientific Journal Video about A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro at JoVE.com

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

a-5-mc-dot-blot-assay-quantifying-dna-methylation-level-chondrocyte

Research

JoVE Journal

Developmental Biology

16.7K Views.  Guangzhou Medical University.  Wij presenteren een methode om DNA-methylering te kwantificeren op basis van de 5-methylcytosine (5-mC) dot blot. We bepalen de 5-mC niveaus tijdens chondrocyten dedifferentiatie. Deze eenvoudige techniek kan gebruikt worden om snel het chondrocytfenotype in ACI-behandeling te bepalen. 

Video: A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Genetics

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many lineages for regenerative medicine. ES cell culture in vitro has long been the subject of widespread concerns. Classically, mouse ES cells are maintained in serum and leukemia inhibitory factor (LIF)-containing medium. However, under serum/LIF conditions, cells show heterogeneity in morphology and the expression profile of pluripotency-related genes, and are mostly in a metastable state. Moreover, cultured ES cells exhibit global hypermethylation, but na&#239;ve ES cells of the inner cell mass (ICM) and primordial germ cells (PGCs) are in a state of global hypomethylation. The hypomethylated state of ICM and PGCs is closely associated with their pluripotency. To improve mouse ES cell culture methods, we have recently developed a new method based on the selectively combined utilization of two small-molecule compounds to maintain the DNA hypomethylated and pluripotent state. Here, we present that the co-treatment of vitamin C (Vc) and PD0325901 can erase about 90% of 5-methylcytosine (5mC) at 5 days in mouse ES cells. The generated 5mC content is comparable to that in PGCs. The mechanistic investigation shows that PD0325901 up-regulates Prdm14 expression to suppress Dnmt3b (de novo DNA methyltransferase) and Dnmt3l (the cofactor of Dnmt3b), by reducing de novo 5mC synthesis. Vc facilitates the conversion of 5mC to 5-hydroxymethylcytosine (5hmC) catalyzed mainly by Tet1 and Tet2, indicating the involvement of both passive and active DNA demethylations. Moreover, under Vc/PD0325901 conditions, mouse ES cells show homogeneous morphology and pluripotent state. Collectively, we propose a novel and chemical-synergy culture method for achieving DNA hypomethylation and maintenance of pluripotency in mouse ES cells. The small-molecule chemical-dependent method overcomes the major shortcomings of serum culture, and holds promise to generate homogeneous ES cells for further clinical applications and researches.

We described in detail two chemical-based protocols for culturing mouse embryonic stem cells. This new method utilizes synergistic mechanisms of promoting Tet-mediated oxidation (by vitamin C) and repressing de novo synthesis of 5-methylcytosine (by PD0325901) to maintain DNA hypomethylation and pluripotency of mouse embryonic stem cells.

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many ...

Biochemistry

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control cellular functions. The reversal of DNA methylation, or DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family of dioxygenases. Although it has been widely reported that aberrant DNA methylation and demethylation are associated with developmental defects and cancer, how these epigenetic changes directly contribute to the subsequent alteration in gene expression or disease progression remains unclear, largely owing to the lack of reliable tools to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution. To overcome this hurdle, we designed a split-TET2 enzyme to enable temporal control of 5-methylcytosine (5mC) oxidation and subsequent remodeling of epigenetic states in mammalian cells by simply adding chemicals. Here, we describe methods for introducing a chemical-inducible epigenome remodeling tool (CiDER), based on an engineered split-TET2 enzyme, into mammalian cells and quantifying the chemical inducible production of 5-hydroxymethylcytosine (5hmC) with immunostaining, flow cytometry or a dot-blot assay. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype&#8722;phenotype relations in various biological systems.

Detailed protocols for introducing an engineered split-TET2 enzyme (CiDER) into mammalian cells for chemical inducible DNA hydroxymethylation and epigenetic remodeling are presented.

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control ...

Chemistry

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

Biology

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Neuroscience

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Chondroprogenitor and Chondrocyte Isolation from Human Articular Cartilage

Isolation of Chondrocytes and Chondroprogenitors Using Fibronectin Adhesion and Migratory Assay

Chondroprogenitor cells (CPCs), recently identified as a distinct subpopulation, exhibit promise due to their mesenchymal properties, heightened chondrogenesis, and limited hypertrophic traits. The enrichment of progenitors is achieved through differential fibronectin adhesion and migration-based explant assays, with Fibronectin Adhesion Assay-derived Chondroprogenitors (FAA-CPs) and Migratory Chondroprogenitors (MCPs) demonstrating superior potential compared to chondrocytes. This article delves into the details of isolating resident cartilage-derived cells, namely chondrocytes and chondroprogenitors. While valuable insights from chondrocyte research contribute to our understanding of cartilage repair, ongoing efforts are directed toward the use of chondroprogenitors and exploring their potential as an alternative therapeutic approach. Additionally, this methodology article provides a detailed step-by-step protocol for isolating three specific cell types from cartilage: chondrocytes, FAA-CPs, and MCPs. By following standardized procedures, this protocol facilitates the successful extraction of these cell subtypes. Grounded in extensive research, the article focuses on the intricate techniques utilized in isolating the different subsets and the optimized culture conditions required to expand and maintain their cultures. The methodology encompasses enzymatic isolation of human articular cartilage-derived chondrocytes, differential fibronectin adhesion following sequential enzymatic digestion, and migration-based explant assays to obtain cartilage-resident cells.

This protocol details the isolation of chondrocytes, Fibronectin Adhesion Assay-derived Chondroprogenitors (FAA-CPs), and Migratory Chondroprogenitors (MCPs) from human articular cartilage. It covers enzymatic digestion, fibronectin adhesion, and migration-based assays for isolating and characterizing these cells.

Chondroprogenitor cells (CPCs), recently identified as a distinct subpopulation, exhibit promise due to their mesenchymal properties, heightened ...

5-Methylcytosine Dot Blot to Determine the Extent of DNA Methylation

Source: Jia, Z. et al., <a href="https://app.jove.com/v/55565">A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro.</a>&#160;J. Vis. Exp.&#160;(2017)
This video describes the technique of dot-blot to determine the level of DNA methylation in human chondrocytes in vitro via the blotting of DNA samples on a nylon membrane and the subsequent visualization using monoclonal antibodies. This helps to determine the chondrocytes&#39; phenotype at various stages in culture.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

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