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

Summary

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.

Abstract

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'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.

Introduction

Autologous chondrocyte implantation (ACI) is a relatively new, state-of-the-art procedure to treat articular cartilage defects^1,2. 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 treatment^3,4. 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' status. Recently, the association between DNA methylation and chondrocyte dedifferentiation has attracted much attention^4,5,6. 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 methylation^7,8. 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 levels^9,10. 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.

Protocol

This study was approved by the Human Ethics Committee of Shenzhen Second People's Hospital.

1. Human Articular Cartilage Tissue Collection and Chondrocyte Culture

1. Preparation of materials
   1. Prepare Dulbecco'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 µm nylon).
2. Human articular cartilage tissue collection
   1. Isolate articular cartilage from the knee joints of donor patients after trauma. Obtain informed consent from all participants.
   2. 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 °C for 12-16 h.
   3. Filter the resulting cell suspension through a cell strainer (40 µm) and wash twice with PBS.
   4. 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.
   5. Culture in an incubator at 37 °C.
3. Monolayer chondrocyte expansion
   1. 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.
   2. Culture chondrocytes in monolayers for up to six passages and assess at passages 1, 2, 3, 4 and 5.

2. Genomic DNA (gDNA) Extraction

1. Collect cells and resuspend in 500 µL lysis buffer (15 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 0.5% SDS, 200 µg/mL RNase A) per 10 million cells. Resuspend cells by pipetting and rapid inversion, and then incubate for 1 h at 37 °C.
2. Add proteinase K at a concentration of 160 µg/mL of cell lysate and invert the mixture vigorously. Incubate 6 h at 55 °C.
3. 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.
4. Centrifuge the sample at room temperature for 5 min at 13,000 × g. Remove the upper aqueous phase and transfer the layer to a fresh tube.
5. Extract with an equal volume of chloroform to remove phenol and transfer the top aqueous phase to a fresh tube.
6. Precipitate DNA by adding 0.1 sample volume of 3 M sodium acetate pH 8.0 and 2 volumes of 100% ethanol.
7. Store the tube at -20 °C overnight to precipitate the gDNA.
8. Centrifuge the sample at 4 °C for 10 min at 16,000 x g to pellet gDNA.
9. Wash the sample three times with 70% ethanol, and centrifuge at 4 °C for 2 min at 13,000 x g.
10. Remove the supernatant carefully and then air-dry. Resuspend the DNA in 10 mM Tris pH 8.0, 0.1 mM EDTA.

3. DNA Methylation Profiling

1. Use a DNA methylation kit to perform the bisulfite conversion reaction using a total of 500 ng of the genomic DNA, according to manufacturer's protocol. Elute in 10 µL of elution buffer (50 ng/µL).
2. Perform the DNA methylation profiling using a commercial kit according to manufacturer's protocol.

4. Dot Blot Analysis

1. Denature the isolated DNA (1 mg per sample) in 0.1 M NaOH for 10 min at 95 °C. Neutralize the DNA with 1 M NH₄OAc on ice, and then dilute two-fold. Spot 2 µL of the serial diluted genomic DNA on an N⁺ membrane.
2. Blot the membrane at 80 °C for 30 min.
3. 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.
4. 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 °C overnight.
5. 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.
6. Wash the membrane for 5 min three times in TBS-T.
7. 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's instructions.

Results

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...

Discussion

Chondrocyte dedifferentiation in vitro severely compromises the outcome of ACI in the treatment of cartilage defect repair^11,12. 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 dedifferentiation^4,6. Thus, it is imperative to...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
DMEM	Gibco Inc.	11965–092	Warm in 37 °C water bath before use
Phosphate-Buffered Saline (PBS)	HyClone Inc.	SH30256.01B	D-PBS, free of Ca2+/Mg2+
FBS	Gibco Inc.	10099-141
0.25% Trypsin/EDTA	Gibco Inc.	25200-056
1% Penicillin-Streptomycin	Gibco Inc.	15140-122
Chloroform	Mallinckrodt	4440
Isoamyl Alcohol	Sigma	I-3643
Phenol	Gibco BRL	15513-039
Proteinase K	Gibco BRL	24568-2
TAE buffer	Bio Whittaker	16-011V
Distilled Water	Gibco BRL	15230-170
1 M Tris-HCl	Biosharp Inc.	BL514A
Tween20	Biotopped Inc.	C58H114O26
BSA	Proliant Inc.	68700
Collagenase, Type II	Sigma-Aldrich	C6885
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Equipment
Hemocytometer	ISOLAB Inc.	075.03.001
Falcon 100 mm dish	Corning	353003
Centrifuge Tubes	TPP AG	91050	Gamma-sterilized
High-speed centrifuge	Eppendorf	5804R
ThermoMixer	MIULAB	MTH-100
Carbon dioxide cell incubator	Thermo scientific	3111
Chemi-imaging Analyse System	UVITEC Cambridge	ALLIANCE