Korean J Physiol Pharmacol.  2020 Nov;24(6):463-472. 10.4196/kjpp.2020.24.6.463.

Direct reprogramming of fibroblasts into diverse lineage cells by DNA demethylation followed by differentiating cultures

• Yang DW
• Moon J
• Ko HM
• Shin YK
• Fukumoto S
• Kim SH
• Kim MS

• KMID: 2507729
• DOI: http://doi.org/10.4196/kjpp.2020.24.6.463

Abstract

Direct reprogramming, also known as a trans-differentiation, is a technique to allow mature cells to be converted into other types of cells without inducing a pluripotent stage. It has been suggested as a major strategy to acquire the desired type of cells in cell-based therapies to repair damaged tissues. Studies related to switching the fate of cells through epigenetic modification have been progressing and they can bypass safety issues raised by the virus-based transfection methods. In this study, a protocol was established to directly convert fully differentiated fibroblasts into diverse mesenchymal-lineage cells, such as osteoblasts, adipocytes, chondrocytes, and ectodermal cells, including neurons, by means of DNA demethylation, immediately followed by culturing in various differentiating media. First, 24 h exposure of 5-azacytidine (5-aza-CN), a well-characterized DNA methyl transferase inhibitor, to NIH-3T3 murine fibroblast cells induced the expression of stem-cell markers, that is, increasing cell plasticity. Next, 5-aza-CN treated fibroblasts were cultured in osteogenic, adipogenic, chondrogenic, and neurogenic media with or without bone morphogenetic protein 2 for a designated period. Differentiation of each desired type of cell was verified by quantitative reverse transcriptase-polymerase chain reaction/western blot assays for appropriate marker expression and by various staining methods, such as alkaline phosphatase/alizarin red S/oil red O/alcian blue. These proposed procedures allowed easier acquisition of the desired cells without any transgenic modification, using direct reprogramming technology, and thus may help make it more available in the clinical fields of regenerative medicine.

Keyword

Cell lineage; Cellular reprogramming; DNA demethylation; Epigenesis, genetic 

Figure

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  Fig. 1 Epigenetic modification in 5-azacytidine (5-aza-CN)-treated NIH-3T3 cells. (A) After 24 h of 5 μM 5-aza-CN treatment, morphologic changes of cells were observed under an inverted microscope. (a) Untreated; (b) 5-aza-CN-treated; (c) incubated in fresh media without 5-aza-CN for an additional 24 h after treatment with 5-aza-CN. (B) After treatment at the indicated concentrations of 5-aza-CN for 24 h, the global DNA methylation patterns determined by using a methylated DNA quantification kit. (C) The expression of pluripotent stem cell markers such as SOX2, KLF4, NANOG and OCT4 genes was examined by conventional (left) and quantitative RT-PCR (RT-qPCR, right) with or without 5 μM 5-aza-CN for 24 h. (D) The protein expression of CD146, mesenchymal stem cell marker and OCT4 was analyzed by flow cytometry with or without 5 μM 5-aza-CN for 24 h, and quantified to a mean fluorescent intensity (MFI) value. Control (Ctrl) means untreated. These data represent three independent experiments. The values shown represent means ± standard errors. SOX2, sex determining region Y(SRY)-box 2; KLF4, Krüppel-like factor 4; OCT4, octamer-binding transcription factor 4. *p < 0.05.

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  Fig. 2 Osteogenic differentiation of 5-azacytidine (5-aza-CN)-treated NIH-3T3 cells. (A) The schematic diagram of this study is based on direct reprogramming principles. After 7 days culture in osteogenic differentiation medium (DMEM supplemented 2% FBS containing 170 μM AA, 5 mM β-GP and 200 ng/ml BMP2), expression of osteogenic marker genes such as alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), Runt-related transcription factor 2 (RUNX2) and osterix (OSX) in 5 μM 5-aza-CN-treated NIH-3T3 cells was determined by RT-qPCR (B), and osteogenic differentiation of 5-aza-CN-treated NIH-3T3 was verified by ALP and alizarin red S (AR-S) staining on days 7 and 14, respectively (C). High magnification images of AR-S staining are shown in the lower panel. (D) Densities of ALP stain were quantified by Scion Image software. AR-S stain was extracted with 10% cetylpyridinium chloride and quantified by measuring the absorbance at 570 nm using an ELISA reader. These data represent three independent experiments. The values shown represent means ± standard errors. *p < 0.05.

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  Fig. 3 Adipogenic and chondrogenic differentiation of 5-azacytidine (5-aza-CN)-treated NIH-3T3 cells. (A) After 7 days culture in adipogenic differentiation medium containing DMEM supplemented 2% FBS with 0.5 mM IBMX, 1 μM DEX, 100 μM, 10 μg/ml insulin and 200 ng/ml BMP2, expression of adipogenic marker genes such as adipocyte protein 2 (AP2), CCAAT-enhancer-binding proteins (C/EBP), peroxisome proliferator-activated receptor gamma (PPARG) in 5 μM 5-aza-CN-treated NIH-3T3 cells was quantified by RT-qPCR. (B) The lipid droplet formations were observed by Oil-Red O staining on day 14 and high magnification images of Oil-Red O staining are shown in the lower panel. (C) After 7 days of chondrogenic differentiation medium culture, expression of chondrogenic marker genes such as aggrecan (ACAN), type II collagen (COL2), SRY-box 9 (SOX9) in 5 μM 5-aza-CN-treated NIH-3T3 cells was quantified by RT-qPCR. (D) The chondrogenic matrix formation was observed by Alcian blue staining on day 14 and high magnification images of Alcian blue staining are shown in the lower panel. These data represent three independent experiments. The values shown represent means ± standard errors. *p < 0.05.

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  Fig. 4 Neurogenic differentiation of 5-azacytidine (5-aza-CN)-treated NIH-3T3 cells. (A, B) After 5 and 14 days culture in neurogenic differentiation medium (NM) containing DMEM supplemented 2% FBS, 2% dimethyl sulfoxide and 100 μM butylated hydroxyanisole, expressions of neuronal specific intermediated filament genes such as neurofilament medium (NFM) and Nestin in 5 μM 5-aza-CN-treated NIH-3T3 cells were quantified by RT-qPCR and Western blot analysis, respectively. Arbitrary densities of proteins were quantified by Scion Image software, as shown in the graph below. (C) After 14 days of neurogenic differentiation medium culture, morphologic changes in 5 μM 5-aza-CN-treated NIH-3T3 cells were observed under an inverted microscope (arrows), and NFM protein expression was visualized by immunofluorescence staining. Ctrl means untreated. These data represent three independent experiments. The values shown represent means ± standard errors. *p < 0.05.

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