Gene expression profiling of chondrogenic differentiation by dexamethasone-conjugated polyethyleneimine with SOX trio genes in stem cells
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During differentiation of stem cells, it is recognized that molecular mechanisms of transcription factors manage stem cells towards the intended lineage. In this study, using microarray-based technology, gene expression profiling was examined during the process of chondrogenic differentiation of human mesenchymal stem cells (hMSCs). To induce chondrogenic differentiation of hMSCs, the cationic polymer polyethyleneimine (PEI) was coupled with the synthetic glucocorticoid dexamethasone (DEX). DEX/PEI could be polyplexed with anionic plasmid DNAs (pDNAs) harboring the chondrogenesis-inducing factors SOX5, SOX6, and SOX9. These are named differentiation-inducing nanoparticles (DI-NPs).
A DI-NP system for inducing chondrogenic differentiation was designed and characterized by dynamic light scattering and scanning electron microscopy (SEM). Chondrogenic induction of hMSCs was evaluated using various tools such as reverse-transcription polymerase chain reaction (RT-PCR), Western blotting, confocal fluorescent microscopy, and immunohistochemistry analysis. The gene expression profiling of DI-NP-treated hMSCs was performed by microarray analysis.
The hMSCs were more efficiently transfected with pDNAs using DI-NPs than using PEI. Moreover, microarray analysis demonstrated the gene expression profiling of hMSCs transfected with DI-NPs. Chondrogenic factors including SOX9, collagen type II (COLII), Aggrecan, and cartilage oligometric matrix protein (COMP) were upregulated while osteogenic factors including collagen type I (COLI) was downregulated. Chondrogenesis-induced hMSCs were better differentiated as assessed by RT-PCR, Western blotting analyses, and immunohistochemistry.
DI-NPs are good gene delivery carriers and induce chondrogenic differentiation of hMSCs. Additionally, comprehensive examination of the gene expression was attempted to identify specific genes related to differentiation by microarray analysis.
KeywordshMSC Dexamethasone PEI DI-NP SOX trio mRNA profiling
Critical aggregation concentration
Collagen type 1
Collagen type 2
Cartilage oligomeric matrix protein
Fluorescence-activated cell sorting
Human mesenchymal stem cell
Mesenchymal stem cell
Molecular weight cut-off
Reverse-transcription polymerase chain reaction
Scanning electron microscopy
- SOX trio
SOX5, SOX6, and SOX9
Gene therapy is a revolutionary way to treat the causes of diseases at the gene level. In addition, it is possible to achieve long-term expression in vivo, and genetic information mutated by genetic recombination can be corrected. Many studies have focused on gene delivery as a promising tool to treat diseases at the genetic level [1, 2]. Genes encoding transcription factors, which regulate signaling pathways related to cell proliferation, growth, differentiation, and death, have been the focus of great interest [3, 4, 5, 6]. The internalization of genes encoding transcription factors related to stem cell differentiation has been extensively studied [7, 8, 9, 10]. Cells have been transfected with genes encoding specific transcription factors in an attempt to enhance differentiation towards the desired lineage and prevent differentiation towards other lineages [11, 12]. In this regard, we designed and synthesized dexamethasone (DEX)-conjugated polyethyleneimine (PEI), called DEX/PEI, as a gene carrier. DEX is a synthetic glucocorticoid and therefore binds to the glucocorticoid receptor in the cytosol via the ligand-receptor complex  after transfection of DEX/PEI. The complex is translocated to the nucleus, resulting in enlargement of the nuclear pore; this is helpful for gene delivery. Furthermore, DEX is already known to be an important factor for mesenchymal stem cells leading to osteoblasts, adipocytes, and chondrocytes , and DEX also alleviates inflammation. By conjugating DEX to PEI, the efficiency of gene delivery could be improved, facilitating induction of differentiation with low cytotoxicity and low effects of inflammation when applied to an in-vivo study .
Cationic PEI of DEX/PEI could be easily complexed with anionic plasmid DNA (pDNA) harboring three master chondrogenesis factors: SOX5 (specifically L-SOX5), SOX6, and SOX9 . SOX9 is expressed in chondrogenic progenitor cells and mature chondrocytes and induces expression of cartilage-related genes such as collagen type 2 alpha 1 and aggrecan . L-SOX5 and SOX6 are excited at the stage of prechondrocytes in the presence of SOX9 and increase cartilage-specific gene expression together with the existence of SOX9 . These DEX/PEI complexes with pDNA of SOX5, SOX6, and SOX9 are termed differentiation-inducing nanoparticles (DI-NPs), and DI-NPs could effectively induce chondrogenic differentiation of stem cells. Mesenchymal stem cells (MSCs) are one of the broadly used stem cell types for repairing cartilage defects [19, 20, 21]. In this study, DI-NPs were applied to MSCs for inducing chondrogenic differentiation. The differentiation of MSCs into chondrocytes has not, however, been overly studied for gene expression profiling using microarray . Through gene expression profiling, it is possible to measure and compare the expression level of mRNAs in differentiation-induced cells and noninduced cells. This allows the identification of genes that are overexpressed or underexpressed in differentiation-induced cells. The expression profile may provide information regarding the level of differentiation of an induced model by a particular tool [23, 24]. Gene expression profiling using microarrays is a major tool for the measurement of levels and patterns of gene expression. It can be used to identify diseases and uncover treatments in clinical medicine. Moreover, analyses of gene expression can be clinically useful for therapeutic application of stem cells toward the treatment of diseases related to cartilage defects.
Preparation of nanoparticles (PEI and DEX/PEI)
Tetramethylrhodamine (TRITC; 1 mg; Sigma Aldrich, St. Louis, MO, USA) dissolved in 3 mL dimethyl sulfoxide (DMSO) was mixed with 500 mg branched PEI (bPEI; 25,000 Da; Sigma Aldrich, St. Louis, MO, USA) dissolved in 10 mL distilled water for 12 h at room temperature. The solution was then dialyzed (molecular weight cut-off (MWCO), 1000 Da) for 2 days in the dark; and TRITC-conjugated bPEI (PEI) were synthesized. TRITC/DEX-conjugated bPEI (DEX/PEI) was synthesized via a conventional carbodiimide reaction. The bPEI (250 mg, 10 μmol) was stirred in 10 mL DMSO until completely dissolved. Then, a mixture of DEX (25 mg, 50.76 μmol; Steraloids, Newport, RI, USA), dicyclohexylcarbodiimide (DCC; 16 mg, 77.55 μmol), and n-hydroxysuccinimide (NHS; 16 mg, 139.02 μmol) prepared in 5 mL DMSO was added to the bPEI solution and incubated for 24 h at room temperature. Subsequently, 1 mg TRITC dissolved in 3 mL DMSO was added dropwise to this solution over 12 h. The final solution was dialyzed (MWCO, 1000 Da) against distilled water for 3 days. The final products were obtained by lyophilization.
Characterization of nanoparticles
First, the size and surface charge of the nanoparticles (NPs) were measured with a Zetasizer Nano ZS apparatus (Malvern, USA). NPs were dispersed with distilled water. Twenty measurements were conducted per sample. The mean diameter and size distribution of NPs was evaluated, and morphology of NPs was visualized by scanning electron microscopy (SEM). A microscope coverglass (18 mm diameter; 0111580) was coated with NPs and dried overnight. The samples were coated with platinum using a PT Sputter (E-1030, Hitachi, Japan) and finally visualized by SEM (S-4700, Hitachi, Japan). The critical aggregation concentrations (CACs) of PEI and DEX/PEI were determined with a fluorescence spectrophotometer (RF-5301PC, Shimadzu, Japan) using Hoechst 33342 as a fluorescent probe. Briefly, a stock solution of Hoechst 33342 (1.4 × 10–3 M) was prepared in double-distilled water. Various concentrations of DEX/PEI (2.4 × 10–4 to 0.5 mg/mL) were added to this solution. The final concentration of Hoechst 33342 was 7.0 × 10−4 M in each case. Fluorescence was measured with excitation and emission wavelengths of 355 and 457 nm, respectively.
The hMSCs, isolated from the bone marrow of a 20-year-old male, were purchased from Lonza Walkersville Inc. (PT-2501) and cultured in alpha minimum essential medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution. For the 3D culture system, the hMSCs were pelleted in a 15-mL conical tube by centrifugation at 1300 rpm for 3 min. The cell pellets were cultured for 3 weeks, changing the medium every 2–3 days, and maintained their ball-like shape.
Triple gene transfection of hMSCs using trio-coated PEI and DI-NPs
The hMSCs were transfected with pDNAs harboring SOX5, SOX6, and SOX9 using the optimal concentrations of PEI and DEX/PEI (trio-coated PEI and DI-NPs) in serum-free medium for 20 min and incubated for 4 h. Thereafter, SOX5/6/9 protein expression was evaluated by FACS, Western blotting, and fluorescence confocal microscopy (LSM 880 META, Zeiss).
Evaluation of the cytotoxicity of trio-coated PEI and DI-NPs
Cytotoxicity was evaluated with a cell counting kit (CCK)8 kit and a Live/Dead kit (Invitrogen, Carlsbad, CA, USA). A total of 2 × 105 hMSCs, treated with trio-coated PEI or DI-NPs, were treated with CCK8 solution for 2 h at 37 °C. The optical density at 450 nm was measured using a spectrophotometer. The hMSCs, treated with trio-coated PEI or DI-NPs, were incubated in a solution containing 2 μM calcein AM (to stain live cells) and 4 μM EthD-1 (to stain dead cells) for the Live/Dead assay, and visualized by fluorescence microscopy (EVOS Fl). The hMSCs were treated with 1, 3, 5, 7, and 10 μg DI-NPs for 4 h. The state of the cells treated with 1 μg DI-NPs was confirmed by FACS.
Evaluation of the cellular uptake of trio-coated PEI and DI-NPs
The hMSCs were seeded onto a microscope coverglass (18 mm diameter; 0111580, Marienfeld) and treated with trio-coated PEI and DI-NPs for 0.5, 1, and 4 h. The uptake of trio-coated PEI and DI-NPs was visualized by fluorescence confocal microscopy (LSM 880 META, Zeiss). The uptake of trio-coated PEI and DI-NPs was also visualized by staining endosomes. The NP-treated hMSCs were exposed to the Cell Light® Reagent BacMam 2.0 (2 μL per 10,000 cells), which stains early endosomes, for 16 h at 37 °C and visualized by fluorescence confocal microscopy.
Evaluation of chondrogenic and osteogenic markers
The mRNA and protein were extracted from hMSCs cultured in two-dimensional and 3D systems at 1, 3, 7, 14, and 21 days after transfection. Thereafter, mRNA and protein expression were evaluated by RT-PCR and Western blotting, respectively.
Total RNA was extracted using TRIzol (Invitrogen), and its quality was determined using a spectrophotometer (NanoDrop 2000) and an Agilent Bioanalyzer™ 2100 system. Thereafter, cRNA was synthesized from 10 ng RNA with the WT Pico Reagent Kit (Affymetrix) according to the manufacturer’s instructions. The samples were hybridized onto the Human Gene 2.0 ST Array (Affymetrix) for 17 h at 45 °C according to the manufacturer’s instructions. The arrays were scanned using a GeneChip Scanner 3000 7G (Affymetrix). After normalization, the genes related to differentiation and proliferation were selected and data processing was performed using GeneSpring GX13.1.1.
Histology and immunofluorescence
The hMSCs cultured in 3D systems were fixed with 4% paraformaldehyde for 1 h. For hMSCs cultured in the 3D system, the pellets were embedded in optimal cutting temperature compound (TISSUE-TEK 4583; Sakura Finetek Inc.) and frozen at −80 °C. The frozen samples were sliced into sections (9 μm thick) using a cryotome (HM 525, MICROM) at −25 °C. All samples were stained with hematoxylin (HHS16, Sigma Aldrich) and eosin (110116, Sigma Aldrich). Staining with Alcian blue (A3157, Sigma Aldrich), Safranin O (HT90432, Sigma Aldrich), and Masson’s trichrome (HT15, Sigma Aldrich) was performed to evaluate chondrogenic differentiation. All staining was performed according to the manufacturer’s instructions. Immunofluorescence was performed under humidified conditions using primary antibodies against SOX9, type 1 collagen (COLI), type 2 collagen (COLII), and matrix metallopeptidase (MMP) 3. Thereafter, the samples were stained with fluorescently labeled secondary antibodies (1:500; Thermo Scientific), incubated with 4,6-diamidino-2-phenylindole (DAPI) for 10 min to stain the nuclei, and visualized by fluorescence confocal microscopy (LSM 880 META, Zeiss).
All data are shown from at least three independent experiments performed in triplicate. Statistical comparisons were carried out by the Student’s t test and one-way analysis of variance (ANOVA). Probability less than 0.05 was considered statistically significant.
Preparation and characterization of trio-coated PEI and DI-NPs
Evaluating cytotoxicity and uptake efficiency of trio-coated PEI and DI-NPs into hMSCs
Evaluating expression efficiency of trio-coated PEI and DI-NPs
The hMSCs transfected with trio-coated PEI or DI-NPs were evaluated by Western blotting and RT-PCR (Fig. 2e). The mRNA and protein expression of SOX5, SOX6, and SOX9 were detected in cells transfected with either gene carrier (Fig. 2e, panels b and c), demonstrating that all three SOX genes were readily delivered into hMSCs, transcribed, and translated. The protein expression of enhanced green fluorescent protein (EGFP)-tagged SOX5, enhanced yellow fluorescent protein (EYFP)-tagged SOX6, and DsRed-tagged SOX9 in hMSCs was evaluated by confocal laser microscopy using specific antibodies. The results demonstrated that SOX5, SOX6, and SOX9 proteins were expressed in hMSCs (Fig. 2f). Merged images showed that all three SOX genes were simultaneously expressed (Fig. 2f, panels d and h). Moreover, the SOX proteins were colocalized, indicating that all three genes were simultaneously delivered into hMSCs. Together, these results demonstrate that fluorescently labeled SOX5, SOX6, and SOX9 were co-delivered into hMSCs using either gene carrier. The uptake of PEI and DI-NPs was examined for a short period (Additional file 1: Figure S4). In present study, the PEI and DI-NPs showed localization in the cytosol of hMSCs in a short time. The hMSCs were transfected with pDNA harboring green fluorescent protein (GFP) using various amounts of PEI or DEX/PEI (Additional file 1: Figure S5). Following transfection with 1, 3, or 5 μg PEI, 20% of the hMSCs were GFP-positive (Additional file 1: Figure S5A, panels a–c). However, 39%, 25%, and 34% of hMSCs were GFP-positive following transfection with 1, 3, and 5 μg DEX/PEI (Additional file 1: Figure S5A, panels d–f). DEX/PEI has the higher transfection efficiency than PEI due to its compacted morphology and ease of endocytosis into cells, resulting from its hydrophobic moiety from dexamethasone. Therefore, 1 μg DEX/PEI was sufficient to deliver GFP pDNA into hMSCs. GFP protein expression was detected by Western blotting (Additional file 1: Figure S5B). GFP expression differed according to the type and amount of gene carrier. The GFP band was denser for hMSCs transfected using DEX/PEI than for those transfected using PEI. GFP protein expression decreased as the amount of DEX/PEI increased. The quantification of GFP expression in hMSCs confirmed that GFP expression decreased as the amounts of PEI and DEX/PEI increased (Additional file 1: Figure S5B). The fluorescence microscopy analysis of GFP expression in hMSCs is presented in Additional file 1 (Figure S6). Consistent with the FACS and Western blot assays, the fluorescence intensity of GFP was higher in hMSCs transfected using DEXPEI than in those transfected using PEI (Additional file 1: Figure S6, panels d and e).
Evaluating the inductive level of chondrogenic differentiation depending on time over 21 days
Evaluating differentiation- and proliferation-related markers in hMSCs transfected with DI-NPs by microarray analysis
Evaluating mRNA expression level of differentiation-related genes in hMSCs transfected with DI-NPs by microarray analysis
Evaluating the inductive level of chondrogenic differentiation in a 3D system
The efficient delivery of chondrogenesis-related genes to stem cells is one of the important aims for the treatment of cartilage defects in stem cell therapy . There are few studies on cell signaling involved in the differentiation of mesenchymal stem cells into chondrocytes, so it is important to study the mechanism of chondrogenic differentiation. Nanoparticles have already been fabricated and studied a lot for this aim, considering biocompatibility, biodegradability, and efficiency of uptake into cells. In this regard, DI-NPs were designed and fabricated for inducing chondrogenic differentiation. DI-NPs are composed of DEX/PEI and pDNA of the SOX trio (SOX5, SOX6, and SOX9).
Although DEX/PEI contains a lower amount of PEI in the same volume compared with PEI, DEX/PEI has a higher transfection efficiency. DEX/PEI improved transfection efficiency compared with PEI alone due to two favorable effects. First, DEX when conjugated to PEI led to nuclear entry of DEX/PEI being more effective in cells. Second, in an aqueous solution, hydrophobic DEX would be recruited in a core and make hydrophilic PEI exist on the surface of the structure. Therefore, the higher density of the positive charge is represented on the surface of DEX/PEI, resulting in better complexation with pDNA. This means that the compressed and stable structure of DEX/PEI is effective in delivering pDNA to MSCs.
In addition, SOX9, constituting a DI-NP, is an essential transcription factor while, though not necessarily required in chondrogenesis, SOX5 and SOX6 cooperate with SOX9 to activate the expression of COLII and Aggrecan . This strongly suggests that the SOX trio proteins fulfill most of their functions in differentiated chondrocytes cooperatively rather than independently of one another.
Here, overexpressed SOX9, along with SOX5 and SOX6 by DI-NPs, increased the expression of COLII, Aggrecan, and COMP. An interaction between these extracellular matrix (ECM) proteins in differentiated chondrocytes provided a proper environment for the progression of chondrogenesis. Moreover, the genetic level of stem cells induced by DI-NPs or those noninduced were screened with genes related to chondrogenic differentiation. Chondrogenic differentiation-related genes (SOX9, COLII, Aggrecan, and COMP) were upregulated and osteogenic differentiation-related genes (COLI) were downregulated. Furthermore, the increase in collagen family genes and the decrease in MMP family genes observed in hMSCs induced chondrogenic differentiation. MMPs are proteolytic enzymes for the degradation of ECM proteins . Thus, reduced MMP3 expression and increased expression of ECM-related genes increased the levels of proteoglycans, suggesting some degree of differentiation into chondrocytes, indicating that DI-NPs induced chondrogenic differentiation of hMSCs.
This study increases the knowledge on SOX5, SOX6, and SOX9, uncovering molecular networks for the regulation of chondrogenesis. It has also raised new questions for further investigation. For instance, we need to set a framework for complementary studies to establish the actions of the SOX trio at the stages of chondrocyte differentiation. We also need to uncover transcription factors that are likely to functionally interact positively or negatively with the SOX trio in chondrocytes. In addition, these data represent a comprehensive examination of gene expression across the process of chondrogenic differentiation and we attempted to identify specific genes for differentiation using microarray analysis. The gene-based investigations presented here, as well as using additional approaches to represent induction of differentiation, will be helpful to understand the processes of differentiation. Furthermore, through detailed studies of microarray analysis and based on a comprehensive understanding of the factors related to the cartilage tissue engineering process, further research is needed on systemic induction of chondrogenic differentiation by controlling the cell proliferation (cell cycle), inhibition of osteogenesis, and promoting induction of chondrogenesis.
The present study demonstrates that DI-NPs can be used to deliver genes into hMSCs. This carrier can be used to develop a nonviral gene delivery system with increased cellular uptake to enhance expression at the mRNA and protein levels. Furthermore, we show that DI-NPs are good tracers for gene delivery and could potentially be used for imaging and tracing using various tools in vitro and in vivo. Additionally, microarray analysis revealed that the DI-NP-transfected hMSCs showed superior expression of genes related to the differentiation and proliferation of cells. Finally, the hMSCs transfected with the trio of SOX genes complexed with DI-NPs were successfully differentiated for chondrogenesis. Thus, the DI-NPs synthesized in the present study will contribute to cartilage tissue engineering.
This work was supported by the National Research Foundation of Korea (NRF), grants funded by the Korean Government (NRF-2017R1A2A1A05001264, NRF-2017M3A9B4031169, NRF-2017M3A9C6061360 and NRF-2018R1D1A1B07040353).
Availability of data and materials
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
SWY was responsible for study planning, management, and manuscript writing. She was further involved in cellular and biological work. HS was involved in fabrication of DEX/PEI, performed analysis of DEX/PEI, and was involved in manuscript writing. JSL, HJO, and HJK were involved in cellular work and analysis of differentiation. K-HP and JSP initiated and supervised this study and were involved in project initiation and management, as well as interpretation and discussion of the results. All authors read and approved the final manuscript.
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